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Tissue ACE inhibition and sodium status in left ventricular dysfunctionWestendorp, Bart

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Tissue ACE Inhibition and Sodium Status in Left

Ventricular Dysfunction

Titelblad en inhoudsopgave versie 1 21 juli 2005

The work described in this thesis was financially supported by Menarini Ricerche SpA, Firenze, Italia Publication of this thesis was financially supported by: AstraZeneca BV, Hope Farms / abdiets (Woerden), Menarini BV, Merck Sharpe & Dohme BV, Pfizer BV, Sanofi-Aventis BV, and Servier Nederland Farma BV. CIP-gegevens koninklijke bibiotheek, Den Haag Westendorp, Bart Tissue ACE inhibition and sodium status in left ventricular dysfunction. Proefschrift Groningen. Met literatuuropgave en samenvatting in het Nederlands ISBN 90-9019792-3 All rights reserved. No part of this publication may be reproduced, or transmitted in any form or by any means, without permission of the author. Typesetting and layout: Bart Westendorp, Groningen Printed by: Febodruk BV, Enschede Cover illustration: ©Maaike van der Meulen, Groningen. The cover illustration symbolizes protection of heart as well as kidneys and vasculature by ACE

inhibitors; this class of drugs was initially designed based on hypotensive peptides extracted from

snake venom.


Rijksuniversiteit Groningen

Tissue ACE Inhibition and Sodium Status in Left

Ventricular Dysfunction

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningen op gezag van de

Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op

maandag 10 oktober 2005 om 16:15 uur

door

Bart Westendorp

geboren op 7 mei 1977 te Heerde


Promotores: Prof. dr. W.H. van Gilst Prof. dr. D.J. van Veldhuisen Copromotores: Dr. R.G. Schoemaker Dr. H. Buikema Beoordelingscommissie: Prof. dr. A.H.J. Danser Prof. dr. G.J. Navis Prof. dr. F. Zijlstra


Paranimfen: Mirjam van Albada Jan Peter Vergeer The research described in this thesis was funded by a grant of the Netherlands Heart Foundation (NHF grant 2000.146). Financial support by the Netherlands Heart Foundation and the Groningen University Institute for Drug Exploration for the publication of this thesis is gratefully acknowledged.


Table of contents

Chapter: Page: 1. General introduction and aim of the thesis. 9 2. Dietary sodium restriction specifically potentiates left 21 ventricular ACE inhibition by zofenopril, and is associated with attenuated hypertrophic response in rats with myocardial infarction. J. Renin Angiotensin Aldosterone Syst. 2004; 5: 27-31

3. Beneficial effects of add-on hydrochlorothiazide in rats with 35 myocardial infarction optimally treated with quinapril. Eur. J. Heart Fail. 2005; in press

4. Hydrochlorothiazide increases plasma and tissue ACE inhibitor 51 drug levels in rats with myocardial infarction: differential effects on lisinopril and zofenopril. Submitted for publication

5. Adverse renal effects of hydrochlorothiazide in rats with 67 myocardial infarction treated with ACE inhibition. Submitted for publication

6. Progressive left ventricular hypertrophy after withdrawal 79 of long-term ACE inhibition following experimental myocardial infarction. Eur. J. Heart Fail. 2005; in press

7. Improvement of EDHF by chronic ACE Inhibition declines 95 rapidly after withdrawal in rats with myocardial infarction. J. Cardiovasc. Pharmacol.; accepted for publication

8. General discussion. 111 Summary. 118 Samenvatting in het Nederlands. 122 Dankwoord. 126 Curriculum vitae and list of publications. 129

Chapter 1

Introduction and Aim of the Thesis.

10

Chapter 1 - Introduction & aim versie 2 11augustus 2005

Chapter 1

Left ventricular dysfunction

This thesis deals with left ventricular (LV) dysfunction. A problematic aspect of this pathology is its progressive character, eventually leading to chronic heart failure (CHF). The end stage - chronic heart failure - is a disease which is characterized by a grave prognosis, with a 5-year survival of ±25%1. Furthermore, heart failure is an immobilizing disease, which is characterized by impaired exercise capacity and edema in lungs and limbs, and is associated with numerous hospitalizations. Currently, as much as 1-2% of the general population in developed countries suffers from heart failure, and its prevalence is increasing2. Figure 1 illustrates the development and progression of LV dysfunction in time, starting with an index event. In the majority of cases this event is myocardial infarction3,4. Myocardial infarction causes a loss of contractile LV tissue, which leads to structural remodeling of the infarct area (scar formation), but also of the remaining viable tissue. The latter initiates a downward spiral of further deterioration of cardiac pump function and remodeling (figure 1). Eventually this leads to the clinical syndrome of chronic heart failure. LV remodeling involves cardiomyocyte hypertrophy, which results in a relative reduction of capillary density and increased oxygen diffusion distance, and a subsequent deterioration of cardiomyocyte metabolism and function. A second key process in LV remodeling is proliferation of fibroblasts and fibrosis, which stiffens the ventricle and impairs cardiac contractility. Thirdly, the progression of LV dysfunction towards chronic heart failure after myocardial infarction is accompanied by increasing peripheral vascular resistance, which is a result of reduced production of endothelium-derived vasodilator substances5,6 and myogenic constriction of resistance arteries7. This may serve as a mechanism to compensate for reduced cardiac output to redistribute blood flow to the organs. However the increase in peripheral resistance may become excessive and actually trigger the progression of LV dysfunction8,9. Excess activation of physiological compensatory regulation systems, in particular the renin-angiotensin-aldosterone system (RAAS), plays a central role in all these processes mediating the development of chronic heart failure. Hence, pharmacological intervention with the RAAS substantially improves prognosis of patients with LV dysfunction.

ACE inhibitor therapy

Intervention with the RAAS by inhibition of the angiotensin I converting enzyme (ACE) is first-line therapy to attenuate the progression of LV dysfunction. ACE inhibitors were initially designed as vasodilator drugs about 25 years ago to treat high-renin hypertensive patients, and to reduce fluid overload symptoms in patients with heart failure. The mechanism by which ACE inhibition attenuates the progression of left ventricular dysfunction was initially thought to be vasodilation, resulting in reduced cardiac workload, as ACE inhibition prevents the degradation of the vasodilating peptide bradykinin, and the formation of the vasoconstricting peptide angiotensin II. However, superiority of ACE inhibitors over directly acting vasodilator

11


Introduction and Aim

drugs to reduce mortality and improve of cardiac function10-12 clearly pointed towards effects beyond vasodilation. By now, a vast amount of evidence has shown that angiotensin II directly triggers cardiomyocyte hypertrophy and cardiac fibrosis on the cellular level, and conversely that RAAS blockade effectively inhibits cardiac and vascular remodeling processes. ACE inhibitors provide protection against end-organ damage in several cardiovascular pathologies: renal failure, diabetic nephropathy, hypertension, LV dysfunction after myocardial infarction, and overt CHF. During the last two decades, numerous landmark trials have unambiguously shown that ACE inhibition improves cardiac function13, prognosis14, and quality of life15 in patients with myocardial infarction and/or chronic heart failure.

Sodium restriction

Although ACE-inhibition slows the gradual progression of myocardial dysfunction towards overt chronic heart failure (CHF), it does not prevent it. Hence, morbidity and mortality remain high, and further optimization of therapy is warranted. Apart from developing new strategies that intervene with remodeling via different mechanisms, one should ask whether current treatment options are used optimally. In this respect, an important potential target could be intervention with the sodium balance. In numerous studies, dietary sodium restriction has been shown to increase the efficacy of ACE-I treatment. This potentiation is consistently found across different clinical an experimental conditions, including proteinuric renal dysfunction, and essential hypertension16-18. Conversely, sodium loading can completely annihilate these

Figure 1. Schematic representation of the progression of remodeling after myocardial

infarction (or another index event). The picture shows alterations in LV dimensions after

myocardial infarction in a cross-sectional view through the ventricles.

12


Chapter 1

therapeutic effects of ACE inhibition19,20. However, intervention with the sodium balance is currently not used as a deliberate strategy to optimize the therapy response to ACE inhibition in patients with LV dysfunction. Before discussing the potential mechanisms underlying interaction between sodium depletion and ACE inhibition, effects of a negative sodium balance per se during LV dysfunction will be addressed.

Sodium depletion during left ventricular dysfunction

There are several strategies to induce a negative sodium balance, of which dietary sodium restriction is the first obvious approach. However, long-term patient compliance with low sodium diets is not always achieved. In clinical practice, sodium depletion with diuretics at a moderate salt intake may be more feasible, since diuretic therapy is easier to maintain. Moreover, approximately 30% of post-MI patients are currently treated with diuretics to reduce fluid overload symptoms. Non-potassium sparing diuretics, notably the loop diuretic furosemide, are the most potent and most frequently used diuretics to reduce fluid overload. Hence in patients with post-MI LV dysfunction this class of diuretics could serve for sodium depletion to optimize ACE-I therapy too. However, based on their longer duration of action and absence of peak diuresis, thiazide diuretics would be the first choice of treatment to use for this therapeutic strategy. Finally, of the different potassium-sparing diuretics, solely aldosterone receptor antagonists are sufficiently potent to promote sustained significant sodium excretion21,22. The direct cardiac effects of these interventions are described below. Dietary sodium restriction or non-potassium sparing diuretics attenuate LV hypertrophy due to hypertension, mainly by mechanical unloading of the heart23. Also in an experimental model for myocardial infarction in combination with hypertension development of LV hypertrophy was shown to be attenuated by treatment with a loop diuretic24. However, in a situation of normal or even decreased blood pressure, - as is generally the case after myocardial infarction - effects of sodium depletion per se on LV remodeling may be minimal. Indeed, Sharpe et al. reported no effects of long-term furosemide treatment on left ventricular function in patients with moderate LV dysfunction after myocardial infarction25. Furthermore neither chronic dietary sodium restriction, nor treatment with hydrochlorothiazide or furosemide showed to affect development of LV hypertrophy in rats with myocardial infarction26-30. It was shown that thiazides and loop diuretics cause venodilation at therapeutic concentrations, but it remains uncertain whether this could beneficially influence the progression of LV dysfunction31-34. Contrary to furosemide, the more recently developed loop diuretic torasemide was shown to beneficially affect LV remodeling. It significantly reduced morbidity and mortality compared to furosemide in the TORIC study, a randomized open-label trial in 1377 patients with heart failure35. Furthermore, the drug was shown to reduce myocardial fibrosis, while furosemide had no effect36. Thus, torasemide may prove to be preferable over furosemide in patients with left ventricular dysfunction, but double-blind randomized trials will be required. The most probable mechanism underlying these beneficial effects of torasemide may be interference with aldosterone. Firstly, it was shown that torasemide directly inhibits aldosterone synthesis in aldosterone-

13



producing cells37. Furthermore torasemide inhibited binding of aldosterone to its receptor38. Aldosterone receptor antagonists effectively attenuate (aldosterone-mediated) LV remodeling and endothelial dysfunction, and reduce morbidity and mortality in patients with post-MI LV dysfunction39 and severe heart failure40. To distinguish between effects of sodium depletion and aldosterone interference, diuretics directly interfering with aldosterone were not studied in the present thesis.

Optimization of ACE inhibition with sodium depletion?

In numerous studies, dietary sodium restriction was shown to augment the response to ACE-I therapy for renal dysfunction and essential hypertension16-18. Despite the substantial amount of clinical and experimental evidence, the mechanisms underlying this potentiation by sodium restriction are poorly understood. The following section provides an overview of potential factors involved. Presumably, activation of the RAAS is required for effective ACE inhibitor therapy. During ACE inhibition salt restriction may cause a shift of production of Ang II towards Angiotensin-(1-7) (Ang-(1-7), see figure 2). Increased production of Ang-(1-7) under

Figure 2. Simplified RAAS scheme showing potential balance of detrimental and beneficial

effects of angiotensin peptides. Activiating the RAAS with sodium restriction during ACE

inhibition may increase Ang-(1-7), shifting the ratio between effects of Ang II and Ang–(1-7).

14


Chapter 1

ACE inhibition has been proposed to underlie at least part of the therapeutic effects. In line with this, Ang-(1-7) appears to act as a physiological antagonist of angiotensin II41, and actually attenuates the progression of heart failure in rats with myocardial infarction42. The observation that reduction of salt intake in humans treated with an ACE-I further increases plasma Ang-(1-7) concentrations43 argues in favor of this mechanism. Secondly, sodium restriction may alter the expression of the Angiotensin II type 1 and 2 (AT1 and AT2) receptors. AT2-receptor stimulation has antihypertrophic and antifibrotic effects, counteracting the detrimental effects of AT1 stimulation44,45 (figure 3). During high dietary salt intake, expression of AT2 receptors decreases46, while AT1 expression increases47. Conversely, dietary sodium restriction could lead to a more favorable AT2/AT1 receptor ratio, causing the remaining Ang II that may be produced despite ACE inhibition48 actually to exert beneficial effects.

Figure 3. A scheme of the RAAS, showing its most biologically relevant peptides and

enzymes. Grey boxes indicate physiologically active angiotensin peptides. Numbers between

brackets indicate the sequence of amino acids in the different angiotensin-derived peptides.

15



Limitations of sodium depletion

Sodium depletion using diuretics is not by definition harmless, and may have unwanted effects. Firstly, thiazide and loop diuretics not only cause sodium depletion, but also increase the excretion of potassium and magnesium. Hypokalemia by chronic treatment can trigger cardiac arrhythmias, and indeed high doses of diuretic therapy have been associated with increased mortality due to ventricular arrhythmias in patients with LV dysfunction49,50. These results should however be interpreted with some caution, as these findings were done in post hoc studies, and diuretic-use is associated with severity of symptoms and cardiac impairment. Severity of cardiac dysfunction may be associated with incidence of ventricular arrhythmias, due to remodeling51. Secondly, diuretic therapy or dietary sodium restriction could cause further activation of the RAAS, which potentially accelerates cardiac remodeling. Indeed furosemide treatment caused accelerated progression towards severe heart failure induced by rapid pacing in pigs. This progression was associated with markedly increased plasma aldosterone concentrations, indicating further RAAS activation52. Treatment with an ACE inhibitor or AT1 receptor antagonist would theoretically block this RAAS-inducing effect of sodium depletion. However, a study in patients with mild-to-moderate heart failure showed that reduced sodium intake caused an increase in plasma aldosterone concentrations, albeit almost all patients were treated with ACE inhibitors53. This could be related to a phenomenon called “aldosterone escape”: high aldosterone concentrations despite ACE-I therapy54. This phenomenon may be caused by angiotensin conversion through chymase. Furthermore, ACE inhibitors can cause K+ retention, which in turn directly triggers aldosterone production. Aldosterone itself can aggravate LV dysfunction failure by causing endothelial dysfunction, fibrosis, and LV hypertrophy55-58. Aldosterone receptor antagonists are in this respect of obvious therapeutic importance, as apart from inducing natriuresis, they attenuate the deleterious effects of aldosterone (escape), and moreover have potassium-retaining effects. However, aldosterone antagonist-induced hyperkalemia occurs frequently, and is a serious concern, as it is associated with an increased risk of cardiovascular events59. Combining low doses of thiazide diuretics with aldosterone receptor antagonist is an option worth further study, as this strategy could establish efficient natriuresis without a net effect on potassium balance60.

Long-term ACE inhibition and its withdrawal

LV dysfunction after myocardial infarction is a structural disease of the heart, and ACE inhibition is not curative. Therefore ACE inhibition therapy should in principle be lifelong. However, about 10% of all patients do not tolerate ACE inhibitors due to serious side effects61. As a consequence ACE inhibitor withdrawal often occurs in clinical practice. In addition, discontinuation may occur because of patient’s incompliance62. Nevertheless, little has been described in literature about the actual consequences of cessation of therapy. This issue deserves attention for several reasons. Firstly, withdrawal of several cardiovascular drugs, such as β-blockers63, nitrates64, and statins65 can cause pronounced rebound effects, requiring stepwise cessation of therapy.

16


Chapter 1

Furthermore, in the Captopril And Thrombolysis Study (CATS), withdrawal of chronic ACE inhibition after the trial period caused a high incidence of ischemia-related events within one month66. As the expression of renin, the rate-limiting step in the RAAS cascade, is drastically increased during ACE inhibition therapy, occurrence of a rebound effect is probable. However, there are also arguments against pronounced detrimental effects of withdrawal. Antihypertensive effects of ACE inhibitors are sustained long after withdrawal in experimental models67,68. Furthermore, the beneficial effects of ACE inhibition on mortality rates decrease over time69,70, and consequences of cessation of chronic therapy may thus be limited.

Aim of the thesis

Summarizing the above, RAAS activation plays a major role in the progression of left ventricular dysfunction towards chronic heart failure. ACE inhibition therapy improves cardiac function and reduces morbidity and mortality. Although ACE-inhibition slows the gradual progression of myocardial dysfunction towards overt chronic heart failure (CHF), it does not prevent it. Apart from developing new strategies that intervene with remodeling via different mechanisms, one should ask whether current treatment options are used optimally. Accordingly, several issues concerning long-term efficacy and optimization of ACE-I therapy in relation to sodium status need further study. Hence, the aim of this thesis is to investigate the following two hypotheses: 1. Sodium depletion can safely be added to ACE inhibition therapy for left ventricular dysfunction after myocardial infarction, and does improve treatment outcome (chapters 2-5). 2. ACE inhibitor therapy still effectively blocks the RAAS after long treatment periods, and discontinuation results in rebound disease progression (chapters 6 and 7).

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Thrombolysis Study (CATS) Investigators. J Am Coll Cardiol. 1997;30:400-405.

67. Paull JR, Widdop RE. Persistent cardiovascular effects of chronic renin-angiotensin system inhibition

following withdrawal in adult spontaneously hypertensive rats. J Hypertens. 2001;19:1393-1402.

20


Chapter 1

68. Richer C, Fornes P, Vacher E et al. Trandolapril's protective effects in stroke-prone spontaneously

hypertensive rats persist long after treatment withdrawal. Am J Cardiol. 1994;73:26C-35C.

69. Hall AS, Murray GD, Ball SG. Follow-up study of patients randomly allocated ramipril or placebo for

heart failure after acute myocardial infarction: AIRE Extension (AIREX) Study. Acute Infarction

Ramipril Efficacy. Lancet. 1997;349:1493-1497.

70. Swedberg K, Kjekshus J, Snapinn S. Long-term survival in severe heart failure in patients treated with

enalapril. Ten year follow-up of CONSENSUS I. Eur Heart J. 1999;20:136-139.

Chapter 2

Dietary Sodium Restriction Specifically

Potentiates Left Ventricular ACE Inhibition

by Zofenopril, and is Associated with

Attenuated Hypertrophic Response in Rats

with Experimental Myocardial Infarction.

Bart Westendorp

Regien Schoemaker

Hendrik Buikema

Dick de Zeeuw

Dirk Jan van Veldhuisen

Wiek van Gilst

Chapter 2 versie 3 10 augustus 2005

Chapter 2

22

Abstract

Background: In patients with myocardial infarction-induced heart failure, angiotensin converting enzyme inhibitors (ACE-I) are proven effective therapy by inhibiting the progression towards overt heart failure. However, prognosis is still very poor, and optimization of therapy is warranted. Antihypertensive and renoprotective effects of ACE-I can be substantially enhanced by dietary sodium restriction. In line with the latter, the aim of the present study was to explore if dietary sodium restriction enhances the cardioprotective efficacy of ACE-I after myocardial infarction. Methods: Rats with myocardial infarction-induced left ventricular dysfunction received ACE-I therapy with zofenopril (5.5 mg kg-1 day-1 orally), either with or without dietary sodium restriction. ACE activity was measured in non-infarcted left ventricular tissue, kidneys, and plasma. Effects on cardiac hypertrophy were examined by means of organ weight/body weight ratios. After blood pressure measurements, functional consequences of therapy were evaluated as left ventricular pressure development in isolated perfused hearts. Results: Blood pressure was reduced after infarction, and further reduced by zofenopril, but not affected by sodium diet. Dietary sodium restriction alone had no effect on any of the measured parameters, zofenopril alone significantly reduced plasma and kidney ACE activity, but not left ventricular ACE activity, nor left ventricular weight/body weight. However, only when ACE-inhibitor therapy was combined with dietary sodium restriction, left ventricular ACE activity was significantly reduced. This effect was paralleled by inhibition of left ventricular hypertrophy. Neither treatment could be associated with effects on the myocardial infarction-induced reduction of in vitro left ventricular function. Conclusions: Effects of ACE inhibition with zofenopril can be potentiated by additional dietary sodium restriction. However, effects were tissue specific, since left ventricular -but not kidney or plasma- ACE activity was affected by the additional dietary sodium restriction. Effects on left ventricular ACE activity were paralleled by reduced left ventricular hypertrophy. Since measured parameters did not indicate adverse side effects, dietary sodium restriction may provide a safe strategy to improve ACE-inhibitor efficacy in patients with infarction-induced left ventricular dysfunction.


Dietary Sodium Restriction Potentiates LV ACE Inhibition

23

Introduction

Chronic activation of the renin-angiotensin-aldosterone system (RAAS) is regarded as one of the major causes of progressive deterioration of left ventricular pump function after acute myocardial infarction (MI). Accordingly, RAAS-inhibition with ACE-inhibitor therapy is associated with better cardiac function1, improved prognosis2, and increased quality of life3. Although ACE-inhibition slows the gradual progression of myocardial dysfunction towards established chronic heart failure, it does not prevent it4-6. Hence, morbidity and mortality remain high, and further optimization of therapy is warranted. One area which has not been explored yet in this context is optimization of ACE-I efficacy by dietary sodium restriction. A substantial load of clinical and experimental evidence shows that dietary sodium restriction can improve the antihypertensive and renoprotective effects of ACE-inhibition in patients with hypertension and renal dysfunction, respectively7-10. Vice versa, sodium loading may completely annihilate the effect of ACE-inhibitors11. In view of the fact that dietary sodium restriction is quite commonly advised to patients with LV dysfunction to reduce fluid retention, surprisingly little has been published about its effect on the efficacy of ACE-inhibitor therapy in post-AMI left ventricular (LV) dysfunction. The aim of the present study, therefore, was to explore the influence of dietary sodium restriction on the therapeutic effects of ACE-inhibitor treatment in rats with myocardial infarction. To this end, we studied the effects on blood pressure and cardiac hypertrophy, as well as ACE activity in plasma, and renal and cardiac tissue after 10 weeks of ACE-I treatment, with and without dietary sodium restriction.

Methods

Experimental protocol Male Wistar rats (Harlan, Zeist, The Netherlands) were subjected to experimental myocardial infarction (MI) by coronary ligation, or were sham operated (t=0). Two weeks after MI-induction (t=2), rats were maintained on a normal sodium diet or switched to a low sodium diet, either with or without zofenopril. Thus, MI-rats were allocated to one of the following treatment regimes: normal sodium (n=10), low sodium (n=6), normal sodium + zofenopril (n=10), low sodium + zofenopril (n=6). Sham-operated rats (n=10) were kept on a normal sodium diet and functioned as control rats for untreated MI. Treatment was maintained for 10 weeks (t=12), after which period rats were anaesthetized with isoflurane (2.0-2.5%). Subsequently, aortic blood pressure was obtained through a cannula in the carotid artery. A blood sample was drawn from the abdominal aorta before hearts were isolated and perfused according to Langendorff for in vitro assessment of ventricular function. Renal and cardiac tissues were washed and weighed, snap frozen in liquid nitrogen and stored at –800 C for future analysis. The University of Groningen committee on animal experiments approved the above study design.


Chapter 2

24

Myocardial infarction Rats were anesthetized with isoflurane (2.0-2.5%) in a mixture of N2O2 (2:1), intubated and mechanically ventilated. Coronary artery ligation was performed as described in detail elsewhere12. Briefly, a left side thoracotomy was performed and the left anterior descending coronary artery was occluded with a 6-0 silk suture, 1-2 millimeters from its origin. In sham-operated animals the suture was placed but not tightened. Subsequently, the thorax was closed and rats were extubated upon spontaneous respiration. The coronary occlusion procedure results in extensive transmural MI, comprising a major part of the LV free wall13. Infarct size was determined by planimetry at mid-ventricular levels in transverse slices, as the percentage of LV circumference14. Rats with infarctions of less than 20% of the LV tissue were excluded from further analysis, since these infarcts are found to be hemodynamically fully compensated13,15. Diet and medication Dietary sodium restriction was achieved by treating rats with food pellets containing 0.05% NaCl. Rats on a normal sodium diet were given food pellets with 0.3% NaCl. Zofenopril treatment was achieved by mixing the drug with rat chow during preparation (Hope Farms, Woerden, The Netherlands). Drug content and distribution in the rat chow were checked at the I.P.A.S. institute (Ligornetto, Switzerland). Analysis of drug content of the food together with the assessment of food intake and body weight revealed that drug intake was similar (on average 5.5 and 5.3 mg. kg-1 body weight. day-1) in both zofenopril groups. Blood pressure measurements After anesthesia with isoflurane (2.0-2.5%), the right carotid artery was catheterized with a polyethylene catheter filled with 0.9% saline with heparin, 5000 U. L-1, connected to a pressure transducer (Statham 23 Db, Gould Instruments, Cleveland, OH). The catheter was advanced into the thoracic aorta, and after stabilization aortic blood pressure was recorded. In vitro left ventricular function In all rats, LV function was measured in on a Langendorff setup, as described previously12. After 500 IU of heparin i.v., the heart was isolated and perfused according to Langendorff. For perfusion, a modified Tyrode solution was used (composition in mmol. L-1: glucose 10, NaCl 128.3, KCl 4.7, NaHCO3 20.2, CaCl2 1.35, NaH2PO4 0.42, MgCl2 1.05, equilibrated with 95% O2 and 5% CO2). Perfusion pressure was maintained at 60 mm Hg, and temperature was kept between 38.0 and 38.5 0C. After 10 minutes of stabilization, LV pressure was measured by means of a catheter placed into LV via the mitral valves and connected to a pressure transducer. Coronary flow was measured through a microprocessor, which controlled the perfusion pressure by adjusting the peristaltic perfusion pump. LV pressure, dP/dt, and heart rate were measured and stored in a computerized database system.



25

In order to ensure that induction of MI resulted in LV dysfunction, LV function was evaluated in sham rats and rats with untreated MI12. Although effects of ACE inhibitors on in vivo LV function are undisputed15-17, they cannot be shown on this in vitro setup18,19. Therefore functional data of the treatment groups were not evaluated. Decreased LV systolic pressure, and maximum rates of contraction and relaxation were significantly reduced in all MI rats as compared too sham operated rats, showing indeed MI-induced LV dysfunction. ACE activity ACE activity in plasma, LV (non-infarcted free wall) and kidney were determined using a method previously described in detail20. In short, tissues were homogenized in a 50 mmol. L-1 K2PO4 buffer at pH 7.5. Of the homogenates 100 µl was pipetted in a 0.5 mol. L-1 K2PO4 buffer. Then the ACE substrate Hippuryl-His-Leu (12.5 nmol. L-1, Sigma) was added and incubated at 370 C for exactly 10 minutes. The conversion of the substrate was stopped by adding 1.45 ml of 280 mmol. L-1 NaOH. Thereafter, 100 µl of 1% phtaldialdehyde was added for the labeling of free His-Leu product. The amount of labeled His-Leu was fluorimetrically determined at excitation and emission wavelengths of 364 and 486 nm, respectively. Control samples were included in which the conversion of substrate was prevented by adding NaOH before the substrate Hippuryl-His-Leu. Moreover, the substrate was added after the incubation period in these control samples. Data analysis Data are presented as mean±S.E.M. Untreated MI-rats on normal sodium were compared to sham control rats using student’s unpaired t-test, as to study the effect of CHF after experimental MI. Subsequently, all groups were compared among each other using oneway-analysis of variances (ANOVA) in combination with least significant difference post-hoc analysis for multiple comparisons (SPSS for Windows Standard version 10.0). Differences were considered statistically significant at a level of p<0.05 (two-tailed).

Results

General characteristics Acute mortality after MI-induction was 26%; none of the sham-operated animals died. After post hoc exclusion of rats with infarcts smaller than 20% from further analysis (15 out of 59), infarct sizes were evenly balanced between the MI groups (table 1). Body weights (BW) were comparable at the start of the protocol, but significantly lower at the end of treatment period in the groups receiving zofenopril.


Chapter 2

26

Table 1. General characteristics of the experimental groups

Sham Myocardial infarction

Control MI MI-LS MI-ZOF MI-ZOF-LS

N 10 10 6 10 6

Infarct size (%) 34.3±3.5 35.4±3.5 35.3±3.6 38.0±3.8

Body weight (g)

at t=0 weeks 273±6 278±3 266±5 277±3 268±4

at t=12 weeks 497±16 482±17 460±25 380±9* 386±13*

Organ weight (g)

Heart

2.03±0.07

2.71±0.24#

2.56±0.16

2.09±0.14*

1.62±0.06*

Left ventricle 1.23±0.03 1.63±0.13# 1.59±0.07 1.28±0.07* 1.01±0.03*

Right ventricle 0.26±0.01 0.37±0.06# 0.37±0.05 0.25±0.02* 0.20±0.01*

Data are mean ± SEM. Infarct-size and organ weights were determined at t=12 weeks after

infarction when rats were sacrificed. Abbreviations: myocardial infarction, MI; dietary sodium

restriction; LS, zofenopril; ZOF. # p<0.05 for MI versus control; * p<0.05 treated versus untreated

MI.

Blood pressure Mean arterial blood pressure was lower after MI (73±4 mmHg), as compared to sham control rats (87±4 mmHg, p=0.008). Dietary sodium restriction per se did not affect blood pressure (71±4 mmHg, p=NS versus untreated MI). Zofenopril treatment significantly reduced blood pressure, independent of sodium diet; 44±3 and 48±3 mmHg for zofenopril + normal sodium and zofenopril + low sodium, respectively (both p<0.001 versus untreated MI). Cardiac hypertrophy MI-induced hypertrophy was observed by a marked increase in total heart weight, which could be attributed to increases in left ventricular as well as right ventricular weight (table 1). Zofenopril treatment, but not dietary sodium restriction per se, prevented this cardiac response. However, when zofenopril treatment was combined with a dietary sodium restriction, left ventricular weight was reduced by an additional 21%. Right ventricular weights showed the same treatment effects (20%), though statistical significance was not reached. Left ventricular hypertrophy induced by MI was only effectively prevented by the combination of zofenopril and sodium restriction (figure 1). When left ventricular hypertrophy, represented by LVW-to-BW ratio, of MI-rats was compared to sham-operated animals, all groups except the low sodium-zofenopril group showed a significant increase. Moreover, LVW-to-BW ratio was significantly lower in rats that received zofenopril + dietary sodium restriction compared with rats on zofenopril alone fig. 1).



27

Figure 1. Effect of treatment with zofenopril (ZOF) and/or low sodium diet (LS) on left ventricuar

ACE activity

Induction of CHF did not result in significantly elevated ACE activity in plasma and tissue (figure 2). However, plasma ACE activity was almost completely inhibited by zofenopril, irrespective of sodium intake. Although absolute values were much higher, a similar pattern was seen in the kidneys: marked inhibition by zofenopril but no additional effects by sodium intake. Interestingly, ACE activity in LV tissue was not reduced by zofenopril in rats that were fed on a normal sodium diet. Only in combination with a low sodium diet, ACE activity was significantly decreased. ACE activity in RV tissue showed a similar pattern (data not shown). LV ACE activity and LVW-to-BW ratio showed a significant correlation (figure 3).

Discussion

We studied the effect of dietary sodium restriction on efficacy of ACE-inhibitor therapy after experimental myocardial infarction in rats. The most important observations were: 1) LV ACE activity was inhibited only when zofenopril treatment was combined with dietary sodium restriction; 2) this effect occurred in heart, but not in plasma or in kidney tissue; 3) the significant reduction of LV ACE activity during combined ACE inhibitor/low sodium diet treatment could be associated with a significant reduction in LVW-to-BW ratio, indicating prevention of hypertrophy; 4) A low sodium diet per se had no effects on any of the measured parameters.

sham MI MI-LS MI-ZOF MI-ZOF-LS

LV

W:B

W (

mg

/g)

0,0

0,5

2,0

2,5

3,0

3,5

4,0#

*

Figure 1. Left ventricular (LV) hypertrophy in rats with experimental myocardial infarction

(MI). Data are mean ± SEM and bars represent the left ventricular weight to body weight ratio

(LVW-to-BW) as a measure of LV hypertrophy. * p<0.05 as indicated; # p<0.05 MI versus

sham


Chapter 2

28


AC

E a

ctiv

ity

(nm

ol H

L/m

L/m

in)

0

100

200

300

400

sham MI MI-LS MI-ZOF MI-ZOF-LSAC

E a

ctiv

ity

(nm

ol H

L/m

L/m

in)

0

20

40

60


AC

E a

ctiv

ity

(nm

ol H

L/m

L/m

in)

0

20

40

60

80

100

**

**

LV

Plasma

Kidney

*

Figure 2. Effect of treatment with zofenopril (ZOF) and/or low sodium diet (LS) on plasma

(top panel), left ventricular (middle panel) -and renal tissue ACE activity (bottom panel) in

rats with experimental myocardial infarction (MI). Data are mean ± SEM and bars represent

the formation of His-Leu from Hippuryl-His-Leu. * p<0.05 as indicated



29

ACE activity In the present study, MI did not increase ACE activity in plasma, nor in renal and LV tissue. Cardiac ACE may be activated in the early stage after induction of heart failure and is related to the degree of LV dysfunction12. Moreover, LV ACE activity appears also related to MI size21. The relatively moderate MI size in the present study may therefore not have increased ACE activity. Moreover, the major area in which ACE activity is elevated post-MI is the infarcted area itself, where mainly fibroblasts display increased activity22, whereas analysis in the present study was restricted to the viable part of the LV free wall. In accordance with the lack of activation after MI, there was no significant inhibition of LV ACE activity due to zofenopril treatment. In previous studies zofenopril did significantly inhibit cardiac ACE activity23, but the applied dose turned out to be lower in the current study. This confirms the importance of optimal dosing, as has been reported before24. Dietary sodium restriction alone did not show any effect on the parameters measured in the present study. This is in general accordance with a previous study of Hodsman and coworkers in rats with myocardial infarction25. The lack of effect of dietary sodium restriction alone on ACE activity in the present study suggests absence of a direct influence of sodium restriction on ACE activity and/or expression. However, other components of the renin angiotensin system may have been affected. In this respect it is interesting to mention that both ACE gene expression26, and angiotensin II type 1 (AT-1) receptor expression may be regulated by sodium intake27-29. The AT-1 receptor is regarded the primary mediator of angiotensin II induced cardiac remodeling30,31.Although not affecting ACE activity when applied alone, when dietary

LV ACE activity (nmol HisLeu/mL/min)

0 20 30 40 50 60 70

LV

W:B

W (

mg

/g)

0,0

2,0

2,5

3,0

3,5

4,0

MI-ZOFMI-ZOF-LS

R2=0.58

p=0.01

Figure 3. Correlation between LV tissue ACE activity and LV weight in zofenopril (ZOF)-

treated rats with experimental myocardial infarction. The solid line represents linear

regression line through individual points.


Chapter 2

30

sodium restriction is added to the zofenopril treatment, LV ACE activity was significantly reduced. Notably, this effect was specific for the LV since no additional ACE inhibition could be shown in plasma or kidneys. It remains unclear why ACE activity in the kidney homogenates was not further inhibited by the combination of dietary sodium restriction and zofenopril, as it was in LV. This may seem paradoxical, since renoprotective effects of ACE inhibition can be enhanced by dietary sodium restriction. However, this improved renoprotection, as measured by proteinuria reduction9, has not yet been shown to be associated with a further decrease in renal ACE activity. Moreover, a possible effect of sodium on ACE activity may be only be present in a situation of tissue damage, i.e. effects becoming only apparent when the tissue RAAS is activated by organ damage. In the current study, kidneys were considered to be intact. Thus, the exact mechanisms underlying the additional cardio- or renoprotective effects of combined dietary sodium restriction and zofenopril treatment is not yet clear and will be subject of further investigation. Cardiac hypertrophy LVW was significantly increased after MI, and neither dietary sodium restriction, nor zofenopril treatment alone did have any effect. Only when the two treatments were combined, LV hypertrophy was effectively prevented. Interestingly, the effects of therapy on LV ACE activity paralleled the effects on LV hypertrophy. Indeed, regression analysis revealed that LV ACE activity in zofenopril-treated rats was linearly related to left ventricular weight (Figure 3), suggesting that LV ACE activity is a major determinant of LV hypertrophy in presence of an ACE inhibitor. A previous study, using a 3 times higher dose of zofenopril and significantly decreasing LV ACE activity as well as LV weight23, support this suggestion. In contrast, high sodium diet abolished the effect of perindopril on blood pressure and cardiac hypertrophy, but cardiac ACE inhibition was found unaltered32. Although in the latter study blood pressure rather than cardiac ACE activity was regarded the determinant of cardiac hypertrophy, in the present study, the effects on hypertrophy were found to be independent of blood pressure – i.e. blood pressure was similarly reduced in the zofenopril groups without additional effects of dietary sodium. It has been reported before that RAAS activation rather than blood pressure determines cardiac hypertrophy after MI33. Clinical implications Dietary sodium restriction is commonly recommended and/or applied to heart failure patients, who most often are on ACE-inhibitor therapy as well. Theoretically this dietary sodium restriction may have a dual effect. On the one hand it may (further) stimulate the RAAS, with its well-known deleterious effects. On the other hand it may improve efficacy of inhibition of the RAAS, either by a similar or by different mechanisms. In a recent study, this dual effect was illustrated by improved survival, but at the same time substantial potentiation of plasma renin activity, in spontaneously hypertensive rats with MI, treated with furosemide in addition to ramipril21.



31

Furosemide, by causing sodium depletion and increased plasma renin activity may have similar effects as dietary sodium restriction. Data from the present study suggest that dietary sodium restriction may be safely applied to MI patients on ACE-inhibitors, in that it specifically potentiates cardiac ACE inhibition leading to further inhibition of cardiac remodeling, and without adverse side effects such as hypotension. Whether dietary sodium restriction will lead to further improvement of cardiac function and/or prognosis when added to ACE-inhibitor treatment in patients with MI-induced heart failure, needs further investigation. Conclusion The present study was aimed to explore whether dietary sodium restriction would influence the effects of ACE-inhibitor therapy with zofenopril in rats with myocardial infarction. Results show that left ventricular-, but not plasma or renal ACE inhibition, was potentiated by additional dietary sodium restriction, indicating a tissue specific effect. Effect on ventricular ACE inhibition was paralleled by effects on cardiac hypertrophy, without effects on blood pressure, suggesting left ventricular ACE activity as a primary determinant of cardiac hypertrophy. Additional studies are needed to examine whether this effect is drug specific, and whether the observed effects can be associated with beneficial effects on in vivo cardiac function and prognosis.

References

1. Kober L, Torp-Pedersen C, Carlsen JE et al. A clinical trial of the angiotensin-converting-enzyme

inhibitor trandolapril in patients with left ventricular dysfunction after myocardial infarction.

Trandolapril Cardiac Evaluation (TRACE) Study Group. N Engl J Med. 1995;333:1670-1676.

2. Flather MD, Yusuf S, Kober L et al. Long-term ACE-inhibitor therapy in patients with heart failure or

left-ventricular dysfunction: a systematic overview of data from individual patients. ACE-Inhibitor

Myocardial Infarction Collaborative Group. Lancet. 2000;355:1575-1581.

3. Brogden RN, Todd PA, Sorkin EM. Captopril. An update of its pharmacodynamic and pharmacokinetic

properties, and therapeutic use in hypertension and congestive heart failure. Drugs. 1988;36:540-600.

4. Cleland JG, Erhardt L, Murray G et al. Effect of ramipril on morbidity and mode of death among

survivors of acute myocardial infarction with clinical evidence of heart failure. A report from the AIRE

Study Investigators. Eur Heart J. 1997;18:41-51.

5. Effect of enalapril on survival in patients with reduced left ventricular ejection fractions and congestive

heart failure. The SOLVD Investigators. N Engl J Med. 1991;325:293-302.

6. Konstam MA, Rousseau MF, Kronenberg MW et al. Effects of the angiotensin converting enzyme

inhibitor enalapril on the long-term progression of left ventricular dysfunction in patients with heart

failure. SOLVD Investigators. Circulation. 1992;86:431-438.


comparison of low and liberal sodium diet in hypertensive patients. J Cardiovasc Pharmacol.

1987;9:743-748.






Chapter 2

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10. Fernandez D, Bolli P, Snedden W et al. Modulation of left ventricular hypertrophy by dietary salt and

inhibition of angiotensin converting enzyme. J Hypertens Suppl. 1988;6:S145-S147.



1685.

12. Pinto YM, de Smet BG, van Gilst WH et al. Selective and time related activation of the cardiac renin-

angiotensin system after experimental heart failure: relation to ventricular function and morphology.

Cardiovasc Res. 1993;27:1933-1938.

13. Pfeffer MA, Pfeffer JM, Fishbein MC et al. Myocardial infarct size and ventricular function in rats. Circ

Res. 1979;44:503-512.

14. Nelissen-Vrancken HJ, Kuizinga MC, Daemen MJ et al. Early captopril treatment inhibits DNA synthesis

in endothelial cells and normalization of maximal coronary flow in infarcted rat hearts. Cardiovasc Res.

1998;40:156-164.

15. Schoemaker RG, Debets JJ, Struyker-Boudier HA et al. Delayed but not immediate captopril therapy

improves cardiac function in conscious rats, following myocardial infarction. J Mol Cell Cardiol.

1991;23:187-197.

16. Mitchell GF, Lamas GA, Vaughan DE et al. Left ventricular remodeling in the year after first anterior

myocardial infarction: a quantitative analysis of contractile segment lengths and ventricular shape. J Am

Coll Cardiol. 1992;19:1136-1144.

17. Pfeffer MA, Braunwald E. Ventricular enlargement following infarction is a modifiable process. Am J

Cardiol. 1991;68:127D-131D.

18. Mill JG, Gomes AP, Carrara AB et al. Influence of chronic captopril therapy on the mechanical

performance of the infarcted rat heart. Pharmacol Res. 1994;29:77-88.

19. Kalkman EA, Saxena PR, Schoemaker RG. Sensitivity to ischemia of chronically infarcted rat hearts;

effects of long-term captopril treatment. Eur J Pharmacol. 1996;298:121-128.

20. Cushman DW, Cheung HS. Concentrations of angiotensin-converting enzyme in tissues of the rat.

Biochim Biophys Acta. 1971;250:261-265.



22. Sun Y, Zhang J, Zhang JQ et al. Renin expression at sites of repair in the infarcted rat heart. J Mol Cell

Cardiol. 2001;33:995-1003.

23. van Wijngaarden J, Pinto YM, van Gilst WH et al. Converting enzyme inhibition after experimental

myocardial infarction in rats: comparative study between spirapril and zofenopril. Cardiovasc Res.

1991;25:936-942.

24. Wollert KC, Studer R, von Bulow B et al. Survival after myocardial infarction in the rat. Role of tissue

angiotensin-converting enzyme inhibition. Circulation. 1994;90:2457-2467.



1988;12:467-472.

26. Kreutz R, Fernandez-Alfonso MS, Liu Y et al. Induction of cardiac angiotensin I-converting enzyme

with dietary NaCl-loading in genetically hypertensive and normotensive rats. J Mol Med. 1995;73:243-

248.



33

27. Schmid C, Castrop H, Reitbauer J et al. Dietary salt intake modulates angiotensin II type 1 receptor gene

expression. Hypertension. 1997;29:923-929.

28. Nickenig G, Strehlow K, Roeling J et al. Salt induces vascular AT1 receptor overexpression in vitro and

in vivo. Hypertension. 1998;31:1272-1277.

29. Ruan X, Wagner C, Chatziantoniou C et al. Regulation of angiotensin II receptor AT1 subtypes in renal

afferent arterioles during chronic changes in sodium diet. J Clin Invest. 1997;99:1072-1081.

30. McEwan PE, Gray GA, Sherry L et al. Differential effects of angiotensin II on cardiac cell proliferation

and intramyocardial perivascular fibrosis in vivo. Circulation. 1998;98:2765-2773.

31. Regitz-Zagrosek V, Fielitz J, Fleck E. Myocardial angiotensin receptors in human hearts. Basic Res

Cardiol. 1998;93 Suppl 2:37-42.

32. Yoshida K, Kohzuki M, Casley DJ et al. Angiotensin-converting enzyme inhibition and salt in

experimental myocardial infarction. J Cardiovasc Pharmacol. 1998;32:357-365.

33. Teisman AC, Pinto YM, Buikema H et al. Dissociation of blood pressure reduction from end-organ

damage in TGR(mREN2)27 transgenic hypertensive rats. J Hypertens. 1998;16:1759-1765.

Chapter 3

Beneficial Effects of Add-on

Hydrochlorothiazide in Rats with Myocardial

Infarction Optimally Treated with Quinapril.

Bart Westendorp

Regien Schoemaker

Hendrik Buikema

Dick de Zeeuw

Frans Boomsma

Wiek van Gilst


36

Chapter 3 - Versie 3 16 augustus 2005

Chapter 3

Abstract

Background: The antihypertensive and renoprotective effects of ACE inhibitor (ACE-I) therapy are enhanced by inducing a negative sodium balance. Whether this strategy also improves outcome of chronic ACE-I treatment after myocardial infarction (MI) is unknown. Therefore, we investigated whether hydrochlorothiazide (HCTZ) or dietary sodium restriction further improves survival in ACE-I-treated rats with MI. Methods: MI was induced by coronary ligation. After 2 weeks rats were randomised to quinapril (QUI), HCTZ added to quinapril (QUI+HCTZ), or low sodium diet added to quinapril (QUI+LS). Survival was monitored for 62 weeks, after which left ventricular (LV) pressures were measured and blood for neurohumoral characterisation was collected. A separate group of rats, subjected to the same procedure, was evaluated after 35 weeks. Results: After 62 weeks, mortality was comparable in all groups. However, survival was improved by HCTZ until 35 weeks. This effect on survival was paralleled by decreased proteinuria and LV end-diastolic pressures in QUI+HCTZ rats at 35, but not 62 weeks. Plasma renin activity was significantly decreased in QUI+HCTZ rats at 35 weeks. Contrary to HCTZ, LS added to QUI caused no benefit. Conclusions: Adding HCTZ, but not LS, to quinapril improved survival, neurohumoral status, and proteinuria during the early chronic phase of experimental post-MI LV dysfunction. Since no adverse effects were observed, HCTZ may safely be used to improve ACE-I therapy.

Beneficial Effects of Add-on Hydrochlorothiazide

37


Introduction

The progression of left ventricular (LV) dysfunction towards overt chronic heart failure (CHF) after myocardial infarction (MI) is associated with progressive cardiac remodelling. Renin angiotensin aldosterone system (RAAS) activation plays a central role in this process, and blocking the RAAS with angiotensin-converting enzyme inhibitors (ACE-I) effectively reduces remodelling and prolongs survival after MI. Still, prognosis post-MI is grim, and optimisation of therapy is necessary1,2. Antihypertensive and renoprotective effects of ACE-I can be enhanced by dietary sodium restriction or co-treatment with diuretics3-5. For heart failure treatment, this has not been investigated. Sodium depletion itself has never been shown to affect the progression of LV dysfunction in terms of LV remodelling6-9 or LV hemodynamic parameters10. However, induction of a negative sodium balance may provide a strategy to enhance the therapeutic efficacy of ACE-I in heart failure. We previously showed that sodium restriction added to ACE-I treatment further attenuated LV hypertrophy in MI-rats, which was associated with augmented inhibition of LV tissue ACE activity11. Whether this also results in improved cardiac function and reduced mortality was addressed in the current study. We evaluated the long-term effects of additional diuretic treatment or a low sodium diet on mortality, cardiac function, neurohumoral activity, and urinary protein excretion in rats with experimental MI on optimal ACE-I therapy. We hypothesised that diuretics and dietary sodium restriction 1) augment the beneficial effects of ACE-I therapy and thus positively affect cardiac function and survival in rats with MI, and 2) exert no adverse effects and can be safely applied in post-MI ACE-I therapy.

Methods

Study design The investigation conforms to the Guide for the Care and Use of Laboratory Animals (Published by the US National Institutes of Health, NIH publication No. 85-23, revised 1996). The animal research committee of the University of Groningen approved this study protocol. Male, Sprague Dawley rats (Harlan, Zeist, The Netherlands), weighing 280±25g were subjected to coronary ligation as previously described12,13. Since the study was aimed at the chronic phase after myocardial infarction, and not at interfering with the early healing process and scar formation, treatment was started 14 days post-MI. Rats were randomly assigned to: quinapril alone (QUI), quinapril + hydrochlorothiazide (QUI+HCTZ), quinapril + low sodium (QUI+LS). Since HCTZ or LS alone do not affect post-MI LV remodelling and function 6-11, we did not include treatment arms on only HCTZ or LS. As the effects of ACE-I on MI rats are well established, we also refrained from including a non-treated MI-group in the survival study. Quinapril (15 mg kg-1 day-1) was mixed through food (Hope Farms, Woerden, The Netherlands). This dose results in optimal ACE-I therapy14-16. HCTZ (50 mg kg-1 day-1) was dissolved in drinking water. This dose causes an increase in diuresis and RAAS activation, but no blood pressure reduction in normotensive rats9. The LS-group was fed

38


Chapter 3

with food pellets containing 0.05% NaCl instead of 0.3% (normal diet) (LS-diet; Hope Farms, Woerden, The Netherlands). To ensure constant drug intakes during the entire study period, concentrations of quinapril and HCTZ were adjusted weekly. This was done by measuring food/water intake and average body weight per cage weekly, and calculating the required drug concentration in food and water (per cage) for the week after. Rats were fed ad libitum, and housed group-wise in clear polyethylene cages in temperature (220 C)- and humidity (50%)-controlled rooms with a 12h light/dark cycle. Study 1: 62 weeks survival Animals surviving surgical procedures (159 out of 320) were allocated to one of three active treatment groups, and monitored - in a blinded fashion - until death or for 62 weeks after onset of therapy. Use of colour-tags ensured appropriate housing and treatment by caretakers. Cages were checked for dead animals at least once daily; tissues of dead rats were collected, weighed and stored for analysis. At 17, 35 and 52 weeks of treatment 24h urine samples were collected for measurement of total urinary protein using a nephelometer (Dade Behring Diagnostic, Marburg, Germany). After the 62-week follow-up period, subgroups of randomly chosen surviving rats were sacrificed for assessment of LV function and neurohumoral activity. Study 2: 35 weeks of treatment In total 90 rats were used for this protocol, designed to study neurohormones and cardiac function during the course of the study period in relation to untreated MI rats. Rats were operated and treated according to the same procedures as in the above study, except that they were sacrificed and studied at 35 weeks after onset of treatment. Assessment of cardiac function Rats were anaesthetised with isoflurane (2%) in a mixture of O2 and N2O (1:2), the carotid artery was cannulated and a pressure tip catheter (Micro-Tip 3French, Millar instruments Inc., Houston TX, USA) advanced into the LV. After registration of LV systolic and diastolic pressures (LVSP and LVEDP), and maximal rates of increase and decrease in LV pressure (+dP/dt and –dP/dt), the catheter was retracted into the aortic arch, and arterial pressures and heart rate were recorded. Subsequently, arterial blood and tissues were collected for further analysis. Neurohumoral measurements Arterial blood was anti-coagulated with EDTA and N-terminal atrial natriuretic peptide (N-ANP), plasma renin activity, and aldosterone, were measured in the plasma as previously described17. Plasma for ACE activity determination was collected separately, and not anti-coagulated with EDTA. ACE activity in the plasma and spared myocardial tissue was determined as described before12. Tissue collection and histology Hearts were rinsed with ice cold NaCl (0.9%), and the atria and right ventricle were removed on ice for determination of LV weights. The apical 1/3 part of the LV was cut-off, spared and infarcted tissues were separated, and frozen in liquid nitrogen for


39


measurement of left ventricular ACE activity. The mid-ventricular slice of the LV was stored in 2% paraformaldehyde for histologic assessment of infarct size on slices stained with Sirius Red/Fast Green, as described previously12. Infarct size was determined as percentage of LV circumference. The mid-ventricular section provides adequate estimation of total LV infarct size18. Only rats with MI sizes larger than 20% of LV were included for analysis, since smaller infarcts do not result in LV dysfunction19,20. Statistical methods The survival study was designed to detect a difference of 20% in 1-year mortality with a power of 0.80, with an expected mortality of 60% in the control group that received quinapril only, based on the early captopril studies21. Survival analysis was done using log rank analysis with pairwise comparison over strata. Functional and neurohumoral parameters were compared using one-way analysis of variances (ANOVA) with least square difference post hoc analysis for multiple comparisons in case of normal distribution of data. Otherwise, a Kruskal-Wallis test was used. Differences were considered significant at the level of 0.05 (two tailed). Bodyweights, food and water intakes were analysed with ANOVA for repeated measurements (general linear model). Data are presented as means ± SEM in case of normal distribution, otherwise in boxplots.

Results

Study 1: 62 weeks survival Of the 159 rats studied, 35 were excluded from survival analysis: MI-size was <20% in 26 rats, infarct size could not be determined in 3 rats; 6 rats developed tumours and were sacrificed prematurely. Remaining group sizes at the onset of treatment were n=44 for quinapril, n=45 for quinapril + HCTZ, and n=35 for quinapril + low sodium diet. Infarct sizes were evenly distributed over the different treatment groups (table 1). Comparison of subgroups based on infarct size irrespective of treatment showed that total mortality was significantly higher in rats with extensive infarcts (>35% of LV) than in rats with moderate (20-35% of LV) infarcts (fig. 1, p=0.02). This confirms that mortality in this study is related to myocardial infarction, in a MI-size-dependent way. The time course of mortality suggests different phases, with a low mortality during the first 35-40 weeks of treatment, followed by a steep increase in mortality in rats with large MI in the late phase of follow-up. Survival was similar in the three treatment groups after the complete follow-up period of 62 weeks (figure 2A). However, the data suggest two different periods with distinct effects of additional treatment (figures 2B and C. During the first 35 weeks survival was significantly improved by additional hydrochlorothiazide (table 1, Figure 2B). Mortality in the groups treated with quinapril only and quinapril + HCTZ was parallel between week 35 and 62 (figure 2C). Survival in the group receiving a low sodium diet in addition to quinapril was intermediate and never significantly different from the other groups.

40


Chapter 3

Table 1. General characteristics of rats included in the survival study, and general characteristics

and hemodynamics of MI rats after chronic ACE-I therapy, and additional effects of HCTZ or

dietary sodium restriction after 35 and 62 weeks.

MI control QUI QUI+HCTZ QUI+LS

Survival study:

n (start treatment) 44 45 35

Infarct size (% of LV) 33.7±1.1 32.0±1.1 34.8±1.4

Mortality

at 35 weeks (%) 11 0 * 3

at 62 weeks (%) 23 20 26

Sacrificed rats:

n wk35 4 6 5 5

wk62 - 11 12 10

Infarct size (% of LV) wk35 26.9±2.5 24.7±1.0 28.2±1.1 29.3±2.6

wk62 - 34.0±2.2 32.7±1.3 36.7±1.6

Hemodynamic

parameters:

MAP (mmHg) wk35 98.3±7.2 72.1±3.0 * 71.5±4.4 * 71.6±3.6 *

wk62 - 71.3±4.5 72.5±2.6 70.0±3.1

LVEDP (mmHg) wk35 14.0±2.3 10.4±1.8 7.3±2.5 # 10.0±2.5

wk62 - 12.4±2.6 12.4±0.9 12.8±1.1

+dP/dt (103 mmHg. s-1) wk35 10.3±0.8 8.4±0.2 * 8.7±0.5 * 8.9±0.4

wk62 - 8.5±0.3 8.1±0.4 7.8±0.2

-dP/dt

(103 mmHg. s-1)

wk35

-8.6±0.6

-6.9±0.6 *

-7.6±0.3

-7.4±0.4

wk62 - -7.1±0.3 -6.9±0.3 -6.6±0.2

Electrolytes:

Plasma Na+ (mmol. L-1) wk35 142.8±4.4 137.4±0.3 136.6±0.5 * 134.5±4.7 *

Plasma K+ (mmol. L-1) wk35 3.9±0.2 4.4±0.1 * 4.4±0.2 * 4.5±0.1*

* indicates p<0.05 versus MI-control. # LVEDP at 35 weeks: QUI+HCTZ p=0.07 versus control.

wk; week of treatment.


41


Body weight gain was significantly reduced during the first 3 weeks in rats treated with quinapril + HCTZ. Thereafter, the difference remained approximately 20 g compared to quinapril only throughout the study (fig. 3A). The reduced weight gain in rats on quinapril + HCTZ was paralleled by a combination of markedly increased drinking and slightly decreased food intake (figs. 3B and C). Water intake remained significantly increased in the HCTZ group during the entire treatment period (fig. 3C). Body weights in rats additionally receiving low sodium diets as compared to quinapril only were almost identical during the first 45 weeks of treatment, but became significantly lower thereafter. After 62 weeks of treatment, no differences in mean arterial blood pressure (MAP), cardiac function (LV systolic and diastolic pressures, +dP/dt and -dP/dt), and LV hypertrophy (cardiac weights) were seen (tables 1 and 2). At comparable urine production during all time points (see legend figure 4), urinary protein excretion was significantly lower in rats treated with quinapril + HCTZ than in rats receiving quinapril only at 17, but not at 35 and 52 weeks. Contrary to HCTZ, dietary sodium restriction did not influence urinary protein excretion. Interestingly, comparison of survival curves of MI-rats based on urinary protein excretion at week 17 irrespective of treatment, suggests a trend of improved survival in the group with protein excretion below average (Figure 4B). Study 2: 35 weeks treatment Mortality within 24 hours after MI-induction in this group was 38%. Of the remaining 53 MI-rats that entered the study, 33 were excluded from analysis for infarct sizes below 20%, leaving the following groups: n=4 untreated MI controls, n=6 for QUI, n=5

Time (weeks)

0 10 20 30 40 50 60

Su

rviv

al (

% r

ats

aliv

e)

0

60

70

80

90

100

MI 20-35% (n=80)

MI >35% (n=44)

*

Figure 1. Influence of infarct size on survival after MI in rats treated with quinapril,

irrespective of co-treatment. Groups were divided into moderate (20-35%, n=80) and large

infarction (>35%, n=44). * p < 0.05 as indicated.

42


Chapter 3

for QUI+HCTZ and n=5 for QUI+LS. MI-sizes were similar in all groups in study 2, but on average slightly smaller than in study 1 (table 1). Table 2. Organ weights of MI rats after long term quinapril therapy, and the additional effects of

hydrochlorothiazide therapy or dietary sodium restriction after 35 and 62 weeks.

MI- control QUI QUI+HCTZ QUI+LS

Body weight (g)

wk35

520±16

457±8 *

440±16 *

460±19 *

wk62 - 487±19 452±10 466±11

Heart weight (mg g-1)

wk35

3.51±0.09

2.98±0.07 *

3.04±0.12 *

2.96±0.10 *

wk62 - 3.27±0.10 3.26±0.08 3.13±0.07

wk35 2.38±0.04 1.97±0.05 * 2.00±0.10 * 1.90±0.09 * Left ventricle weight

(mg g-1) wk62 - 2.03±0.04 2.07±0.05 2.06±0.04

All organ weights were corrected for body weight. *indicates p<0.05 versus MI-control.

After 35 weeks, mean arterial blood pressure was markedly decreased by quinapril treatment, but not further affected by hydrochlorothiazide or dietary sodium restriction. LVEDP in rats treated with HCTZ in addition to quinapril was decreased by

Time (weeks)

0 5 10 15 20 25 30 35 40 45 50 55 60

% R

ats

aliv

e

0

70

75

80

85

90

95

100

Time (weeks)35 40 45 50 55 60

% R

ats

aliv

e

0

7580859095

100

A)

B) Time (weeks)0 5 10 15 20 25 30 35

% R

ats

aliv

e

0

85

90

95

100 *

C)

Figure 2. Survival after experimental MI in rats treated with quinapril, and effects of

additional hydrochlorothiazide or low sodium diet. A) Survival after 35 weeks was

significantly improved by HCTZ added to quinapril, as denoted by *. B) and C) exhaustion

analysis of mortality between week 0-35 and 35-62, with number of rats alive after 35 weeks

reset to 100% in C).


43


50% compared with untreated MI controls and 30% compared with quinapril alone (table 1). Both +dP/dt and –dP/dt were similar in the 3 groups treated with quinapril. At 35 weeks, heart and left ventricular weights were significantly reduced by quinapril, and additional hydrochlorothiazide or low sodium diet (table 2) did not modify this reduction. Neurohormones in studies 1 and 2 Neurohormones were studied after 35 and 62 weeks of treatment (figures 5 and 6). At 35 weeks, plasma N-ANP (indicative for LV stress), ACE activity, and aldosterone were significantly decreased in quinapril treated rats compared to untreated MI-rats. From 35 to 62 weeks, N-ANP, ACE activity, and aldosterone showed an increasing trend in all groups, suggesting progression of LV dysfunction despite ACE inhibition therapy.

Time (weeks)

0 10 20 30 40 50 60

Wat

er in

take

(m

L/r

at/d

ay)

0

10

20

30

40

50

60

70

Time (weeks)

0 10 20 30 40 50 60

Bo

dy

wei

gh

t (g

)

0

300

350

400

450

500

Time (weeks)

0 10 20 30 40 50 60

Fo

od

inta

ke (

g/r

at/d

ay)

014

15

16

17

18

19

20

A)

B) C)

nd

*

**

Figure 3. Body weights, water and food intake of MI rats treated with quinapril (QUI), and

effects of additional HCTZ or dietary sodium restriction. Error bars in C) are omitted for

reasons of clarity. nd: no data. * p<0.05 vs. QUI.

44


Chapter 3

Additional treatment with hydrochlorothiazide or dietary sodium restriction did not modify these effects of quinapril, except for plasma aldosterone being significantly higher in rats co-treated with low sodium than with quinapril alone at 62 weeks. LV ACE activity was comparable in all groups both at 35 and 62 weeks of treatment. Quinapril caused a profound increase in plasma renin activity at 35 weeks, which tended to be decreased again after 62 weeks treatment. Interestingly, this increase was markedly reduced after co-treatment with hydrochlorothiazide, but not with dietary sodium restriction. After 62 weeks, plasma renin activity no longer differed among the treatment groups. Slightly increased plasma K+ levels in quinapril-treated rats at 35 weeks were not affected by co-treatment. In contrast, plasma Na+ concentrations were not significantly affected at 35 weeks in rats treated with quinapril alone, as compared to those untreated, but significantly decreased in those rats additionally treated with

Uri

nar

y p

rote

in (

mg

/24h

)

0

2

4

6

8

10

12

14

16

18

Time (weeks)

0 20 25 30 35 40 45 50 55 60

Su

rviv

al (

% r

ats

aliv

e)

0

10

75

80

85

90

95

100

low urinary protein (n=63)high urinary protein (n=59)

A) B)*

week 17 week 35 week 52

QUI (n=33)QUI+HCTZ (n=35)QUI+LS (n=31)

†

†

Figure 4. Urinary protein excretion during chronic quinapril therapy, and the effects of

additional hydrochlorothiazide or dietary sodium restriction. A) Bar graphs showing

significantly decreased proteinuria at 17 and 35 weeks in QUI+HCTZ rats, and no differences

at 52 weeks; only rats alive at 52 weeks were included in the analysis to avoid bias by

mortality. Urine production was comparable and decreased over time in all groups: at 17

weeks 27±1, 27±1, and 29±2; at 35 weeks 17±2, 18±1, and 23±2; at 52 weeks 15±1, 20±1, and

18±1 mL for QUI, QUI+HCTZ, and QUI+LS, respectively. B) Survival curves suggesting

predictive value of urinary protein levels. Rats were divided into 2 groups: below and above

total average 24h-protein excretion for each treatment group. The group with lower urinary

protein excretion showed a trend towards improved survival (no significance). * p<0.05 vs.

QUI, † p<0.05 vs. QUI+LS.


45


hydrochlorothiazide or low sodium – which is in accordance with our study aim to induce a negative sodium balance.

Discussion

The aim of this study was to investigate whether a negative sodium balance enhances cardioprotective effects of high-dose ACE inhibitor therapy after MI. We studied long-term effects of additional hydrochlorothiazide or dietary sodium restriction on survival, cardiac function, and neurohormones in rats with MI-induced LV dysfunction receiving quinapril. Hydrochlorothiazide Overall mortality during ACE-I therapy was low compared to similar survival studies with experimental heart failure21,22. Since only high-dose ACE-I treatment exerts a profound effect on long-term survival after MI23, the low mortality rate in the present study indicates ACE-I treatment in accordance with our aim; to study sodium depletion on top of optimal ACE-I therapy. Survival was not improved by HCTZ at the complete follow up period of 62 weeks. However, when looking at the survival curves in figures 1 and 2 in more detail, different phases could be identified. Survival curves based on infarct size (figure 1) show two distinct phases: comparable mortality in rats with moderate and extensive MI in the early phase (± week 1-40), whereas curves diverged rapidly in the late phase (± week 40-62). Add-on HCTZ therapy significantly improved survival until 35 weeks, but had no beneficial effect on survival between week 35 and 62. Interestingly, several other animal studies on RAAS inhibition with long follow-up periods show a biphasic pattern as well21,23,24. Captopril only improved early survival in rats with large infarcts, but predominantly late survival in rats with moderate infarct sizes21. In a clinical setting, the beneficial effects of ACE-I therapy with enalapril were most pronounced during the early treatment phase25, and may decrease over time26,27. Taken together, these findings suggest different stages of post-MI dysfunction, both time- and infarct-size related, with distinct effects of ACE-I treatment. It may hence be not surprising that in our study effects of additional HCTZ treatment follow these phases. The exact mechanism underlying improvement of ACE-I therapy with hydrochlorothiazide is not clear. As dietary sodium restriction had no beneficial effects, despite similar reduction in plasma Na+ levels, favourable effects of HCTZ cannot be explained by sodium depletion only. A hemodynamic effect may play a role, since hydrochlorothiazide has direct vasodilator effects on vascular smooth muscle 28. LVEDP after 35 weeks tended to be lower in rats treated with HCTZ on top of quinapril, which could reflect decreased venous return. Interestingly, parallel to the observed survival benefit at 35 but not 62 weeks, plasma renin activity after 35 but not 62 weeks of treatment was markedly reduced by HCTZ added to quinapril. From the point of view that diuretic therapy generally causes volume depletion and consequently increased plasma renin activity9, this observation is surprising. An association between increased plasma renin activity and mortality in post-MI patients was shown in several studies 29-

46


Chapter 3

31. The most important regulators of renin activity post-MI are decreased cardiac output and sympathetic activation, which both are associated with LV dysfunction and remodelling. Renin itself is not known to have direct cellular effects during post-infarct cardiac remodelling. However, HCTZ alone decreases sympathetic activation in rats with MI7. Thus, the decrease in plasma renin is likely to be an indicator for improved therapy outcome rather than a mediator of HCTZ effects.

pg

mL

-1

0

50

100

150

200

250

300

Plasma renin activity

ng

An

g1

mL

-1 h

-1

0

50

100

150

200

250

MI-Controln=4

QUIn=6

QUI+HCTZn=5

QUI+LSn=5

Plasma N-ANP

pm

ol m

L-1

0

1

2

3

nm

ol m

L-1

min

-1

0

10

20

30

40

50

60

LV ACE activity

nm

ol m

L-1

min

-1

0

5

10

15

20

25

35 weeks treatment

*

*

*

*

Plasma aldosterone

Plasma ACE activity

†

Figure 5. Circulating and tissue RAAS after 35 weeks treatment with QUI, and effects of

additional hydrochlorothiazide or low sodium diet. * p<0.05 vs. MI untreated, † p<0.05 vs.

QUI.


47


In addition to improved survival during the first phase of the study, total urinary protein excretion was significantly decreased in rats treated with HCTZ. This further supports the early beneficial effects of additional HCTZ treatment. MI causes a progressive loss of renal function which affects prognosis32. Furthermore in humans urinary albumin excretion, even at non-proteinuric levels, is predictive for cardiovascular mortality33. As the degree of proteinuria reduction by ACE inhibition


ng

An

g1

mL

-1 h

-1

0

20

40

60

80

100

QUIn=11

QUI+HCTZn=12

QUI+LSn=10

pg

mL

-1

0

50

100

150

200

250550600

Plasma N-ANP

pm

ol m

L-1

0

1

2

3

nm

ol m

L-1

min

-1

0

10

20

30

40

50

60

LV ACE activity

nm

ol m

L-1

min

-1

0

5

10

15

20

25

62 weeks treatment

*

Plasma aldosterone

Plasma ACE activity

Figure 6. Circulating and tissue RAAS after 62 weeks treatment, and effects of additional

hydrochlorothiazide or low sodium diet. * p<0.05 vs. QUI.

48


Chapter 3

was found to be predictive for clinical long-term cardioprotection34, we postulate that any reduction in urinary protein excretion may be considered to be indicative for improved ACE-I therapy. Dietary sodium restriction Dietary sodium restriction did not positively affect quinapril treatment in this study, despite a reduction in plasma Na+. We previously reported that addition of dietary sodium restriction to ACE-I therapy resulted in significantly reduced cardiac hypertrophy and ACE activity11. However, this previous study was performed using zofenopril at a relatively lower (possibly sub-optimal) dose, which could explain differences between the two studies. One concern is that dietary sodium restriction caused a twofold increase in plasma aldosterone levels in survivors at 62 weeks of treatment. This may be considered deleterious, since high aldosterone levels were associated with clinical events in heart failure patients29. Clinical safety / feasibility Although often applied in clinical practice, the use of diuretics in patients with LV dysfunction is controversial. Major concerns are severe hypotension and hypokalemia causing arrhythmia-associated deaths in patients with LV dysfunction35. Hence, we chose to treat rats with hydrochlorothiazide, which has less pronounced effects than loop diuretics, and indeed adding hydrochlorothiazide did not affect blood pressure or plasma K+ levels. Thus, our results indicate that HCTZ may be safely added to long-term quinapril treatment. In summary, we observed that additional hydrochlorothiazide treatment further improved survival during quinapril therapy in the first 35 weeks of chronic treatment after experimental MI in rats. This improvement in survival was associated with decreased urinary protein excretion, and improved parameters of left ventricular filling pressure and decreased RAAS activation. In contrast, dietary sodium restriction added to quinapril did not show any beneficial effects. As the rise in circulating aldosterone levels by dietary sodium restriction may be detrimental, the benefit of long-term dietary sodium restriction on post-MI outcomes could be questioned. Since no adverse effects of the combined therapy were observed, diuretics added to ACE inhibition can be a safe strategy to improve prognosis of post-MI LV dysfunction.

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Cooperative Studies Group. Circulation. 1993;87:VI40-VI48.

32. Hillege HL, van Gilst WH, van Veldhuisen DJ et al. Accelerated decline and prognostic impact of renal

function after myocardial infarction and the benefits of ACE inhibition: the CATS randomized trial. Eur

Heart J. 2003;24:412-420.

33. Hillege HL, Janssen WM, Bak AA et al. Microalbuminuria is common, also in a nondiabetic,

nonhypertensive population, and an independent indicator of cardiovascular risk factors and

cardiovascular morbidity. J Intern Med. 2001;249:519-526.

34. de Zeeuw D, Remuzzi G, Parving HH et al. Albuminuria, a therapeutic target for cardiovascular

protection in type 2 diabetic patients with nephropathy. Circulation. 2004;110:921-927.

35. Cooper HA, Dries DL, Davis CE et al. Diuretics and risk of arrhythmic death in patients with left

ventricular dysfunction. Circulation. 1999;100:1311-1315.

Chapter 4

Hydrochlorothiazide Increases Plasma or

Tissue ACE Inhibitor Drug Levels in Rats with

Myocardial Infarction: Differential Effects on Lisinopril and Zofenopril.

Bart Westendorp

Regien Schoemaker

Wiek van Gilst


Hendrik Buikema

52


Chapter 4

Abstract

Background: Sodium depletion with diuretics augments the efficacy of ACE inhibitor (ACE-I) therapy for hypertension and renal dysfunction, and possibly for LV dysfunction after myocardial infarction. Underlying mechanisms may involve altered ACE-I pharmacokinetics. We hypothesized that the diuretic hydrochlorothiazide (HCTZ) causes increased steady-state levels of the ACE-inhibitors lisinopril (LIS) and zofenopril (ZOF) in rats with myocardial infarction. Methods: Rats were subjected to coronary ligation to induce myocardial infarction. After 1 week, rats were randomized to 50 mg/kg/day HCTZ or control treatment for 3 weeks. The last week, rats received LIS or ZOF in equipotent dosages (3.3 and 10 mg/kg/day, respectively. Rats were sacrificed at Tmax after the last dose of ACE-I, and tissues were collected for analysis of drug concentrations. Results: LIS concentrations in plasma were significantly increased by HCTZ, at unchanged tissue concentrations. This increase could be fully explained by decreased renal function, as evidenced by increased plasma creatinine levels (LIS-HCTZ; 82±5 versus LIS 61±5 µM, p<0.001). In contrast, zofenoprilat levels in kidney and non-infarcted LV were markedly increased by HCTZ, whereas plasma concentrations were unchanged. Although HCTZ tended to increase plasma creatinine in zofenopril-treated rats as well, this increase was less pronounced (ZOF-HCTZ; 61±3 versus ZOF 54±2 µM, p=0.15). Conclusions: HCTZ increases steady-state ACE-I drug levels, most likely by affecting their renal clearance. Notably, the lipophilic ACE-I zofenopril accumulated in tissue, whereas the hydrophilic lisinopril increased in plasma. Whether combining different ACE inhibitors with HCTZ translates into distinct clinical profiles requires further study.

53


Hydrochlorothiazide Increases ACE Inhibitor Levels

Introduction

Left ventricular dysfunction after myocardial infarction (MI) is characterized by progressive cardiac remodeling eventually leading to chronic heart failure. Activation of the renin angiotensin aldosterone system is thought to play a central role in this process. Consequently, angiotensin-converting enzyme inhibitor (ACE-I) therapy effectively prevents this remodeling and reduces mortality, although the therapeutic effects of ACE inhibitors may be at least partially independent from inhibition of the ACE enzyme in itself. Angiotensin II levels can even be elevated during ACE inhibition therapy1,2. Diuretic-induced sodium restriction can enhance the effects of ACE-I in renoprotective and antihypertensive therapy3-6. Whether this can be extended to cardioprotection by ACE inhibition is less well established, but experimental data from animal studies suggest that adding a diuretic may improve ACE-I treatment in the early chronic phase after myocardial infarction7-9. The mechanism underlying diuretic-induced enhanced efficacy of ACE-I therapy is unknown, but a pharmacokinetic interaction may play a role. Interestingly, we previously observed in MI-rats augmented inhibition of cardiac ACE activity by zofenopril during dietary sodium restriction, whereas dietary sodium intake in itself had no effect on ACE activity10. Potentially, sodium restriction affected pharmacokinetics of the ACE-I, for instance by affecting its renal clearance. Accordingly, we studied the influence of hydrochlorothiazide (HCTZ) on steady-state plasma, cardiac and renal tissue ACE-I drug levels and ACE activity after ACE-I therapy in an experimental setting of LV dysfunction after MI in rats.

Methods

Study design The present study was performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. The animal research committee of the University of Groningen approved the study protocol. Male Sprague Dawley rats were subjected to coronary artery ligation (n=163) or sham operation (n=9), as described before11. MI-rats were randomly allocated to one of five experimental groups. One group of MI-rats, as well as the group of sham-operated rats, received no active treatment (i.e. untreated control groups). The other four groups of MI-rats were allocated to one of four different treatment regimens, as summarized in figure 1. We chose to design a protocol with established HCTZ treatment preceding the start of ACE-I treatment. Thus, after a recovery period of one week, MI-rats were randomized to either HCTZ or control treatment. HCTZ was dissolved in the drinking water to achieve a final dosage of 50 mg/kg/day. We and others previously showed that in rats with MI this dose results in diuresis and RAAS activation without blood pressure reduction7,12. As initiation of HCTZ affects water intake, HCTZ was initiated two weeks before ACE-I therapy, to ensure stable water intake and HCTZ dosing at the onset of ACE inhibition.

54


Chapter 4

Against this background of established HCTZ-treatment, ACE-I treatment with either lisinopril or zofenopril was started at day 21, i.e. 3 weeks after induction of MI. The treatment regimen of zofenopril (10 mg/kg/day) and lisinopril (3.3 mg/kg/day) was based on previous experiments in our laboratory13,14. The dose of lisinopril was chosen relative to zofenopril based on the clinical defined daily doses for both ACE-I (10 and 30 mg, for lisinopril and zofenopril, respectively; ATC index, 1998). Until day 26, a steady-state of both ACE-I was achieved by dissolving appropriate amounts of drug in the drinking water. Steady-state is achieved after 4-5 half-lives, which is in total 20-35 hours for zofenopril and and 48-60 hours for lisinopril15. The required amounts of food and water were determined as guided by measurements of body weight and daily water intake. This was done by measuring water intake and average body weight per cage weekly, and calculating the required drug concentration in the drinking water (per cage) for the week after. On days 27 and 28 of the study protocol, the ACE-I as well as HCTZ was administered by means of oral gavage (total volume of 1 µl/g body weight nitrocellulose containing the drugs, or without the drugs in case of untreated controls) as to ensure a synchronized, accurate drug intake in all rats. During the last night before sacrifice, all rats were fasted. Harvesting of tissues and plasma At the day of sacrifice, rats were terminated exactly at Tmax after administration of zofenopril (0.5 hours) or lisinopril (4 hours)15. Tmax was found to be unaffected by HCTZ,

Figure 1. Study scheme. (A) One week after recovery of myocardial infarction (MI), rats were

instituted on hydrochlorothiazide (HCTZ, 50 mg/kg/day in drinking water) or vehicle. Two

weeks thereafter, additional ACE-inhibitor (ACE-I) treatment was started with either

lisinopril or zofenopril (3.3 and 10 mg/kg/day) in drinking water. (B) During the last two days

before sacrifice, drugs were administered by means of oral gavage to ensure synchronized and

accurate drug intake in all rats. (C) After receiving the last dose of ACE-I at four weeks post-

MI, rats were sacrificed at Tmax, i.e. 0.5 and 4 hours for zofenopril and lisinopril, respectively.

55



both for zofenoprilat (unpublished studies at Menarini Ricerche, Firenze) and lisinopril16. Rats were anaesthetized with isoflurane (2.0-2.5%), heparin (1000 IE) was injected into the tail vein for anticoagulation, arterial blood was drawn from the abdominal aorta and collected in separate tubes for analysis of plasma ACE activity and ACE-I drug levels; one mL of arterial blood was mixed with N-ethylmaleimide (5mg/mL) to prevent oxidation of zofenoprilat. Tubes were immediately centrifuged at 1600G for 10 minutes at 4° C. Subsequently, plasma was frozen in liquid nitrogen, and stored at -80° C until assay. After blood collection, rats were perfused with saline to remove remnant blood, as to avoid contribution from the blood compartment in assessment of tissue drug levels and tissue ACE activity. To this end, 10 mL cold 0.9% NaCl solution was gently injected into the aorta, and a hole was pinched into the vena cava inferior to let out the rinsing solution. Thereafter, organs were quickly removed, weighed, divided and frozen in liquid nitrogen for measurement of either tissue concentrations of zofenopril/zofenoprilat or lisinopril, or tissue ACE activity. For measurement of renal drug levels and ACE activity, small pieces of cortical tissue were used. In case of cardiac measurements, both ACE activity and drug concentrations were measured in small pieces of non-infarcted free LV wall. Moreover, a mid-ventricular slice of the LV was stored in 2% paraformaldehyde for histological assessment of infarct size using planimetry on Sirius Red/ Fast Green-stained slides as described previously11. Only rats with MI sizes comprising over 20% of the LV were included for analysis.

Drug measurements Before shipment, tissues were homogenized on dry ice in a 0.5 M K2PO4 buffer containing 5 mg/mL N-ethylmaleimide to prevent oxidation of zofenoprilat. For measurement of lisinopril concentrations, homogenates were shipped on dry ice to Analytical Laboratories R&D, Berlin-ChemieLab (Berlin, Germany) for HPLC analysis. The determination of lisinopril and internal standard (Enalapril-Diketopiperazin DKP) in rat plasma was performed by means of a validated HPLC-MS/MS analytical method. The lower limit of quantification (LOQ) for lisinopril was 2.0 ng/mL of rat plasma The lisinopril and the internal standard were extracted from rat plasma by a solid-phase extraction method. Briefly, after addition of 0.2 N HCl to the samples, the samples were vortexed and transferred into a solid phase extraction cartridge (Waters Oasis 30 mg), that had been conditioned with 1 mL methanol and 1 ml HPLC water. After loading, the cartridge was washed with 1 mL water and 1 mL methanol/ water (5:95; v/v). Subsequently, both drugs were eluated with 3 x 1 mL methanol. The methanolic eluates were evaporated to dryness; the residue was redissolved in mobile phase and injected into the chromatographic system. The HPLC-MS/MS system consisted of a model 200 solvent delivery pump (Perkin Elmer) equipped with an autosampler Gilson 234, a column oven Agilent (G1316A), an HPLC-column (Symmetry Shield RP-8, 2.1 x 150 mmn and 5 µm particle size (Waters)), an HPLC precolumn (Symmetry Shield RP-8, 2.1 x 10 mmn and 3,5 µm particle size

56


Chapter 4

(Waters), an API 2000 tandem mass spectrometer (PE Biosystems) and a computer equipped with Analyst 1.3 Software. For measurement of zofenopril and zofenoprilat samples were shipped on dry ice to the research lab of Menarini Richerche (Roma, Italy). The assay was performed by liquid chromatography coupled with tandem mass spectrometry as described previously17. Briefly, analytes were extracted by liquid-liquid extraction with toluene. The organic phase was separated, dried, reconstituted with 200 µL of a methanol/water mixture (1:1), and injected through an autosampler. The extract was chromatographed on a reverse phase column coupled to a triple quadripole mass spectrometer. ACE activity ACE activity in the plasma and spared myocardial tissue was determined according to the Hip-His-Leu method, as has been described before11. In short, tissues were homogenized in a 50 mM KPO4 buffer. Of the homogenates 100 µl was pipetted in a 0.5 M K2PO4 buffer. Then the ACE substrate Hippury-His-Leu 12.5 nM (Sigma) was added and incubated at 370 C for exactly 10 minutes. The conversion of the substrate was stopped by adding 1.45 ml 280 mM NaOH. Thereafter, 100 µl phtaldialdehyde was added for the labeling of free His-Leu. The amount of labeled His-Leu was fluorimetrically determined at excitation and emission wavelenghts of 364 and 486 nm, respectively. Control samples were included in which the conversion of substrate was prevented by adding NaOH before the substrate Hippuryl-His-Leu. Statistical analysis For comparison of zofenopril and lisinopril effects in the infarct model, ANOVA with post hoc least square difference correction was performed. For analysis of drug levels in MI rats, group averages of HCTZ+ACE-I were compared to ACE-I alone with a student’s t-test in case of normal distribution. If distribution was not normal, log transformation was used to achieve normality. If log transformation did not result in a normal distribution, a non-parametric Mann-Whitney test was used. Levels of zofenoprilat in some of the LV and kidney tissue samples were undetectably low. In case of detection problems, zofenoprilat levels were given the value of the detection limit in statistical analysis, to be sure not to overestimate any difference.

Results

General characteristics Mortality during the first 24 hours was 33% after MI induction; during the rest of the follow-up period, none of the rats died. None of the rats died after sham operation. After exclusion of data from rats with infarcts smaller than 20% of the LV circumference, infarct sizes were comparable in the MI groups, except for a small, yet significant difference between rats with lisinopril + HCTZ and lisinopril only (table 1). Both zofenopril and lisinopril significantly reduced body weight gain, as compared to untreated MI-rats. HCTZ further reduced body weight gain, in both zofenopril- and lisinopril-treated rats (table 1).

57



Table 1. Effects of experimental myocardial infarction and of 10 mg/kg/day zofenopril and 3.3

mg/kg/day lisinopril on LV hypertrophy.

No treatment MI - Lisinopril MI - Zofenopril

Sham MI Vehicle HCTZ Vehicle HCTZ

N= 9 7 14 12 15 12

BW (g) 358 ± 6 357 ± 9 336 ± 6 *† 317 ± 3 *†‡ 344 ± 8 297 ± 4 *†

∆ BW (g) 21 ± 5 34 ± 8 15 ± 2 † 2 ± 1 *†‡ 13 ± 2† 5 ± 3 *†§

Infarct size (%) - 30 ± 3 26 ± 1 32 ± 2 ‡ 29 ± 2 28 ± 2

Plasma K+ 3.8±0.1 3.8±0.1 4.4±0.1*† 4.6±0.1*† 4.3±0.1*† 3.9±0.1§

LV:BW (mg/g) 2.4 ± 0.1 2.9 ± 0.1 * 2.5 ± 0.0 † 2.5 ± 0.1 † 2.5 ± 0.0 † 2.5 ± 0.1 †

Data are shown as mean±sem. The ∆-BW is the difference in bodyweight (g) between time of

operation and sacrifice, giving an indication of treatment on body weight development. Infarct

size is expressed as percentage of infarct-to-total left ventricular circumference.* p<0.05 versus

Sham; † p<0.05 versus MI, ‡ p<0.05 versus MI+lisinopril; § p<0.05 versus MI+zofenopril. HCTZ;

hydrochlorothiazide. LV:BW; left ventricle-to-body weight ratio. Both lisinopril and zofenopril treatment alone caused a moderate, but highly significant increase in plasma K+ concentrations. HCTZ treatment did not alter this increase in lisinopril-treated rats, whereas HCTZ in zofenopril-treated rats normalized plasma K+ concentrations . MI resulted in LV hypertrophy, as indicated by increased LV/BW ratios in untreated MI rats compared to sham-operated rats. Zofenopril and lisinopril treatment similarly reduced LV/BW ratios, without additional effects of HCTZ (table 1). Drug concentrations Plasma lisinopril concentrations were significantly higher (60%, p=0.03) in rats instituted with HCTZ as compared to those not, whereas lisinopril concentrations in cardiac and renal tissue were not different (figure 2a). Plasma levels of the prodrug zofenopril were very low (approximately 4.5% of total plasma zofenopril plus zofenoprilat levels) compared to the plasma levels of zofenoprilat, but neither plasma zofenopril (152±14 vs. 125±15 ng/mL, p=ns) nor plasma zofenoprilat levels (figure 2b) differed between rats treated with HCTZ and vehicle, respectively. Contrary to lisinopril, however, tissue levels of the active metabolite zofenoprilat were significantly higher in rats treated with HCTZ as compared to those not; 3-fold and 20-fold increases in median values for LV and renal tissue, respectively (figure 2c). Finally, the prodrug zofenopril could not be detected in these tissues.

58


Chapter 4

Kidney function To asses whether alterations in drug levels could be explained by altered renal drug clearance, kidney function was determined, as measured by plasma creatinine concentrations. Myocardial infarction alone did not alter plasma creatinine levels. Lisinopril alone caused a moderate increase in plasma creatinine, and treatment with HCTZ caused a significant further increase (figure 3). Treatment with zofenopril alone did not significantly increase plasma creatinine. Zofenopril treatment in rats treated

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Figure 2. Plasma, left ventricle (LV) and kidney tissue ACE-inhibitor drug concentrations in

myocardium infarcted rats instituted either or not with hydrochlorothiazide (HCTZ), after

treatment with either (A) lisinopril (3.3 mg/kg/day) or (B) zofenopril (10 mg/kg/day). Boxes

delineate 25th and 75th percentiles, lines within boxes represent medians, and whiskers

represent 10th and 90th percentiles, respectively. * p<0.05 as indicated.

59



with HCTZ caused a moderate increase in plasma creatinine (p<0.05 versus untreated sham- and MI-rats). The HCTZ-induced increase in plasma lisinopril was related to a decrease in renal function, as is shown by the highly significant correlation between plasma creatinine and lisinopril concentrations (figure 4A). Tissue concentrations of lisinopril were positively correlated with plasma creatinine as well (figure 4C). Trends were similar for LV and kidney. Plasma concentrations of zofenoprilat were not significantly associated with creatinine (figure 4B). However kidney as well as LV concentrations of zofenoprilat were significantly correlated with plasma creatinine (figure 4D). ACE activity Both ACE inhibitors caused a nearly complete reduction (±90%) in plasma ACE activity compared to untreated MI- and sham-rats, and this effect did not differ between rats treated with or without HCTZ (figure 5). Renal ACE activity tended to be slightly decreased in MI-rats compared to sham operated rats. ACE-I treatment further decreased ACE activity in the kidneys, and consequently all ACE-I treated groups displayed significantly decreased renal ACE activity compared to the group of untreated sham-rats. As all ACE-I treated groups displayed very low renal ACE activity no differences were observed between MI-rats treated with or without HCTZ. LV ACE activity was significantly increased in MI-rats compared to untreated sham-controls, but both ACE-I failed to reduce LV ACE activity, regardless whether or not rats were treated with HCTZ (figure 5). No significant correlation between ACE-I drug levels (neither zofenoprilat nor lisinopril) and ACE activity was observed in plasma or tissue.

Sham MI-control MI MI-HCTZ MI MI-HCTZ

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Figure 3. Renal function, as indicated by plasma creatinine concentration, in rats with

myocardial infarction, and effects of lisinopril and zofenopril, alone or treated with the

diuretic hydrochlorothiazide. * p<0.05 versus sham, † p<0.05 versus MI, ‡ p<0.05 versus

lisinopril alone. HCTZ; hydrochlorothiazide.

60


Chapter 4

D) Zofenoprilat

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Figure 4. Relation between kidney function and plasma ACE–I concentrations in rats with

myocardial infarction, either or not treated with hydrochlorothiazide. HCTZ;

hydrochlorothiazide. A) Lisinopril concentrations in plasma were highly correlated with

plasma creatinine concentrations. B) Zofenoprilat and creatinine concentrations in plasma did

not correlate significantly. C) and D) Correlation between plasma creatinine and (kidney)

tissue concentrations of zofenoprilat.

61



A) Plasma ACE


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Figure 5. ACE activity in plasma, left ventricle, and kidney in rats with myocardial infarction,

and effects of the ACE inhibitors zofenopril and lisinopril, alone or combined with

hydrochlorothiazide. Data are shown in boxplots in case data were not normally distributed.

HCTZ; hydrochlorothiazide. * p<0.05 versus sham; † p<0.05 versus MI control.

62


Chapter 4

Discussion

Aim of the present study was to investigate the effects of hydrochlorothiazide (HCTZ) on steady-state plasma, cardiac- and renal tissue ACE-I drug levels and ACE activity after ACE-I therapy in an experimental setting of LV dysfunction after MI in rats. ACE-I accumulation by diuretic treatment Lisinopril, the lysine derivate of enalaprilat, is highly hydrophilic, does not require metabolic transformation to become active, and is cleared unchanged via the kidneys. We found that plasma drug levels of this ACE-I were higher in rats treated with HCTZ. This was explained by decreased renal clearance of lisinopril, as plasma concentrations of lisinopril showed a highly significant relation with plasma creatinine, a measure for glomerular filtration rate (GFR). Most likely, volume depletion accounted for this decrease in GFR, thereby resulting in drug accumulation. Others have reported no or marginal interactions in terms of clearance between HCTZ and lisinopril16,18, but note that these were all studies testing effects of HCTZ on plasma lisinopril concentrations after one single dose. To our knowledge, this study is the first to address the effects of HCTZ on steady-state ACE-I levels in a setting of repeated drug dosing. Infarct sizes were significantly different between lisinopril- and lisinopril + HCTZ-treated rats. However this difference in infarct size between the two lisinopril groups does not interfere with our findings, as we found no relation between infarct size and ACE-I concentrations. We also tested effects of HCTZ on ACE-I concentrations in sham-operated rats (data not shown for reasons of clarity), and results were very similar, confirming that presence of myocardial infarction did not play a role in the observed effects of HCTZ. To compare ACE inhibitors with different kinetic properties, we also investigated the effects of HCTZ on zofenopril/zofenoprilat drug levels. Zofenopril is a lipophilic ACE-I prodrug which undergoes hydrolyzation to its active metabolite zofenoprilat in tissue. Plasma prodrug levels represented only 4.5% of the total circulating levels of zofenopril plus zofenoprilat in the present study, indicating a near total conversion of zofenopril into its active metabolite. Zofenoprilat is hydrophobic, and cleared predominantly via the kidneys (±70%), but also via the bile and feces15. In contrast to lisinopril, plasma zofenoprilat levels were only marginally increased by HCTZ, whereas tissue zofenoprilat levels increased markedly, indicating ACE-I drug accumulation in the tissue. These findings may be related to volume depletion by addition of HCTZ to zofenopril treatment. Rather than leading to increased plasma levels, the lipophilic nature of zofenopril would favor drug penetration of the ACE-I into the tissue, with a subsequent tissue accumulation of the active metabolite zofenoprilat19. Note that we observed a significant correlation between plasma creatinine and tissue zofenoprilat concentrations, potentially as both depend on volume status. Surprisingly, zofenoprilat concentrations in plasma did not increase significantly along with tissue concentrations during HCTZ treatment. Thus, mechanisms independent of volume status may be involved as well in the observed accumulation of zofenoprilat in renal and cardiac tissue.

63



Relevance/implications of increased drug levels. Increased ACE inhibitor levels were not associated with a significant reduction in ACE activity in the current study. In case of plasma and kidney, this may be explained by maximal inhibition as a result of high dosing: both zofenopril and lisinopril caused nearly complete inhibition of circulating and renal ACE. For cardiac ACE activity, we observed no effect of zofenopril or lisinopril, and also no effects of HCTZ treatment. This finding is consistent with previous studies employing the rat coronary ligation model of LV dysfunction post-MI10,20,21. An explanation may be upregulation of cardiac ACE expression under ACE inhibition22, although this has not unambiguously been shown for cardiac tissue8,23,24. It has long been recognized that the local rather than the circulating RAAS is involved in pathophysiology of cardiovascular disease25. Thus, increasing the tissue penetration of the ACE-I may potentiate its beneficial effects. Although we found no further reduction of ACE activity, the HCTZ-induced increase in tissue zofenoprilat levels may have local beneficial effects independent from ACE inhibition per se. Firstly, increased levels of tissue ACE-I could lead to further reduction in oxidative stress. Zofenoprilat contains a sulphydryl-group, which may scavenge reactive oxygen species, thereby reducing inflammation and increasing NO bioavailability13,26,27. Secondly, ACE-inhibitors have zinc-chelating properties, which may interfere with cardiac remodeling via reduction of matrix metalloproteinase activity28-31.

Renal effects HCTZ on lisinopril versus zofenopril We used equipotent dosages of ACE inhibitors, based on previous results13, and the ratio between clinically used dosages. Dose equivalence was illustrated by similar reductions in LV hypertrophy and ACE activity, and a comparable rise in serum K+. Notably, effects of HCTZ treatment on zofenopril and lisinopril were differential, substantiating the view that not all ACE inhibitors are by definition interchangeable within their class32,33. Firstly, we observed differential effects of HCTZ on lisinopril and zofenoprilat concentrations, as discussed above. Furthermore, we observed that HCTZ decreased (i.e. normalized) plasma K+ levels in zofenopril- but not lisinopril-treated rats. We have no explanation for this finding, but as plasma electrolyte disturbances are of great importance in patients with heart failure34, this matter deserves further study. Combining HCTZ with lisinopril increased plasma creatinine concentrations. Although plasma creatinine may not be the most accurate indicator of renal function, it corresponds with a substantial drop in glomerular filtration rate GFR by ±25% compared to lisinopril monotherapy and ±50% compared to untreated rats. Importantly, decreased renal function by combining RAAS inhibition with diuretic treatment has also been reported in humans35. This effect of combining ACE inhibition with HCTZ treatment could have clinical implications, as even mildly impaired renal function is strongly and independently associated with worsened prognosis after MI36.

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Chapter 4

Conclusion In the present study diuretic treatment with HCTZ significantly influenced steady-state plasma and tissue ACE-I drug levels in rats with experimental MI. The effect of HCTZ differed for the different ACE-I employed, resulting in increased ACE-I drug levels in the plasma in case of the hydrophilic ACE-I lisinopril versus increased ACE-I drug levels in renal and cardiac tissue in case of the lipophilic ACE-I zofenopril. Increased tissue ACE-I drug levels may contribute to the enhanced organ-protective effects of ACE-I therapy. However, decreased renal function by combining HCTZ with ACE inhibition may have adverse effects.

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15. Subissi A, Evangelista S, Giachetti A. Preclinical profile of zofenopril: an angiotensin converting

enzyme inhibitor with peculiar cardioprotective properties. Cardiovasc Drug Rev. 1999;17:115-133.

16. Swaisland AJ. The pharmacokinetics of co-administered lisinopril and hydrochlorothiazide. J Hum

Hypertens. 1991;5 Suppl 2:69-71.

17. Marzo A, Dal Bo L, Mazzucchelli P et al. Pharmacokinetics and pharmacodynamics of zofenopril in

healthy volunteers. Arzneimittelforschung. 1999;49:992-996.

18. Laher MS, Mulkerrins E, Hosie J et al. The effects of age and renal impairment on the pharmacokinetics

of co-administered lisinopril and hydrochlorothiazide. J Hum Hypertens. 1991;5 Suppl 2:77-84.

19. Ranadive SA, Chen AX, Serajuddin AT. Relative lipophilicities and structural-pharmacological

considerations of various angiotensin-converting enzyme (ACE) inhibitors. Pharm Res. 1992;9:1480-

1486.



21. Hirsch AT, Talsness CE, Smith AD et al. Differential effects of captopril and enalapril on tissue renin-

angiotensin systems in experimental heart failure. Circulation. 1992;86:1566-1574.

22. Schunkert H, Ingelfinger JR, Hirsch AT et al. Feedback regulation of angiotensin converting enzyme

activity and mRNA levels by angiotensin II. Circ Res. 1993;72:312-318.

23. Kelly MP, Kahr O, Aalkjaer C et al. Tissue expression of components of the renin-angiotensin system in

experimental post-infarction heart failure in rats: effects of heart failure and angiotensin-converting

enzyme inhibitor treatment. Clin Sci (Lond). 1997;92:455-465.

24. Samani NJ, Cumin F, Kelly M et al. Expression of components of the RAS during prolonged blockade at

different levels in primates. Am J Physiol. 1994;267:E612-E619.

25. Dzau VJ, Bernstein K, Celermajer D et al. Pathophysiologic and therapeutic importance of tissue ACE: a

consensus report. Cardiovasc Drugs Ther. 2002;16:149-160.

26. Cominacini L, Pasini A, Garbin U et al. Zofenopril inhibits the expression of adhesion molecules on

endothelial cells by reducing reactive oxygen species. Am J Hypertens. 2002;15:891-895.

27. Evangelista S, Manzini S. Antioxidant and cardioprotective properties of the sulphydryl angiotensin-

converting enzyme inhibitor zofenopril. J Int Med Res. 2005;33:42-54.

28. Sorbi D, Fadly M, Hicks R et al. Captopril inhibits the 72 kDa and 92 kDa matrix metalloproteinases.

Kidney Int. 1993;44:1266-1272.

29. Hayashidani S, Tsutsui H, Ikeuchi M et al. Targeted deletion of MMP-2 attenuates early LV rupture and

late remodeling after experimental myocardial infarction. Am J Physiol Heart Circ Physiol.

2003;285:H1229-H1235.

30. Reinhardt D, Sigusch HH, Hensse J et al. Cardiac remodelling in end stage heart failure: upregulation of

matrix metalloproteinase (MMP) irrespective of the underlying disease, and evidence for a direct

inhibitory effect of ACE inhibitors on MMP. Heart. 2002;88:525-530.

31. Sakata Y, Yamamoto K, Mano T et al. Activation of matrix metalloproteinases precedes left ventricular

remodeling in hypertensive heart failure rats: its inhibition as a primary effect of Angiotensin-

converting enzyme inhibitor. Circulation. 2004;109:2143-2149.

32. Furberg CD, Psaty BM. Should evidence-based proof of drug efficacy be extrapolated to a "class of

agents"? Circulation. 2003;108:2608-2610.

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33. Furberg CD, Pitt B. Are all angiotensin-converting enzyme inhibitors interchangeable? J Am Coll

Cardiol. 2001;37:1456-1460.

34. Macdonald JE, Struthers AD. What is the optimal serum potassium level in cardiovascular patients? J

Am Coll Cardiol. 2004;43:155-161.

35. Esnault VL, Ekhlas A, Delcroix C et al. Diuretic and Enhanced Sodium Restriction Results in Improved

Antiproteinuric Response to RAS Blocking Agents. J Am Soc Nephrol. 2005;16:474-481.

36. Sorensen CR, Brendorp B, Rask-Madsen C et al. The prognostic importance of creatinine clearance after

acute myocardial infarction. Eur Heart J. 2002;23:948-952.

Chapter 5

Adverse Renal Effects of Hydrochlorothiazide

in Rats with Myocardial Infarction Treated

with ACE Inhibition.

Bart Westendorp

Inge Hamming

Gerjan Navis

Harry van Goor

Hendrik Buikema

Wiek van Gilst

Regien Schoemaker

68

Chapter 5 - Versie 1 29 juli 2005

Chapter 5

Abstract

Background: Diuretics reduce fluid retention and can potentiate the response to ACE inhibition (ACE-I). However, volume depletion during ACE-I can also have adverse renal structural and functional effects. We studied renal effects of adding diuretic to ACE-I after myocardial infarction (MI), i.e. a condition with intrinsically normal kidneys. Methods: MI was induced in rats by coronary ligation. After 2 weeks, rats were randomized to ACE-I quinapril alone (QUI, n=34) or with add-on hydrochlorothiazide (QUI+HCTZ, n=46). Survival was monitored for 14 months. Plasma creatinine (pCr) was measured at 4 months. Subgroups were sacrificed to study renal morphology after 8 and 14 months. In addition, untreated MI rats were studied at 8 months. Kidneys were studied for interstitial damage, myofibroblast transformation and fibrosis, and macrophage influx. Results: At 4 months, pCr was increased by 40% in QUI+HCTZ compared to QUI (46 vs. 33 µmol/L, p=0.001). Though 14-month mortality was similar in QUI+HCTZ and QUI, stratification based on pCr showed increased mortality in the tertile with highest pCr (p=0.03, Log rank). Remarkably, add-on HCTZ caused severe renal interstitial lesions, i.e. tubular dilatation and fibrosis. Interstitial SMA was increased at 8 and 14 months, and coincided with collagen deposition and macrophage influx. HCTZ did not affect blood pressure or plasma K+. In rats with QUI monotherapy or untreated MI renal structure was normal. Conclusions: Adding HCTZ to ACE-I detrimentally affected not only renal function, but also renal structure in rats with MI. As decreased renal function was associated with increased mortality, adverse renal effects of volume depletion by adding HCTZ to ACE-I may exert unfavorable effects on long-term prognosis after MI.

69



Introduction

The progression of left ventricular (LV) dysfunction towards overt chronic heart failure (CHF) after myocardial infarction (MI) is associated with progressive cardiac remodeling. Activation of the renin angiotensin aldosterone system (RAAS) plays a central role in this process, and blocking the RAAS with angiotensin-converting enzyme inhibitors (ACE-I) effectively reduces remodeling and prolongs survival after MI. Diuretics are often chronically added to ACE-I treatment to prevent fluid retention, although clinical trials to show their effect on mortality are lacking. On one hand, a diuretic-induced negative sodium balance generally elicits an optimal therapeutic effect of ACE inhibition1-3. On the other hand, diuretics may have adverse effects, i.e. electrolyte disturbances and renal function loss. We previously reported improved survival and cardiac function by add-on diuretic therapy with hydrochlorothiazide in rats with MI in the early chronic phase4. However, this survival benefit was not maintained during prolonged long-term follow-up (14 months). Addition of diuretic treatment to ACE inhibition may have adverse renal structural and functional effects, which may in turn affect long-term survival after MI, as poor renal function is strongly and independently associated with worsened long-term prognosis after MI5. Addition of a diuretic to ACE inhibition however may decrease renal function6. In the present study we investigated the long-term effects of adding a diuretic to ACE inhibition on renal morphology and function in relation to prognosis. We hypothesized that hydrochlorothiazide added to ACE inhibition in rats with myocardial infarction causes a sustained decrease in renal function and alterations in renal morphology, which is associated with worsened long-term survival.

Methods

The investigation conforms to the Guide for the Care and Use of Laboratory Animals (Published by the US National Institutes of Health, NIH publication No. 85-23, revised 1996). The animal research committee of the University of Groningen approved this study protocol. Male, Sprague Dawley rats (Harlan, Zeist, The Netherlands), weighing 280±25g were subjected to coronary ligation as previously described7. Since the study was aimed at the chronic phase after myocardial infarction, and not at interfering with the early healing process and scar formation, treatment was started 14 days post-MI. Rats were randomly assigned to: quinapril (QUI, 15 mg kg-1 day-1), quinapril + hydrochlorothiazide (HCTZ), quinapril + low sodium diet (LS). Quinapril was mixed through food (Hope Farms, Woerden, The Netherlands). This dose results in optimal ACE-I therapy8,9. HCTZ (50 mg kg-1 day-1) was dissolved in drinking water. This dose causes RAAS activation and an increase in diuresis, but no blood pressure reduction in rats with myocardial infarction10. The LS-group was fed with food pellets (Hope Farms, Woerden, The Netherlands containing 0.05% NaCl instead of 0.3% (normal diet) (LS-diet;). To ensure constant drug intakes during the entire study period,

70


Chapter 5

concentrations of quinapril and HCTZ were adjusted weekly. This was done by measuring food/water intake and average body weight per cage weekly, and calculating the required drug concentration in food and water (per cage) for the week after. Rats were fed ad libitum, and housed group-wise in clear polyethylene cages in temperature (220 C)- and humidity (50%)-controlled rooms with a 12h light/dark cycle. Study 1- Renal function and mortality Rats were allocated to one of the three active treatment groups, and survival was monitored - in a blinded fashion – for a period of 14 months after onset of therapy. Use of color-tags ensured appropriate housing and treatment by caretakers. Cages were checked for dead animals at least once daily; tissues of dead rats were collected, weighed and stored for analysis. After 4 months of treatment 1 mL blood was obtained from the retro-orbital plexus under isoflurane anesthesia. The blood was anti-coagulated with EDTA, centrifuged at 1600G for 10 minutes at 4°C, and stored at -80°C until measurement of plasma creatinine concentrations according to routine clinical methods. Study 2 - Renal morphology during and after chronic treatment Rats were operated and treated according to the same procedures as in the above study, and sacrificed 8 months after onset of treatment to study renal morphology. In addition, subgroups of randomly chosen surviving rats from the study described above were sacrificed for assessment of renal morphology after the 14-month follow-up period. Tissue processing Rats were anesthetized with isoflurane (2%) in a mixture of O2 and N2O (1:2), the carotid artery was cannulated and a pressure tip catheter (Micro-Tip 3French, Millar instruments, Houston TX, USA) advanced into the aortic arch, and arterial blood pressure was recorded. Subsequently, arterial blood and tissues were collected for further analysis. Blood was drawn from the abdominal aorta, anti-coagulated with EDTA, centrifuged at 1600G for 10 minutes at 4°C, and stored at -80°C until assay. Plasma K+ and Na+

concentrations were determined according to routine clinical methods. Hearts and kidneys were rinsed with ice cold NaCl (0.9%), and fixed in 2% paraformaldehyde. The kidneys were cut longitudinally in two parts. The fixated tissues were then processed for paraffin embedding according to standard procedures. LV infarct size was determined, on sections stained with Sirius Red/Fast Green, as percentage of LV circumference, as described before7. Only rats with MI sizes larger than 20% of LV were included for analysis, since smaller infarcts are fully compensated, and do not result in LV dysfunction. Kidney histology Routine morphology was evaluated using periodic acid shift-stained sections by a qualified pathologist. Renal interstitial α-smooth muscle actin (α-SMA) was detected in 3-µm paraffin sections with a mouse monoclonal antibody. First, the antibody was incubated for 60 minutes, and subsequently its binding was detected with peroxidase (PO)-labeled

71



rabbit anti-mouse antibody for 30 minutes. The expression of interstitial α-SMA was quantified by computerized morphometry (50 fields/kidney, magnification 200x); Glomeruli and vessels were excluded from measurement by tracing them with a cursor along Bowman’s capsule or the vessel wall. The medulla was not evaluated. Renal interstitial macrophage infiltration was detected using a mouse anti-rat monoclonal antibody against ED1 (Serotec, Oxford, England). Subsequently sections were incubated with PO-labeled secondary antibody. Peroxidase activity was visualized by 3-amino-9-ethylcarbazol (AEC). Interstitial macrophages were counted using morphometry; per kidney, 50 fields were evaluated. Statistics Data are presented as mean ± standard error of the mean. Survival analysis was done using log rank analysis with pairwise comparison over strata. Other parameters were compared using oneway analysis of variances (ANOVA) with least square difference post hoc analysis for multiple comparisons. Differences were considered significant at the level of 0.05 (two tailed).

Results

Study 1 - Renal function and mortality To investigate whether renal function affects mortality in rats with MI, we analyzed long-term survival based on plasma creatinine concentrations assessed after 4 months of treatment (figure 1A). Stratification of groups into tertiles based on plasma creatinine concentrations, regardless of treatment, showed significantly increased mortality in the highest tertile after 14 months treatment, as compared to the group with lowest plasma creatinine concentrations. The middle tertile showed intermediate survival. The three groups stratified on plasma creatinine had similar infarct sizes; 32±1, 34±1, and 33±1 % of LV, for low, intermediate and high plasma creatinine, showing that the extent of myocardial damage was no confounder in the relation between renal function and long-term survival. The effects of treatment on renal function are shown in figure 1B. The three treatment groups had comparable infarct sizes: 34±1 %, 32±1 %, and 33±1 %, for quinapril, quinapril + HCTZ, and quinapril + LS, respectively. Addition of HCTZ to quinapril caused a ~40% increase in plasma creatinine concentrations after 4 months of treatment (p=0.001, figure 1B). Notably, average plasma creatinine concentrations in the HCTZ-treated rats corresponded with the highest tertile in the survival analysis described above. Dietary sodium restriction did not influence plasma creatinine concentrations. Study 2 – Renal morphology Table 1 shows general characteristics of the rats sacrificed for pathological analyses after 8 and 14 months of treatment. Comparison of untreated and quinapril-treated rats at 8 months showed a marked reduction in systolic blood pressure (p<0.005) and an increase in plasma K+ concentrations (p<0.05). Addition of hydrochlorothiazide or low sodium diet to quinapril did not further alter blood pressure or plasma K+ concentrations, neither at 8 nor at 14 months. Plasma Na+ concentrations were slightly lower in rats

72


Chapter 5

treated with HCTZ or dietary sodium in addition to quinapril when compared to quinapril alone. Treatment groups were well balanced for infarct size (table 1).

Table 1. General characteristics of rats included in histological measurements, after 8 and 14

months treatment.

MI-Quinapril MI

Month Control + HCTZ + LS Control

8 6 5 5 4 N=

14 11 12 10 -

8 25±1 28±1 29±3 27±3 MI size (% of LV)

14 34±2 33±1 37±2 -

8 4.4±0.1 * 4.4±0.2 * 4.4±0.2 * 3.9±0.2 Plasma K+ (mmol/L)

14 4.7±0.2 4.5±0.2 5.0±0.1 -

8 137.4±0.3 136.6±0.5 * 134.5±2.1 * 142.8±4.4 Plasma Na+ (mmol/L)

14 131.1±0.9 130.3±0.4 130.6±0.6

8 92±2 * 91± 3 * 89±4 * 125±9 SBP (mm Hg)

14 94±5 94±3 91±3 -

* p<0.05 versus untreated control. HCTZ; Hydrochlorothiazide, LS; Low sodium diet, SBP; systolic

blood pressure at termination.

Time (weeks)0 20 40 60

Rat

s al

ive

(%)

0

50

60

70

80

90

100

Creatinine <31 µmol/L (n=33)Creatinine 31-43 µmol/L (n=32)Creatinine >43 µmol/L (n=33)

p = 0.025

Creatinine measurement

Plasma creatinine

µmo

l/L

0

10

20

30

40

50

60

QUI (n=28)QUI + HCTZ (n=41)QUI + LS (n=29)

**

A) B)

Figure 1. Renal function during ACE inhibition after myocardial infarction, and relation with

mortality. A) Survival curves represent groups stratified into tertiles based on plasma

creatinine. B) Effect of add-on HCTZ and dietary sodium restriction on renal function as

reflected by plasma creatinin concentrations. ** p<0.005 versus QUI.

73



A)

C)

D)

F)

G)

1). 2).

B) E)

Figure 2. Renal tubular degeneration and increased expression of the fibrosis marker α-smooth

muscle actin (brown) by addition of hydrochlorothiazide to ACE inhibitor therapy in rats with

myocardial infarction. A) 8 Months untreated MI; B-D) 8 months treatment with QUI,

QUI+HCTZ, and QUI+LS, respectively; E-G 14 months treatment with QUI, QUI+HCTZ, and

QUI+LS, respectively. 1) tubular dilatation, 2) infiltration of inflammatory cells.

A) Untreated - 8 months

B) QUI - 8 months

C) QUI+HCTZ - 8 months

D) QUI+LS - 8 months

E) QUI - 14 months

F) QUI+HCTZ - 14 months

G) QUI+LS - 14 months

74


Chapter 5

Quinapril treatment did not result in abnormal renal morphology, except for proximal arteriolar wall thickening (not shown). However, addition of HCTZ to quinapril treatment had profound effects on tubulo-interstitial morphology. Most notably marked tubular dilatation and degeneration of tubular cells occurred (figure 2). This was accompanied by myofibroblast transformation, as indicated by significantly increased α-SMA expression (figures 2 and 3). Furthermore, marked peritubular interstitial fibrosis was confirmed by intense collagen-positive staining (with Sirius Red) around the dilated tubuli (figure 4, right panel). Fibrosis coincided with inflammation, as shown by significantly increased interstitial macrophage influx (figures 2 and 3). Comparison between quinapril-treated and untreated MI rats showed that the quinapril per se or combined with dietary sodium restriction did not cause any sign of tubulo-

B) macrophage infiltration

QUI QUI+HCTZ QUI+LS MI QUI QUI+HCTZ QUI+LS

no

. per

fie

ld o

f vi

ew

0

10

20

30

40

8 months treatment 14 months treatment

A) α-smooth muscle actin

QUI QUI+HCTZ QUI+LS MI QUI QUI+HCTZ QUI+LS

% s

urf

ace

area

0

1

2

3

4

5

*

*

8 months treatment 14 months treatment

*

Figure 3. Effects of sodium depletion on parameters representing renal interstitial fibrosis in

rats with myocardial infarction, after 8 and 14 months treatment with ACE inhibitor

quinapril, and. α-SMA; interstitial alpha-smooth muscle actin. * p<0.05 versus quinapril

treated control.

75



interstitial damage. These interstitial lesions were also not observed in rats treated with a low sodium diet in addition to quinapril.

Discussion

We studied the effects of long-term add-on diuretic treatment or dietary sodium restriction to ACE inhibition on kidney structure and function in rats with myocardial infarction. Main observation of this study was that hydrochlorothiazide caused a marked increase in plasma creatinine concentrations, and renal tubular degeneration and interstitial fibrosis in rats treated with the ACE inhibitor quinapril. In contrast, dietary sodium restriction did not have these effects. As reduced kidney function was associated with increased mortality in the current study, adverse renal effects of adding HCTZ treatment to ACE inhibition may hence overwhelm the favorable effects of combination therapy on cardiac function and survival on the long term. This would explain why diuretic treatment failed to improve long–term outcome of ACE inhibition in rats with myocardial infarction, despite early beneficial effects on cardiac function and survival 4. Renal effects of add-on HCTZ Plasma creatinine concentrations were determined as a measure for renal function. The increase in plasma creatinine concentration caused by addition of HCTZ to quinapril may have been caused by volume depletion, resulting in hypoperfusion of the kidneys6. This especially holds true for LV dysfunction, which is intrinsically characterized by decreased cardiac output and low renal perfusion11. The mechanism underlying the tubulo-interstitial abnormalities cannot be determined from the current study. The tubular lesions described in the current study could reflect hypoxia-induced damage as a result of renal hypoperfusion after volume depletion by combining HCTZ and ACE inhibitor treatment. However, this explanation is unlikely, as we saw no tubular degeneration in rats treated with quinapril only or quinapril + low sodium diet, whereas the effects of these regimens on blood pressure in these rats were as pronounced as in rats treated with add-on HCTZ. Furthermore, low renal perfusion by ACE-I is generally reflected by prominent afferent arteriolar wall thickening8, but morphometry analysis did not show further afferent wall thickening in HCTZ-treated rats (data not shown). Alternatively, direct effects of HCTZ on the tubular cells may have caused the lesions reported in the present study. Low sodium diet did not influence renal morphology; indicating that the effects of HCTZ are independent of sodium status. This is in accordance with a previous study, which showed that a comparable dose of HCTZ caused degeneration and apoptosis of distal tubular cells and interstitial infiltration of inflammatory cells in normotensive, sodium-repleted rats12. It was postulated that this effect was caused by complete inhibition of Na+ entry or by increased Ca2+ entry into these cells. In theory, the interstitial lesions could also have been caused by diuretic-induced hypokalemia13. However, this is unlikely for the current study, as we found no effect of HCTZ on plasma K+ concentrations.

76


Chapter 5

Renal function and post-MI survival This is to our knowledge the first experimental animal study showing a relation between renal function and prognosis after myocardial infarction. This finding is of particular interest when considering the mechanisms underlying this cardio-renal interaction. It is firmly established that kidney function is strongly associated with long-term prognosis in patients with left ventricular dysfunction after myocardial infarction14-17. It has been suggested that the relation between mild renal impairment and progression of cardiac disease in human patients is non-causal, and that both are the consequence of traditional cardiovascular risk factors, such as atherosclerosis and hypertension. However, these confounding factors are not present in our experimental model. Hence, decreased GFR (reflected by increased plasma creatinine concentrations) itself may be a risk factor triggering development of heart failure. Accordingly cardiomyocyte apoptosis, and reduced capillary/cardiomyocyte ratios have been observed in rats with mild renal impairment18,19. Furthermore rats with mild renal impairment by nephrectomy showed reduced ischemia tolerance, through a yet unresolved mechanism, but independent of confounding effects of hypertension, sympathetic overactivity, and salt retention20. Hence, a decrease in renal function caused by addition of HCTZ to ACE inhibition may on the long-term affect survival after myocardial infarction. Study limitations The current study does not show a causal relation between the HCTZ-induced decrease in renal function and long-term mortality. However, it shows that this combination causes on average an increase in plasma creatinine concentrations to an extent that is associated with increased long-term mortality. The dose of hydrochlorothiazide used in the present study (50/mg/kg/day) is much higher than the dose generally given to patients with LV dysfunction (±0.5-1 mg/kg/day). However, as drug absorption and kinetics are different in rat and man, this does not mean that we used a toxic, supra-pharmacological dose of HCTZ. Furthermore, previous studies showed that this dose causes no blood pressure reduction and

Figure 4. Collagen staining of renal cortical tubuli. A) Typical example of normal tubuli, B)

Tubular degeneration by HCTZ+quinapril treatment coincided with collagen deposition.

A) B)

77



hypokalemia in rats with myocardial infarction10, and much higher dosages were tolerated in long-term toxicological studies21-23. As we did not include a group treated with HCTZ monotherapy, it cannot be determined whether the renal functional and morphological abnormalities are caused by HCTZ per se, or by the combination of HCTZ with quinapril. Nevertheless, as all patients with LV dysfunction post-MI should be treated with RAAS inhibition, primary aim of our studies was to investigate whether diuretic therapy can be safely applied in a setting mimicking the clinical situation. Conclusion Adding the diuretic hydrochlorothiazide to ACE inhibitor treatment detrimentally affected not only renal function, but also renal structure in rats with myocardial infarction. As decreased renal function was associated with increased mortality, adverse renal effects of volume depletion by adding HCTZ to ACE-I may exert unfavorable effects on long-term prognosis after MI.

References



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2. Heeg JE, de Jong PE, van der Hem GK et al. Efficacy and variability of the antiproteinuric effect of

ACE inhibition by lisinopril. Kidney Int. 1989;36:272-279.

3. Buter H, Hemmelder MH, Navis G et al. The blunting of the antiproteinuric efficacy of ACE

inhibition by high sodium intake can be restored by hydrochlorothiazide. Nephrol Dial Transplant.

1998;13:1682-1685.

4. Westendorp B, Schoemaker RG, Buikema H et al. Beneficial effects of add-on hydrochlorothiazide

in rats with myocardial infarction optimally treated with quinapril. Eur J Heart Fail. 2005.

5. Sorensen CR, Brendorp B, Rask-Madsen C et al. The prognostic importance of creatinine clearance

after acute myocardial infarction. Eur Heart J. 2002;23:948-952.

6. Esnault VL, Ekhlas A, Delcroix C et al. Diuretic and enhanced sodium restriction results in

improved antiproteinuric response to RAS blocking agents. J Am Soc Nephrol. 2005;16:474-481.

7. Pinto YM, de Smet BG, van Gilst WH et al. Selective and time related activation of the cardiac

renin-angiotensin system after experimental heart failure: relation to ventricular function and

morphology. Cardiovasc Res. 1993;27:1933-1938.

8. MacDonald JR, Susick RL, Jr., Pegg DG et al. Renal structure and function in rats after

suprapharmacologic doses of quinapril, an angiotensin-converting enzyme inhibitor. J Cardiovasc

Pharmacol. 1992;19:282-289.

9. Schaison FH, Fernando Ramirez-Gil J, Ciferri S et al. Acute and long-term dose-response study of

quinapril on hormonal profile and tissue angiotensin-converting enzyme in Wistar rats. J

Cardiovasc Pharmacol. 1996;28:11-18.

10. Kohzuki M, Kanazawa M, Yoshida K et al. Cardiomegaly and vasoactive hormones in rats with

chronic myocardial infarction: long-term effects of chlorothiazide. Clin Sci (Lond). 1996;90:31-36.

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11. Zelis R, Sinoway LI, Musch TI et al. Regional blood flow in congestive heart failure: concept of

compensatory mechanisms with short and long time constants. Am J Cardiol. 1988;62:2E-8E.

12. Loffing J, Loffing-Cueni D, Hegyi I et al. Thiazide treatment of rats provokes apoptosis in distal

tubule cells. Kidney Int. 1996;50:1180-1190.

13. Riemenschneider T, Bohle A. Morphologic aspects of low-potassium and low-sodium nephropathy.

Clin Nephrol. 1983;19:271-279.

14. Sarnak MJ, Levey AS, Schoolwerth AC et al. Kidney disease as a risk factor for development of

cardiovascular disease: a statement from the American Heart Association Councils on Kidney in

Cardiovascular Disease, High Blood Pressure Research, Clinical Cardiology, and Epidemiology and

Prevention. Circulation. 2003;108:2154-2169.

15. Sorensen CR, Brendorp B, Rask-Madsen C et al. The prognostic importance of creatinine clearance

after acute myocardial infarction. Eur Heart J. 2002;23:948-952.

16. Gibson CM, Pinto DS, Murphy SA et al. Association of creatinine and creatinine clearance on

presentation in acute myocardial infarction with subsequent mortality. J Am Coll Cardiol.

2003;42:1535-1543.

17. Tokmakova MP, Skali H, Kenchaiah S et al. Chronic kidney disease, cardiovascular risk, and

response to angiotensin-converting enzyme inhibition after myocardial infarction: the Survival

And Ventricular Enlargement (SAVE) study. Circulation. 2004;110:3667-3673.

18. Amann K, Tyralla K, Gross ML et al. Cardiomyocyte loss in experimental renal failure: prevention

by ramipril. Kidney Int. 2003;63:1708-1713.

19. Amann K, Wiest G, Zimmer G et al. Reduced capillary density in the myocardium of uremic rats--

a stereological study. Kidney Int. 1992;42:1079-1085.

20. Dikow R, Kihm LP, Zeier M et al. Increased infarct size in uremic rats: reduced ischemia tolerance?

J Am Soc Nephrol. 2004;15:1530-1536.

21. Lijinsky W, Reuber MD. Pathologic effects of chronic administration of hydrochlorothiazide, with

and without sodium nitrite, to F344 rats. Toxicol Ind Health. 1987;3:413-422.

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Chapter 6

Progressive Left Ventricular Hypertrophy after Withdrawal of Long-term ACE

Inhibition Following Experimental Myocardial Infarction.

Bart Westendorp

Regien Schoemaker

Hendrik Buikema

Frans Boomsma


Wiek van Gilst

80


Chapter 6

Abstract

Background: Although discontinuation of chronic ACE-I therapy after MI is common in clinical practice, some clinical studies reported an increased incidence of ischemia-related events after withdrawal. To further address this issue, we assessed hemodynamic, neurohormonal and vascular consequences of withdrawing long-term ACE inhibitor (ACE-I) treatment after experimental myocardial infarction (MI). Methods: Rats were subjected to coronary ligation to induce MI, and received quinapril (15 mg/kg/day) from 2 weeks to 14 months post-MI. Subsequently, surviving rats were randomized to sacrifice at 0, 4, and 6 weeks after ACE-I withdrawal. Rats were studied for signs of heart failure, hemodynamics and cardiac function, neurohormones, and vascular endothelial function. Results: After discontinuation of ACE-I treatment, plasma aldosterone levels increased between 0-4 weeks without further increment thereafter, suggesting persistent RAAS activation. Acetylcholine-induced aortic relaxation was impaired both at 4 and 6 weeks, indicating rapid and sustained development of endothelial vasodilator dysfunction after withdrawal. Moreover, 24% of the rats developed heart failure signs (edema, dyspnea), and 3 rats died, all within 4 weeks after withdrawal. Significantly increased N-ANP levels and lung weights at 4, but not at 6 weeks suggest a transient volume overload. Finally, LV/body weight ratios significantly increased between 0-4 as well as 4-6 weeks, indicating progressive LV hypertrophy. Conclusions: The observed alterations after withdrawing long-term post-MI quinapril treatment in the present study may account for an increased risk for ischemic events. By that our findings highlight the potentially harmful effects associated with abrupt discontinuation of long-term post-MI ACE inhibition, and imply careful clinical consideration in this matter.

LV Hypertrophy after ACE Inhibitor Withdrawal

81


Introduction

Chronic activation of the RAAS is regarded as one of the major causes of progressive deterioration of left ventricular pump function after myocardial MI. Accordingly, ACE-I therapy after MI remains the mainstay to inhibit the progression to LV dilatation and heart failure. In contrast to the well-studied beneficial effects of ACE inhibition therapy itself, remarkably little has been published about the consequences of its withdrawal. ACE-I withdrawal often occurs in clinical practice, mostly due to intolerance (±10% of all patients)1,2. In the CATS trial, post-MI patients were randomized to captopril or placebo for 12 months, followed by 1-month placebo. The captopril-withdrawn patients showed a high incidence of ischemia-related events within one month after withdrawing treatment3. The exact mechanism behind this finding is unknown, but it may be explained by a rebound phenomenon. Withdrawal of several cardiovascular drugs, such as β-blockers4, nitrates5, and statins6 can cause pronounced rebound effects, requiring stepwise cessation of therapy. Therefore, the current study aimed to further investigate the consequences of withdrawing chronic ACE-I therapy in rats with MI. We hypothesized that the protective effects of ACE inhibitors on cardiac function and morphology, and endothelial function in post-MI treatment decline shortly after withdrawal.

Methods

Study design The investigation conforms to the Guide for the Care and Use of Laboratory Animals (Published by the US National Institutes of Health, NIH publication No. 85-23, revised 1996), and the animal research committee of the University of Groningen approved the study protocol. The study is an extension of one of our previous study on long-term ACE inhibition after myocardial infarction7. Male Sprague Dawley rats (Harlan, Zeist, The Netherlands), weighing 280±25g were subjected to coronary ligation as previously described8. Briefly, rats were anesthetized with isoflurane 2.0-2.5% in oxygen, after which rats were intubated and ventilated with this gas mixture. Subsequently, a left-sided thoracotomy was performed and the left anterior descending coronary artery was occluded with a 6-0 silk ligature 1-2 mm after the bifurcation. Mortality within the first 24 hours after surgery was 51%. Two weeks after coronary ligation, rats were assigned to treatment with quinapril (QUI) in a dose of 15 mg kg-1 day-1, mixed through food. Body weights and food intake were measured, and concentrations of quinapril in the chow were adjusted weekly. The rats were housed in clear polyethylene cages (4-5 per cage). The rooms were temperature- (220 C) and humidity- (50%) controlled and had a 12h light-dark cycle. Treatment was maintained for 14 months. At the end of the follow-up period, none of the surviving rats showed overt signs of chronic heart failure (dyspnea, edema). Rats were randomly subjected to 0 (n=10), 4 (n=12) or 6 (n=6) weeks quinapril withdrawal, before rats were sacrificed for assessment of LV function, neurohormones, and endothelial function.

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LV function Rats were anaesthetized as described above, the carotid artery was cannulated and a pressure tip catheter (Micro-Tip 3French, Millar instruments Inc., Houston TX, USA) connected to a PC with appropriate software (Millar instruments, Germany) was advanced into the left ventricle and used for determination of heart rates, and left ventricular systolic and diastolic pressures (LVSP and LVEDP). As indices for global contraction and relaxation, we determined the maximal rates of increase and decrease in LVP (dPdtmax and dPdtmin), corrected for LV developed pressure. Subsequently, the catheter was retracted into the aortic arch, and arterial pressures were recorded. Neurohormone measurements After LV function measurements, arterial blood was collected, anti-coagulated with EDTA, and centrifuged at 4000 RPM for 10 minutes at 4 °C. Plasma was stored at -80 °C until assay. Plasma for ACE activity determination was collected separately, and not anti-coagulated with EDTA. ACE activity in plasma was determined according to the Hippuryl-His-Leu method, as has been described in detail before8. For other plasma neurohormone measurements, samples were transported on dry ice to the Core Laboratory at the University Hospital Dijkzigt, Rotterdam, The Netherlands, where all measurements were performed as previously described9. Plasma renin activity (PRA) was measured by determining the amount of angiotensin I generated from angiotensinogen with an in-house radioimmunoassay. Concentrations of N-terminal atrial natriuretic peptide (N-ANP) and aldosterone in plasma were measured with commercially available radioimmunoassays from Biotop (Oulu, Finland) and DPC (Los Angeles, CA, USA), respectively. Tissue processing & histology Hearts were excised and rinsed with ice cold NaCl (0.3%). The atria and right ventricle were removed on ice for determination of left ventricular weights. The apical 1/3 part of the LV was cut off, scar tissue and spared myocardium were separated and snap frozen in liquid nitrogen for measurement of ACE activity in the spared myocardium. A midventricular slice of the LV was fixated in 2% paraformaldehyde for histological analysis. Infarct size was determined by histology according to methods described before10. In brief, the sections were embedded in paraffin, and 5 µm slices were cut and stained with Sirius red/Fast green. Infarct size was determined as the percentage of scar to total LV circumference. The midventricular section provides adequate estimation of total left ventricular infarct size11. Only rats with MI sizes exceeding 20% of LV were included in analysis, since smaller infarcts are hemodynamically fully compensated in this model12,13. As an index of LV dilatation midventricular LV cavity areas were measured by planimetry. For quantification of cardiac fibrosis, left ventricular collagen volume fraction was measured by dividing Sirius Red-positive area by total myocardial area within a given field14. Per rat, 10 subendocardial fields were analyzed; the collagen-rich infarct-border and perivascular zones were not analyzed.


83


Endothelial function Aorta segments were used to assess endothelial function, as previously described15. After excision of the heart, the thoracic aorta was removed, immediately cleaned of adhering tissue, and cut into rings of 2 mm in length. Subsequently, rings were mounted on a contraction set-up connected to and isotonic displacement transducer. The aorta sections were placed in an organ bath filled with Krebs buffer (containing in mmol/L: NaCl (120.4), KCl (5.9), CaCl2 (2.5), MgCl2 (1.2), glucose (11.5), and NaHCO3 (25.0)). The organ baths were continuously gassed with 95% O2/5 % CO2, and kept at a temperature of 370 C. After a stabilization period of at least 30 minutes, rings were contracted with 60 mM/L KCl to check for viability. Again, rings were washed and stabilized. Subsequently, rings were precontracted with 1µmol/L phenylephrine (PE), and responses to increasing concentrations of acetylcholine (ACh, 10-8 mol/L to 10-4 mol/L) were determined. Finally, maximal endothelium-independent vasodilatation to the exogenous NO donor sodium nitrite was determined (SN, 10-2 mol/L). This method provides a relevant measure of endothelial function16. Statistical methods Data are presented as means±SEM in case of normal distribution, otherwise in box plots. Groups were compared using one-way analysis of variances (ANOVA) with least squared differences post hoc analysis for multiple comparisons in case of normal distribution. When parameters were not normally distributed, differences were compared with a Kruskal-Wallis test followed by Mann-Whitney tests for individual group comparisons. Differences were considered significant at the level of 0.05 (two tailed).

Results

General characteristics A total of 44 rats with MI>20% was subjected to quinapril treatment for 14 months, during which 13 rats (28%) died. None of the 31 rats alive after the treatment period showed overt signs of heart failure. Of these 31 rats, 10 were (randomly) sacrificed immediately to establish LV function and neurohormones under ACE-I therapy (0 weeks of withdrawal), and 21 rats were subjected to 4 or 6 weeks quinapril withdrawal. Five of the rats subjected to withdrawal developed signs of heart failure (edema, dyspnea), and 3 of those died prematurely (after 4, 6, and 26 days, respectively). At sacrifice, average MI sizes were similar in the 3 different groups (table 1). Similarly, body weights were not significantly different. LV remodeling Parameters of LV remodeling are summarized in figure 1. Withdrawal caused a marked progression of cardiac hypertrophy, as indicated by LV weights/body weight ratios. LV/body weight ratios were significantly increased after 4 weeks, and further increased significantly between 4 and 6 weeks of therapy withdrawal.

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Chapter 6

Table 1. Basal characteristics of sacrificed rats with MI after 62 weeks chronic quinapril therapy,

or quinapril followed by 4 and 6 weeks therapy withdrawal.

Chronic QUI

(n=10)

4 weeks after QUI

withdrawal (n=12)

6 weeks after QUI

withdrawal (n=6)

Body weight 492±19 523±27 478±26

Infarct size (% of LV) 34.0±2.3 31.8±1.5 30.0±3.2

No significant differences were observed. QUI; quinapril 15 mg/kg/day

We found no evidence for interstitial fibrosis; likely, up to 4 weeks after withdrawal, collagen deposition paralleled LV hypertrophy, resulting in unchanged collagen volume fractions whereas between 4 and 6 weeks collagen deposition was lagging behind hypertrophy, as indicated by reduced collagen volume fractions.

LV weight

mg/

g bo

dy w

eigh

t

0,0

2,0

2,2

2,4

2,6

2,8

Chronic QUI4 weeks QUI withdrawal6 weeks QUI withdrawal

LV collagen density

Vol

ume

dens

ity p

erce

ntag

e (%

)

0

2

4

6

8

10

LV cavity area

mm

2

0

10

20

30

40

*

**

** †

†

Figure 1. Effects of 4 and 6 weeks quinapril withdrawal on cardiac remodeling in rats with

myocardial infarction. * p<0.05 versus chronic QUI, ** p<0.005 vs chronic QUI, † p<0.05 vs 4

weeks of withdrawal.


85


LV cavity areas, indicating LV dilatation, were significantly increased at 4 weeks, but normalized after 6 weeks of withdrawal (figure 1), suggesting a transient phase of volume overload. LV function LV end-diastolic pressures tended to increase after 4 weeks and normalize at 6 weeks of withdrawal (figure 2). In parallel, plasma concentrations of N-ANP, representing atrial and left ventricular wall stretch, showed a significant increase after 4, but not 6 weeks of withdrawal (figure 3). Lung wet weights, indicating edema, followed the same time

dPdtmax

103 . m

m H

g/se

c

0

8

9

10

11

12LV systolic pressure

mm

Hg

0

20

80

100

120

140

dPdtmin

103 . m

m H

g/se

c

0

7

8

9

10

11

12

** **

Mean arterial pressure

mm

Hg

0

20

60

80

100

120

Chronic QUI4 weeks QUI withdrawal 6 weeks QUI withdrawal

Heart rate

103 . m

m H

g/se

c

0

260

280

300

320

340

*

LV end-diastolic pressure

mm

Hg

0

5

10

15

20** **

*

Figure 2. LV function during chronic quinapril treatment and after 4 and 6 weeks of

withdrawal in rats with myocardial infarction. *p<0.05 and ** p<0.005 versus chronic

quinapril.

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Chapter 6

pattern. The significant correlation between lung weights and N-ANP levels indicates formation of lung edema due to cardiac overload (figure 3). Mean arterial blood pressures (MAPs) were low in the rats sacrificed during quinapril therapy, and 4 weeks of withdrawal caused a marked increase in MAP (figure 2), that was sustained until 6 weeks. This increase in mean arterial pressure was paralleled by increased maximum LV systolic pressures after 4 and 6 weeks of withdrawal. However, maximum rates of contraction and relaxation corrected for LV developed pressure (dPdtmax and dPdtmin, respectively) were significantly decreased after quinapril withdrawal. Heart rates had not changed significantly after both 4 and 6 weeks of withdrawal. Endothelial function Endothelial function was assessed by measuring responsiveness to acetylcholine in aorta rings placed in a contraction set-up. Dose-response curves show that acetylcholine-dependent relaxation was significantly impaired as early as 4 weeks after quinapril withdrawal (figure 4A), with no further deterioration occurring between 4 and 6 weeks.

Plasma [N-ANP]

0 2 4 6 8 10

Lu

ng

wei

gh

t

0

3

4

5

6

7

8

p = 0.01

R2 = 0.23

Plasma [N-ANP]

pmol

/mL

0

1

2

3

4

5


*

Lung weight

mg/

g bo

dyw

eigh

t

0,0

3,0

3,5

4,0

4,5

5,0

5,5*

Figure 3. Circulating N-terminal A-type natriuretic peptide (N-ANP) levels during chronic

quinapril therapy and effects of withdrawal. Right panel: significant correlation between N-

ANP levels and lung weights, suggesting that lung wet weights indicate lung edema as a result

of volume overload. Dotted lines indicate 95% confidence interval for correlation between N-

ANP and lung weights. * p<0.05 versus chronic quinapril.


87


Endothelium-independent relaxation was also impaired, as evidenced by a decreased maximum response to the exogenous NO donor sodium nitrite after both 4 and 6 weeks of withdrawal, which indicates declined vascular smooth muscle reactivity to NO (figure 4B). Renin-angiotensin-aldosterone system Plasma renin activity was markedly reduced after withdrawal (figure 5). LV ACE activity, but not plasma ACE activity tended to increase after withdrawal. Nonetheless, production of aldosterone, the end product of the RAAS cascade, was markedly increased after 4 weeks of therapy withdrawal. From 4 to 6 weeks, RAAS-parameters did not change.

Discussion

Although discontinuation of ACE inhibitor therapy after MI is common in clinical practice, consequences of withdrawing chronic therapy were hardly studied. We established the effects of withdrawing long-term quinapril treatment in rats with MI. Left ventricular remodeling In patients with chronic heart failure blood pressure was reported to rise quickly after captopril withdrawal17. Accordingly, in the present study arterial blood pressure

Acetylcholine (mol/l)

1e-8 1e-7 1e-6 1e-5 1e-4

% o

f PE

pre

cont

ract

ion

0

10

20

30

40

50

60

70

80

90

100 Chronic QUI 4 weeks QUI withdrawal 6 weeks QUI withdrawal

**

Maximum dilation to sodium nitrite

% d

ilata

tion

afte

r P

E p

reco

ntra

ctio

n

0

10

20

30

40

50

60

70

80

90

100

**

*

A) B)

†

Figure 4. Endothelial function in rats chronically treated with quinapril, and effects of 4 and 6

weeks of withdrawal. A) Dose-response curves to acetylcholine. B) Maximum endothelium-

independent vasodilatation after the NO donor sodium nitrite. * p<0.05 versus chronic

quinapril, ** p<0.005 vs chronic QUI, † p<0.05 vs 4 weeks of withdrawal. PE; phenylephrine

(1 µmol/L).

88


Chapter 6

increased to normotensive levels after 4 weeks quinapril withdrawal, and remained stable. LV systolic pressures showed the same pattern. These findings might paradoxically suggest improved cardiac performance after discontinuation of treatment. However, increased arterial blood pressures also impose an increased afterload to the already functionally impaired heart. Moreover, increased LV systolic function implies increased cardiac oxygen demand. Taken together, this could increase the risk for ischemic events after ACE-I withdrawal18,19. Furthermore, (initially compensatory) LV hypertrophy was progressive in nature with further increase in LV mass between 4 and 6 weeks after withdrawal, whereas LV performance did not further increase in this period. Moreover, maximum rates of contraction and relaxation, corrected for LV developed pressure, were significantly decreased after both 4 and 6 weeks of withdrawal, suggesting impaired contractile function. Hence, progressive LV hypertrophy without concomitant improvement in LV function in the present study should be regarded as a maladaptation.


ng A

ng I.

mL-1

0

20

40

60

80

100


Plasma ACE activity

nmol

His

Leu.

mL-1

. min

-1

0

10

20

30

40

50

60

Plasma aldosterone

pg. m

L-1

0

200

400

600

800

1800

1900

LV ACE activity

nmol

His

Leu.

mL-1

. min

-1

0

10

20

30

40

*

****

*

Figure 5. Circulating and cardiac renin angiotensin system in rats with myocardial infarction

after 62 weeks chronic quinapril therapy, and the effects of 4 and 6 weeks of withdrawal.

* p<0.05 and ** p<0.005 versus chronic quinapril.


89


The hypertrophic response observed after quinapril withdrawal may have been the direct result of increased arterial blood pressure, as well as increased local Angiotensin II (Ang II) formation. The latter seems well in line with the increased LV tissue ACE activity after quinapril withdrawal in the present study. Increased cardiac Ang II levels may also lead to interstitial fibrosis20, which in its turn is associated with altered diastolic LV function21. Therefore we checked for fibrosis by determining total interstitial collagen volume percentages. We did not observe an increase in collagen, but even a significant decrease between 4 and 6 weeks of withdrawal. As collagen was determined as percentage of cardiac tissue, interstitial collagen deposition likely kept pace with cardiomyocyte hypertrophy during the first 4 weeks, whereas from 4 to 6 weeks it was lagging behind, resulting in a relative dilution of collagen. This indicates that quinapril withdrawal did not result in fibrosis during the first 6 weeks after discontinuation of quinapril treatment. Transient volume overload Withdrawal was associated with signs indicative for increased cardiac preload, such as increased plasma N-ANP, LV cavity, LVEDP, and lung weight, at 4, but not at 6 weeks of withdrawal. We refer to this as (transient) volume overload. Accordingly, 5 of 21 rats (24%) developed signs of heart failure (edema, dyspnea), all within the first 4 weeks after withdrawal. In addition, all 3 deaths during the withdrawal phase occurred during these first 4 weeks. Clinical data on the induction of a transient volume overload after ACE-I withdrawal in a setting of LV dysfunction are indefinite. A double-blind withdrawal study in patients with chronic heart failure (continued quinapril versus placebo) reported a gradual worsening of clinical status, starting at 4 to 6 weeks after withdrawal 22. However, many of these patients already showed volume overload symptoms while on quinapril. Furthermore results may have been obscured by diuretic treatment. Our findings seem more consistent with a sub study of SOLVD, in which LV chamber sizes rapidly increased to pre-treatment levels during a 3-week withdrawal period that was preceded by 33 months of active ACE-I treatment. This was paralleled by high incidence of major adverse events 23, including myocardial infarction and unstable angina. Notably, LV filling pressures and N-ANP did not increase progressively, but normalized to some extent after 6 weeks withdrawal. The reason is not clear, but it may be associated with progressive LV hypertrophy between 4 and 6 weeks. The resulting increased mass of contractile LV may deal more adequately with increased cardiac workload after ACE-I withdrawal. Endothelial dysfunction Endothelial dysfunction with increased tendency for coronary vasospasm and acute coronary thrombotic processes is associated with an increased risk for ischemic events, and could well have contributed to the adverse events mentioned above. In agreement with this, endothelium-dependent relaxation of the aorta in response to acetylcholine was considerably impaired as soon as 4 weeks after withdrawal. In this experimental model, MI generally does not result in clearly detectable (aortic) endothelial dysfunction before 8-10 weeks15,16,24. The underlying mechanism is thought

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Chapter 6

to include impaired NO activity through oxidative stress, mediated by activation of the RAAS after MI25. The notion that profound endothelial dysfunction was already present 4 weeks after withdrawal may reflect rebound RAAS activation after withdrawal, resulting in accelerated endothelial dysfunction in the current study. Maximal endothelium-independent relaxation to the NO donor SN was impaired as well. This further underlines the role of increased vascular oxidative stress causing reduced activity of NO, regardless whether NO is from endogenous or exogenous origin. In accordance with this, angiotensin 2 infusion was reported to attenuate the response to nitroglycerin, which could be prevented when oxidative stress was reduced by co-treatment with superoxide dismutase26. RAAS activation Ang II exerts a negative feedback on both renin27 and ACE28 expression. As a consequence, ACE-I-induced decreases in tissue Ang II levels result in markedly increased renin production29,30. Accordingly, withdrawal of quinapril treatment could initially cause an overshoot in RAAS activity and blood pressure28. In the current study, plasma ACE activity was unchanged after both 4 and 6 weeks. This could not be attributed to incomplete ACE inhibition at the time of withdrawal, as we showed previously that this dose results in optimal ACE inhibition7. However, tissue -rather than circulating- ACE inhibition reflects effective ACE-I therapy8,31. LV ACE activity tended to be similarly increased after 4 and 6 weeks of withdrawal. Moreover, plasma aldosterone had on average more than doubled, but to a similar extent after 4 and 6 weeks. Although rebound RAAS activation could have occurred within the first 4 weeks after withdrawal, as in this period mortality and signs of fluid retention were observed, our results suggest sustained rather than rebound activation of the renin-angiotensin- aldosterone system after withdrawal. The occurrence of a transient rebound effect after ACE-I withdrawal may depend on e.g. type and severity of underlying disease, ACE-I dosage, and duration of therapy before withdrawal32,33. Study limitations The three groups were harvested at different time points after coronary ligation. This strategy allowed us to randomize rats individually to one of the three different groups, instead of per cage. This strategy could result in a confounding factor regarding the time course of heart failure development. However, the order of magnitude of difference in time point of sacrifice (weeks) is limited compared to the total treatment period (months). Moreover, comparison with MI-rats treated with the same dose of quinapril for 8 months showed that LV function and hypertrophy were hardly changing between 8 and 14 months7, indicating that a time effect of heart failure development independent of quinapril withdrawal would be rather minor. We intended to sacrifice the withdrawal groups of rats at regular time intervals, i.e. 4 and 8 weeks. However, we observed clear manifestations of heart failure and a trend of increased mortality within the first weeks after withdrawal, and to anticipate that high mortality would lead to a very limited group size, we decided to sacrifice rats already after 6 weeks.


91


Samples for neurohormone measurements were obtained after invasive measurement of hemodynamics. This measurement of hemodynamics, as well as the anesthesia required for this procedure, could potentially interfere with neurohormone measurements. However, since all rats were treated according to the same procedure, and in general predictable differences were observed, reliable comparison between experimental groups seems feasible. Conclusion and clinical implications This study demonstrates that withdrawal of chronic ACE inhibition post-MI resulted in 1) progressive LV hypertrophy at persistent RAAS activation, 2) increased arterial blood pressures, 3) endothelial dysfunction, 4) a transient phase of volume overload early after withdrawal, in association with heart failure symptoms and increased mortality. Together, these processes may increase the risk for ischemic events in post-MI patients early after ACE-I withdrawal. Although extrapolation of animal data to patients has its difficulties, our data are consistent with the scarce clinical evidence of effects of ACE-I withdrawal3,23. Thus, abrupt discontinuation of post-MI ACE inhibition after long-term treatment should be considered very carefully, as it can be potentially harmful.

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8. Pinto YM, de Smet BG, van Gilst WH et al. Selective and time related activation of the

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activation and prognosis in patients with chronic heart failure. Eur Heart J. 1998;19:753-760.

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remodeling after experimental myocardial infarction: a comparison with captopril. J

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DNA synthesis in endothelial cells and normalization of maximal coronary flow in infarcted

rat hearts. Cardiovasc Res. 1998;40:156-164.

12. Schoemaker RG, Debets JJ, Struyker-Boudier HA et al. Delayed but not immediate captopril

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13. Pfeffer MA, Pfeffer JM, Fishbein MC et al. Myocardial infarct size and ventricular function

in rats. Circ Res. 1979;44:503-512.

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deposition in non-infarcted myocardium during remodeling after coronary artery ligation in

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15. Buikema H, Monnink SH, Tio RA et al. Comparison of zofenopril and lisinopril to study the

role of the sulfhydryl-group in improvement of endothelial dysfunction with ACE-

inhibitors in experimental heart failure. Br J Pharmacol. 2000;130:1999-2007.

16. Gschwend S, Buikema H, Henning RH et al. Endothelial dysfunction and infarct-size relate

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17. Nicholls MG, Ikram H, Espiner EA et al. Hemodynamic and hormonal responses during

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20. De Mello WC. Heart failure: how important is cellular sequestration? The role of the renin-

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changes in diastolic function of infarcted rat hearts. Cardiovasc Res. 2000;46:316-323.

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converting enzyme inhibitor withdrawal in chronic heart failure: a double-blind, placebo-

controlled study of quinapril. The Quinapril Heart Failure Trial Investigators. J Am Coll

Cardiol. 1993;22:1557-1563.

23. Konstam MA, Rousseau MF, Kronenberg MW et al. Effects of the angiotensin converting

enzyme inhibitor enalapril on the long-term progression of left ventricular dysfunction in

patients with heart failure. SOLVD Investigators. Circulation. 1992;86:431-438.


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24. Teerlink JR, Clozel M, Fischli W et al. Temporal evolution of endothelial dysfunction in a

rat model of chronic heart failure. J Am Coll Cardiol. 1993;22:615-620.

25. Bauersachs J, Bouloumie A, Fraccarollo D et al. Endothelial dysfunction in chronic

myocardial infarction despite increased vascular endothelial nitric oxide synthase and

soluble guanylate cyclase expression: role of enhanced vascular superoxide production.

Circulation. 1999;100:292-298.

26. Rajagopalan S, Kurz S, Munzel T et al. Angiotensin II-mediated hypertension in the rat

increases vascular superoxide production via membrane NADH/NADPH oxidase activation.

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27. Muller MW, Todorov V, Kramer BK et al. Angiotensin II inhibits renin gene transcription

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28. Schunkert H, Ingelfinger JR, Hirsch AT et al. Feedback regulation of angiotensin converting

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Chapter 7

Improvement of EDHF by Chronic ACE

Inhibition Declines Rapidly after Withdrawal

in Rats with Myocardial Infarction.

Bart Westendorp

Regien Schoemaker

Wiek van Gilst

Hendrik Buikema

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Chapter 7

Abstract

Introduction: Heart failure after myocardial infarction (MI) is associated with endothelial dysfunction. There is conflicting evidence on the exact nature of this endothelial dysfunction, and how endothelium-dependent vasodilation is affected by angiotensin-converting enzyme inhibitor (ACE-I) therapy. Furthermore, consequences of acute ACE-I withdrawal are largely unknown. Therefore, we studied the contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to the effects of ACE-I therapy and its withdrawal on endothelial function in MI-rats. Methods: Rats were subjected to coronary ligation to induce MI, and were assigned to quinapril or vehicle from 2 weeks to 8 months post-MI. In parallel, MI-rats treated for 14 months with quinapril were subjected to treatment withdrawal for 0, 4, and 6 weeks. Acetylcholine (ACh)-induced relaxation and underlying endothelium-derived mediators were studied in isolated aortic rings. Results: Long-term quinapril (8 months) resulted in markedly improved endothelium-dependent vasodilation in rats with myocardial infarction, which could be attributed to marked improvement in non-NO/prostanoids-mediated relaxation (i.e. EDHF). After 14 months of follow-up, maximum vasodilation was still preserved by quinapril. Withdrawal after 14 months treatment caused significantly impaired ACh-induced EDHF-mediated relaxation within 4 weeks. A marked reduction in EDHF mediated relaxation caused this impairment. NO-mediated relaxation was unaffected. Discussion: These findings highlight the importance of EDHF impairment in development of endothelial dysfunction after myocardial infarction, and the possibility to improve EDHF-mediated vasodilation with chronic ACE inhibitor therapy. In addition, withdrawal of chronic ACE inhibition after MI should be considered carefully, as profound endothelial dysfunction may develop rapidly.

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Improvement of EDHF by Chronic ACE Inhibition

Introduction

The progression of left ventricular (LV) dysfunction to chronic heart failure after myocardial infarction (MI) is associated with increased peripheral vascular resistance, which is thought to be the combined result of neurohormonal activation as well as endothelial dysfunction1,2 and alterations in myogenic tone of resistance arteries3. However, there is conflicting evidence on the exact nature of endothelial dysfunction in chronic heart failure after MI4-9. Endothelium-dependent relaxation is mainly mediated by nitric oxide (NO), vasoactive prostaglandins, and endothelium-derived hyperpolarizing factor (EDHF). Alterations of these individual components may be explained by e.g. severity of LV dysfunction, and differences between vessel beds. Angiotensin-converting enzyme inhibitors (ACE-I) effectively inhibit the progression of LV dysfunction towards overt heart failure. Importantly, ACE inhibition beneficially affects the blood vessels - predominantly by improvement of endothelial function10. However it has not been fully elucidated which mediators of endothelium-dependent vasodilation are improved by ACE-I therapy. Conversely, little is known about the consequences of acute therapy withdrawal hereon. Interestingly in this context, the antihypertensive effects of ACE inhibition may be sustained for a prolonged period of time after withdrawal of therapy11,12. However, in spontaneously hypertensive rats, ACE-I withdrawal dissociated between sustained blood pressure lowering after 4 weeks versus loss of improvement of endothelial function at the same moment13. Hence, in a setting of chronic ACE inhibition after MI, such a rapid loss of the vasoprotective effects of ACE-I after withdrawal may impose a considerable early risk of adverse events. Previous experimental findings in rats with MI14 as well as clinical observations15,16 showed the occurrence of endothelial dysfunction and ischemia-related events within weeks after cessation of therapy. The aim of the current study was to examine the effects of long-term treatment with the ACE-I quinapril, and its subsequent withdrawal on different mediators of endothelium-dependent relaxation in rats with myocardial infarction.

Methods

Study design The investigation conforms to the Guide for the Care and Use of Laboratory Animals (Published by the US National Institutes of Health, NIH publication No. 85-23, revised 1996), and the animal research committee of the University of Groningen approved this study protocol. Male Sprague Dawley rats (Harlan, Zeist, The Netherlands), weighing 280±25g were subjected to coronary ligation to induce MI, or sham surgery, as previously described17. Mortality within the first 24 hours after surgery was 51% for MI rats and 0% for sham rats. Two weeks after coronary ligation, rats were randomized to quinapril (15 mg kg-1 day-1, mixed through food), or vehicle. After 8 months of treatment part of the rats were sacrificed. In the remaining rats treatment was continued up to 14 months.

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For the withdrawal study, surviving rats after 14 months of therapy were randomized into 3 groups, which were sacrificed at: 1) end of therapy; 2) 14 months of therapy + 4 weeks withdrawal; 3) 14 months of therapy + 6 weeks withdrawal. This study is an extension of a previous survival study on optimization of ACE-I therapy in rats with MI, and the rationale for the duration treatment for 8 and 14 months was driven by considerations provided in that publication18. Moreover, we intended to sacrifice groups of rats at regular time intervals (i.e. 4 and 8 weeks) after withdrawal. However, as we observed clear manifestations of heart failure and a trend of increased mortality within the first weeks, and to anticipate that high mortality would lead to a very limited group size, we decided to sacrifice rats already at 6 weeks after withdrawal instead of 8 weeks. Blood pressure At the time of sacrifice, rats were anaesthetized as described above, the carotid artery was cannulated and a pressure tip catheter (Micro-Tip 3French, Millar instruments Inc., Houston TX, USA) connected to a 486 PC equipped with an AD converter and appropriate software (Millar instruments, Germany) was advanced into the aortic arch, and arterial blood pressure was recorded. Plasma N-ANP After blood pressure measurements, arterial blood was collected, anti-coagulated with EDTA, and centrifuged at 4000 RPM for 10 minutes at 4 °C. Plasma was stored at -80 °C until assay. Samples were transported on dry ice to the Core Laboratory at the University Hospital Dijkzigt, Rotterdam, The Netherlands, where all measurements were performed as previously. Concentrations of N-terminal atrial natriuretic peptide (N-ANP) in plasma were measured with a commercially available radioimmunoassay from Biotop (Oulu, Finland). Tissue processing/histology Hearts were excised and rinsed with ice cold NaCl (0.3%). The atria and right ventricle were removed on ice for determination of left ventricular weights. A midventricular slice of the LV was stored in 2% paraformaldehyde for histological measurements. Infarct size was determined by histology according to methods described before19. In brief, the sections were embedded in paraffin, and 5 µm slices were cut and stained with Sirius red/Fast green. Size of the infarct was determined by planimetry, and was expressed as the percentage of scar to total LV circumference. The midventricular section provides adequate estimation of total left ventricular infarct size20. Only infarcted rats with MI sizes larger than 20% of LV were included in analysis, since smaller infarcts are reported to be hemodynamically fully compensated in this model21,22; accordingly rats from the present study showing MI<20 % could not be distinguished from sham rats. Aorta experiments After excision of the heart, the thoracic aorta was removed, immediately cleaned of adhering tissue, and cut into rings of 2 mm in length. Subsequently, rings were mounted on a setup for measurements of isotonic displacements at 1.4 g preload. The

99



aorta sections were situated in an organ bath containing Krebs buffer: (in mmol L-1) NaCl (120.4), KCl (5.9), CaCl2 (2.5), MgCl2 (1.2), glucose (11.5), and NaHCO3 (25.0). The organ baths were continuously gassed with a mixture of 95% O2 and 5% CO2, and kept at a temperature of 37.50 C. After a stabilization period of at least 1 h, rings were primed with 60 mM KCl followed by repeated washing and renewed stabilization. Subsequently, rings were precontracted with 1 µmol L-1 phenylephrine (PE), and relaxation responses to increasing concentrations of acetylcholine (ACh, 10 nmol L-1 to 0.1 mmol L-1) were determined. Apart from total response to ACh, relaxation was measured in presence of 0.1 mM NG-mono-methyl-L-arginine (L-NMMA) to determine NO-mediated vasorelaxation. In pilot experiments at our lab, it was established that this concentration was sufficient to prevent NO-mediated relaxation, as increasing dose or adding another NO-inhibitor (0.1 mM L-NAME) did not further inhibit ACh relaxation. Relaxation in presence of L-NMMA was measured with and without addition of 10µmol/L indomethacin to verify contribution of vasoactive prostanoids. The remaining ACh-evoked relaxation in the presence of L-NMMA and indomethacin was considered an estimate of EDHF contribution to total ACh relaxation, in accordance with previous studies demonstrating its complete abrogation in the additional presence of charybdotoxin and apamin9,23,24. Incubations with L-NMMA and indomethacin were started 30 minutes prior to precontractions with PE; neither of these two drugs induced vasoconstriction or –dilation within these 30 minutes. After the dose-response curves to ACh, the maximum response to the NO donor sodium nitrite (SN, 10 mmol L-1) was determined. Drugs Krebs buffers and drug solutions were freshly prepared daily. All compounds were purchased from Sigma (St. Louis, MO, USA). Statistics Data are presented as means±SEM in case of normal distribution, otherwise as median and range. In case of normal distribution, groups were compared using one-way analysis of variances (ANOVA) with least squared differences post hoc analysis for multiple comparisons in SPSS 10.0. In case of non-normal distribution, a Kruskall-Wallis test with subsequent Mann-Whitney tests for individual group comparison was performed. Dose-response curves were compared with ANOVA for repeated measurements. Differences were considered significant at the level of 0.05 (two tailed). The area under the curve for each concentration-response curve was calculated using SigmaPlot 8.0, and expressed in arbitrary units to express the total response to ACh, and response in presence of L-NMMA, to express the NO-and EDHF-mediated relaxations (figure 2).

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Results

General characteristics The groups of MI-rats sacrificed after 8 months of treatment with quinapril or vehicle, respectively, had comparable infarct sizes (table 1). Groups of MI-rats treated for 14 months with quinapril and sacrificed at 0, 4, and 6 weeks after withdrawal, respectively, were also balanced for infarct size, although the latter three groups all tended to have larger infarct sizes than the 8 month-groups. Systolic blood pressure of MI-rats was significantly reduced after 8 months of quinapril treatment as compared to those untreated, and was similar after 8 and 14 months of quinapril treatment. Mean systolic blood pressures had increased back to baseline after 4 weeks of withdrawal, and did not change between 4 and 6 weeks. Untreated MI-rats showed increased left ventricle:body weight (LV:BW) ratios and elevated plasma N-ANP concentrations, demonstrating development of left ventricular hypertrophy and heart failure. Quinapril treatment restored these parameters after 8 months of therapy (table 1). LV:BW ratios were comparable after 8 and 14 months quinapril, but increased significantly after withdrawal. N-ANP concentrations were higher after 14 than 8 months of quinapril, and tended to increase further after withdrawal. After 8 months, a trend towards increased plasma aldosterone levels in rats with MI compared to sham rats was observed. Eight months quinapril treatment significantly reduced aldosterone levels. After 14 months quinapril still effectively reduced aldosterone levels, as indicated by the marked and significant increase after both 4 and 6 weeks of withdrawal. Table 1. General characteristics, and cardiac function of sacrificed rats with myocardial

infarction after 8 and 14 months chronic quinapril therapy, and the effects of 4 and 6 weeks

withdrawal.

dPdt values are shown in (103.mm Hg/sec)/mmHg, and were corrected for systolic pressures. N-

ANP; N-terminal Atrial Natriuretic Peptide, pmol/mL. * p< 0.05 versus 8 months sham, † p<0.05

versus 14 months QUI

8 months 14 months

Sham MI MI +

QUI

MI +

QUI

MI + QUI

4 weeks

withdrawal

MI + QUI

6 weeks

withdrawal

N 5 4 6 4 9 5

Infarct size (%LV) 0±0 27±3 25±1 31±4 32±2 32±4

SBP (mm Hg) 133±12 125±9 92±2 * 100±7 137±9 † 139±7 †

LV:BW (mg/g) 2.1±0.1 2.4±0.1 * 2.0±0.1 2.1±0.1 2.4±0.1† 2.6±0.1 †

Plasma N-ANP 0.8±0.1 1.9±0.2 * 0.8±0.1 2.6±0.4 3.8±0.7 3.6±0.8

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Total endothelium-dependent vasodilation to ACh Eight months after onset of treatment, the concentration-response curve for endothelium-dependent relaxation to ACh was significantly decreased in untreated MI-rats as compared to untreated age-matched sham-controls (left panel figure 1A); when calculated as the AUC the decrease in total relaxation to ACh amounted 40% (figure 2A). Quinapril significantly improved total relaxation to ACh; not only when compared to untreated MI-rats, but also when compared to untreated sham control rats (figures 1A and 2A). The improved response to ACh after 8 months of quinapril was also maintained after 14 months of treatment (right panel figure 1A). However, subsequent cessation of ACE-I treatment significantly impaired total relaxation to ACh, with similar reductions at 4 and 6 weeks after withdrawal (figures 1A and 2A). The worsened response to ACh after ACE-I withdrawal could not be restored by acute incubation of aorta rings with an ACE-I (10 µM quinapril) or an AT1-receptor antagonist (1 µM candesartan, data not shown). Endothelial mediators in ACh-induced relaxation A similar pattern of group differences in concentration-response curves at 8 months after onset of treatment was seen when responses were studied in the combined presence of L-NMMA and indomethacin to block production of NO and vasoactive prostanoid, respectively. These findings indicate a reduced contribution of a non-NO non-prostanoid factor to ACh-induced relaxation in untreated MI-rats, interpreted as reduced EDHF (left panel figure 1B); when calculated as the AUC the decreased EDHF contribution in total relaxation to ACh amounted approximately 30%, compared to untreated age-matched sham-controls (figure 2A). In contrast, the L-NMMA-sensitive contribution in total relaxation to ACh was similar in all groups, indicating that NO-contribution was neither affected by MI, nor by quinapril treatment (figure 2A). Furthermore, the indomethacin-sensitive contribution was negligible in all groups, indicating that vasoactive prostanoids played no role major role in ACh-induced relaxation in the aorta. Treatment of MI-rats with quinapril for 8 months significantly improved the contribution of EDHF in total ACh-induced relaxation, both when compared to untreated MI-rats as well as untreated sham rats, and this improvement appeared still preserved after 14 months of quinapril treatment. In accordance with that, subsequent cessation of ACE-I therapy caused a rapid and marked deterioration in EDHF, with similar reductions at 4 and 6 weeks after withdrawal (figure 1B). Note that the contribution of EDHF to total ACh-induced relaxation after ACE-I withdrawal in quinapril treated MI-rats was as low as in untreated MI-rats at 8 months after infarction (figure 2A). Endothelium-independent vasodilation Maximum endothelium-independent relaxation to the exogenous NO donor sodium nitrite (SN) at 8 months follow-up was similar in untreated sham- and MI-rats (figure 3). Quinapril significantly increased the response to SN at 8 months, and this effect was

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Chapter 7

8 Months

Acetylcholine (mol/L)

1e-8 1e-7 1e-6 1e-5 1e-4

% o

f PE

pre

cont

ract

ion

0

20

40

60

80

100

MI

MI+QUI

A) Total relaxation

Sham

14 Months QUI & withdrawal


1e-8 1e-7 1e-6 1e-5 1e-40

20

40

60

80

100 Chronic QUIChronic QUI + 4 weeks withdrawalChronic QUI + 6 weeks withdrawal

8 Months


1e-8 1e-7 1e-6 1e-5 1e-4

% o

f PE

pre

cont

ract

ion

0

20

40

60

80

100

14 Months QUI & withdrawal


1e-8 1e-7 1e-6 1e-5 1e-40

20

40

60

80

100

Chronic QUIQUI + 4 weeks withdrawalQUI + 6 weeks withdrawal

MI

MI+QUI

B) Relaxation in presence of L-NMMA and indomethacin

Sham

*

*

*

*

†

†

Figure 1. Maximum acetylcholine-induced vasodilation after 8 months MI, effects of long-

term quinapril treatment, and of 4 and 6 weeks of therapy withdrawal in aorta segments of rats

with MI. A) Total relaxation, B) Relaxation in presence of L-NMMA and indomethacin to

block NO- and prostaglandin-mediated vasorelaxation. Responses were calculated as

percentage of precontraction with 10-6 mol. L-1 phenylephrine. L-NMMA: L-methyl arginin

ester. *: p<0.05 vs. sham. †: p<0.05 versus 14 months quinapril.

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maintained at 14 months. Four weeks after withdrawal, maximum SN-mediated vasodilation had significantly decreased. Between 4 and 6 weeks of withdrawal, maximum vasodilation to SN further decreased significantly. Note that such alterations in vascular vasodilator responsiveness may partially account for the observed reduced response to ACh in MI-rats without (or withdrawn from) quinapril treatment. Nevertheless, correcting the ACh response for the maximum response to SN in an attempt to take this into account did not alter the pattern: the contribution of EDHF in ACh-induced relaxation was still significantly impaired in untreated MI-rats and those in which ACE-I had been withdrawn (see inset figure 2B). Responses to ACh were corrected for differences in response to SN by considering the response to SN as 100% relaxation, and subsequently recalculating ACh-induced vasodilation as a percentage of the difference between PE and SN response.

Discussion

The current study evaluates effects of chronic ACE-I therapy and its withdrawal on endothelial function in rats with myocardial infarction. Main findings of this study are that long-term ACE inhibition with quinapril improved non-NO non-prostanoid

A)

Total (NO) (Prostanoids) (EDHF)

AU

C (

arb

itra

ry u

nit

s)

0

2

4

6

8

10

8 months sham8 months MI untreated8 months MI + QUI14 months MI + QUI 14 months MI + QUI - 4 weeks withdrawal14 months MI + QUI - 6 weeks withdrawal

*

B)

Corrected for SN-responseL-NMMA/indomethacin resistant

AU

C (

arb

itra

ry u

nit

s)

0

1

2

3

4

5

6

*

L-NMMA sensitive

Indomethacin-sensitive

L-NMMA/indomethacin-

resistant

*

*

*

† †

† †

† †

Figure 2. Contributions of different mediators of acetylcholine-induced aortic vasodilation

during chronic quinapril treatment, and after 4 and 6 weeks of therapy withdrawal. Data are

represented as area under the dilatation curve (AUC). EDHF-contribution was calculated as

AUC in presence of L-NMMA and indomethacin; NO-contribution was calculated by

subtracting AUC of total relaxation from relaxation in presence of L-NMMA. *: p<0.05 versus

sham, † p<0.05 versus 14 months QUI.

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Chapter 7

mediated (i.e. EDHF-mediated) endothelium-dependent vasorelaxation and that within 4 weeks of withdrawal this beneficial effect completely disappeared. General characteristics Blood pressure was considerably decreased by quinapril, and after 4 weeks of withdrawal, this effect had reversed. The antihypertensive effect of chronic ACE inhibition is generally sustained for weeks after withdrawal, although some studies in hypertensive rats also show lack of sustained blood pressure reduction25,26. Hence it has been suggested that ACE-I dose and duration of therapy may determine whether or not a rebound blood pressure increment occurs shortly after withdrawal, where the higher dosages and longer treatment periods may be associated with more pronounced blood pressure increases26,27. Moreover, in the present study, as the rats were supposed to be initially normotensive or hypotensive due to MI, and the rather quick reversal of blood pressure reduction after withdrawal may as well be related to the disease model; MI versus hypertension. Endothelium-dependent vasodilation Combined data from literature suggest that endothelial function is progressively impaired during LV dysfunction post-MI, and ACE-I therapy results in improved endothelial function. However, the effects of ACE inhibition on the contribution of the different mediators of endothelium-dependent vasodilation are not undisputed. In the present study the ACE-inhibitor quinapril restored the contribution of non-L-NMMA/indomethacin-mediated endothelium-dependent vasorelaxation. This remaining component of relaxation, insensitive to L-NMMA (NO) and indomethacin (prostanoids), is likely to be EDHF-mediated. Although the exact nature of EDHF is not completely elucidated, and may be potassium ions28, electric signaling via gap junctions29, or epoxyeicosatrienoic acids30, all EDHFs exert their vasodilating effect through opening of Ca2+-dependent K+ (KCa) channels. Importantly, previous studies

maximum SN-induced vasodilation

% d

ilati

on

aft

er P

E p

reco

ntr

acti

on

0

60

70

80

90

100

8 months sham8 months MI untreated 8 months MI + QUI14 months MI + QUI 14 months MI + QUI - 4 weeks withdrawal14 months MI + QUI - 6 weeks withdrawal

‡

*

†

†

Figure 3. Vasodilation in response to the nitric oxide donor sodium nitrite (SN, 10 mmol/L).

Response is depicted as percentage of relaxation after a precontraction with 1 µmol/L

phenylephrine (PE). *: p< 0.05 versus sham, †: p<0.05 versus 14 months QUI, ‡: p<0.05 versus

4 weeks withdrawal.

105



demonstrated complete abrogation of the L-NMMA plus indomethacin-resistant vasodilation in the presence of the KCa-blockers charybdotoxin and apamin9,23,24. Involvement herein of a NO pool generally insensitive to eNOS blockade cannot be fully excluded, although little is known about such NO-pools. However, involvement of a specific non-L-NMMA-sensitive pool of NO seems very unlikely, as experiments from a previous study showed that adding another NO-inhibitor (0.1 mM L-NAME) did not further inhibit ACh relaxation9. Previous studies showed that EDHF contributes substantially to endothelium-dependent relaxation, not only in large conductance arteries, but also in coronary arteries and small resistance arteries31-33. As alterations in the latter vessels may play a central role in increasing peripheral vascular resistance and hence development and progression of LV dysfunction, EDHF is an interesting potential therapeutic target. However, the involvement of EDHF in endothelial dysfunction during chronic heart failure has gained little attention yet and is still controversial. One study reports increased EDHF compensating for reduced NO-mediated vasorelaxation in isolated mesenteric arteries during experimental heart failure5. The discrepancy with our present results may be explained by use of different vessels and particularly the much shorter follow-up time of 4-8 weeks after MI induction in that study, as endothelial dysfunction develops over longer periods of time in the rat MI model34. Indeed, our results are in general accordance with a study with a longer follow-up in the same experimental model showing that aortic endothelial dysfunction was due to impaired EDHF9. The exact mechanism underlying EDHF improvement by quinapril and subsequent deterioration after withdrawal cannot be determined from the present study. Blood pressure reduction by ACE inhibition may play a role, but EDHF was also impaired in untreated MI rats compared to sham. As blood pressures were similar between these latter two groups, alterations in endothelial function independent of blood pressure are more likely to underlie the observed effects of quinapril. Rather, local vascular RAAS effects may be involved. Our findings are in line with previous findings showing that RAAS blockade with either ACE inhibition or angiotensin II receptor antagonists improved EDHF in age-related endothelial dysfunction35-37. The link between RAAS (blockade) and EDHF is unknown. However, angiotensin II inhibits opening of KCa channels through AT1 receptor activation38,39. Thus RAAS inhibition would augment vasodilatation mediated via KCa channels. Furthermore RAAS inhibition could have decreased sub-endothelial thickening, thereby improving transfer of EDHF to the vascular smooth muscle cells in the media36. Withdrawal of long-term quinapril treatment resulted in endothelial dysfunction within 4 weeks. This aortic endothelial dysfunction observed after cessation of quinapril was also explained by loss of EDHF-mediated vasodilation. Again, increased RAAS activity after withdrawal of ACE inhibition14,40 is likely to underlie the deterioration in EDHF-mediated vasodilation.

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Chapter 7

Endothelium-independent vasodilation Responsiveness of the aorta to the NO donor sodium nitrite (SN) was increased by quinapril, whereas myocardial infarction itself had no significant effect. The underlying mechanism cannot be determined from the present study did not directly asses responsiveness of the vascular smooth muscle in absence of endothelium. Possibly, ACE inhibition improved cGMP signaling in vascular smooth muscle cells36, by increased soluble guanylyl cyclase expression41. Part of the improvement in ACh-induced vasodilation by ACE inhibition could thus be explained by effects on the smooth muscle level. However, correcting the ACh response for the maximum response to SN in an attempt to take this into account did not alter the pattern: the contribution of EDHF in ACh-induced relaxation was still significantly impaired in untreated MI-rats and those in which ACE-I had been withdrawn. These findings suggest that changes in smooth muscle responsiveness to vasodilators are unlikely to fully account for the improvement of EDHF-mediated responses by quinapril. Study limitations The groups analyzed at 8 months had on average smaller infarctions than the groups analyzed at 14 months and subjected to 0, 4, and 6 weeks withdrawal, and this hampers direct comparison in time. The above differences in MI-size may be attributed to infarct expansion due to chronic increased filling pressures as a consequence of progressive LV dysfunction. The three withdrawal-groups (0, 4, 6 weeks) were harvested at different time points after coronary ligation. This could be a confounding factor when comparing different time points in the course of heart failure development. However, the order of magnitude of difference in time points of sacrifice (weeks) is limited compared to the total treatment period (>1 year). Conclusion Long-term ACE inhibition in rats with myocardial infarction markedly improved non-NO/prostanoid-mediated (i.e. EDHF) endothelium-dependent vasorelaxation. Within four weeks after treatment withdrawal this beneficial effect completely disappeared. These findings highlight the potential importance of EDHF impairment in development of endothelial dysfunction after myocardial infarction, and the possibility to improve EDHF-mediated vasodilation with chronic ACE inhibitor therapy. In addition, withdrawal of ACE inhibition in patients with LV dysfunction after MI should be considered carefully, as profound endothelial dysfunction develops rapidly and could account for adverse effects of ACE-I withdrawal seen in patients with post-MI LV dysfunction. The mechanism by which quinapril may enhance EDHF requires further study.

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Chapter 8

General Discussion.

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Chapter 8

Introduction

Excess activation of the RAAS plays a central role in the progression of left ventricular dysfunction towards chronic heart failure after myocardial infarction. This system becomes activated to maintain sufficient cardiac output, and improve the clinical status of the patient in an acute setting. However, chronic activation of the RAAS mediates progressive cardiac remodeling. During the last two decades, inhibiting the RAAS with ACE inhibitors unambiguously proved to improve cardiac function and prognosis after myocardial infarction. Accordingly, ACE inhibition is the cornerstone of therapy to prevent the progression of left ventricular dysfunction. Despite its undisputed benefits, ACE-inhibition slows down, but does not prevent the gradual progression of myocardial dysfunction towards overt chronic heart failure (CHF). Furthermore, not all patients show an optimal therapy-response to ACE inhibition. Hence, morbidity and mortality remain high, and further optimization of therapy is warranted. One potential target could be intervention with the sodium balance. In numerous studies, dietary sodium restriction was shown to augment the response to ACE-I treatment across different clinical and experimental conditions, including proteinuric renal dysfunction, and essential hypertension1-6. However, this was not investigated yet for treatment of LV dysfunction and heart failure, and this was therefore the topic of the first part of this thesis.

Optimization of ACE inhibition with a low sodium diet?

Despite the substantial amount of clinical and experimental evidence, the mechanisms underlying this potentiation of ACE-I therapy by sodium restriction are poorly understood. It has been suggested that activation of the RAAS is required for effective ACE inhibitor therapy7, because during ACE inhibition RAAS activation with sodium depletion may cause a shift of production of Ang II towards Ang-(1-7). Increased production of Ang-(1-7) under ACE inhibition has been proposed to underlie at least part of the therapeutic effects. In chapter 2 we showed increased left ventricular ACE inhibition and LV hypertrophy reduction with dietary sodium restriction added to ACE-I therapy. The hypothesis of increased RAAS activity and Ang-(1-7) levels could not be tested in that study. In chapter 3 however, we observed no further increase in plasma renin activity by dietary sodium restriction in addition to ACE inhibition after long-term treatment. Thus it is questionable whether sodium depletion during ACE inhibition in rats with myocardial infarction results in further activation of the RAAS on the long term, explaining the lack of improvement of cardiac function and mortality. Notably, these studies investigated effects of sodium restriction on ACE inhibition after an extensively long follow-up period; also in treatment of renal diseases the effects of reducing sodium intake on efficacy of ACE inhibition are documented only for intermediate parameters, i.e. proteinuria, blood pressure, and tissue ACE activity. However, based on our results, effects of sodium restriction on ACE inhibition on hard endpoints (morbidity, mortality) during a follow-up in the order of months to years should be studied in all clinical conditions for which ACE-I treatment is used.

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Beneficial effects of diuretic treatment

Contrary to dietary sodium restriction, diuretic treatment with hydrochlorothiazide improved early chronic survival during ACE inhibitor therapy (Chapter 3). As hydrochlorothiazide added to ACE inhibition caused a decrease in plasma renin activity compared to ACE inhibition alone after 8 months of treatment, the early chronic survival benefit of hydrochlorothiazide was not caused by further RAAS activation. Rather, the explanation for this observation may be either a potentiation of ACE inhibition, or a direct effect of hydrochlorothiazide. HCTZ-mediated venodilation resulting in reduced preload (as shown by reduced LVEDP) could account for such a direct effect. Alternatively, a pharmacokinetic explanation could underlie the interaction between sodium depletion and ACE inhibitors. Interestingly, we found an interaction between HCTZ and two different ACE inhibitors: in chapter 4 we describe that diuretic treatment caused accumulation of ACE inhibitors in either plasma or cardiac and renal tissue, depending on the lipophilicity of the ACE-I. The idea for a kinetic interaction already arose from results in chapter 2, where it was shown that ACE-I therapy caused a more pronounced reduction in tissue ACE activity under sodium depleted conditions. This, while sodium restriction per se does not influence tissue ACE activity8. This finding suggests that during sodium depletion tissue concentrations of ACE inhibitors can be increased, reflecting improved tissue drug penetration. Presumably, a volume depletion effect may underlie the accumulation, either directly, or via decreased renal clearance of the drug. Combining diuretic treatment with ACE inhibition may result in volume depletion, causing a decline in renal perfusion. As a consequence glomerular filtration rate, the primary determinant of renal function, decreases9-12. Increasing tissue levels of ACE inhibitors with diuretics may achieve maximal cardioprotection. Notably, tissue ACE activity during therapy is strongly associated with the severity of organ damage13,14. However, the decrease in renal function caused by addition of a diuretic to ACE inhibition may eventually have a detrimental impact on prognosis after myocardial infarction, as is discussed in the following section.

Diuretics and long-term prognosis

In chapters 4 and 5 it was shown that hydrochlorothiazide added to ACE inhibtion caused a marked increase in plasma creatinine concentrations, indicating reduced glomerular filtration rate. Furthermore, renal interstitial fibrosis and tubular degeneration were seen in rats with myocardial infarction treated with the combination of HCTZ and the ACE inhibitor quinapril, but not quinapril monotherapy. Although a direct causal relation between the HCTZ-induced decrease in renal function and long-term survival was not shown, these observations indicate that HCTZ added to ACE inhibition decreases renal function to an extent that could be associated with increased long-term mortality. It is firmly established that kidney function is negatively influencing long-term prognosis in patients with left ventricular dysfunction after myocardial infarction15-17, but the mechanisms underlying this interaction are still

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incompletely understood. It has been suggested that the relation between mild renal impairment and progression of cardiac disease in humans is non-causal, and that both are the consequence of traditional cardiovascular risk factors, such as atherosclerosis and hypertension18,19. However, these confounding factors are not present in our experimental MI model. Thus, decreased GFR (reflected by increased plasma creatinine concentrations) itself may be a risk factor triggering development heart failure. Accordingly, unfavorable cardiac changes related to remodeling, such as cardiomyocyte dropout20, increased cardiomyocyte/capillary ratios21, and impaired energy metabolism22 have been observed in rats with (mild) renal impairment. Furthermore rats with mild renal impairment by nephrectomy showed reduced ischemia tolerance, via a yet unresolved mechanism, but independent of confounding effects of hypertension, sympathetic overactivity, and salt retention23. Such effects of mild renal function could eventually result in unfavorable effects on mortality during prolonged follow-up. The studies described in this thesis suggest that in HCTZ added to ACE inhibition in experimental LV dysfunction improves intermediate parameters, but that on the long-term, these beneficial effects are offset. Thus, it deserves recommendation that further studies on the effects of diuretics and ACE inhibition on LV dysfunction as well as renal failure and hypertension involve hard end-points and have extended follow-up.

Long-term cardiovascular protection by ACE inhibition

In addition to direct cardiac effects, ACE inhibition has several beneficial effects on the structure and function of blood vessels that may at least partially underlie its cardioprotective effects. Increased peripheral vascular resistance, which is thought to be the combined result of endothelial dysfunction24,25 and increased myogenic tone of resistance arteries26, is a hallmark of LV dysfunction. Although ACE inhibition is known to improve endothelial function, it is not undisputed which components of endothelium-dependent vasodilation are improved27-

32. These components include nitric oxide (NO), vasoactive prostanoids, and endothelium-derived hyperpolarizing factor (EDHF). Chapter 7 shows that development of endothelial dysfunction in rats with myocardial infarction was largely explained by a decline in EDHF-mediated endothelium-dependent vasorelaxation, and that ACE inhibition could restore this EDHF. Although the exact nature of EDHF is not completely elucidated, and may be K+33, electric signaling via gap junctions34, or epoxyeicosatrienoic acids35, all EDHFs exert their vasodilating effect through opening of Ca2+-dependent K+ (KCa) channels. Previous studies showed that EDHF contributes substantially to endothelium-dependent relaxation, not only in large conductance arteries, but also in coronary arteries and small resistance arteries36,37. Our findings confirm the identification of EDHF as a potential therapeutic target to prevent the progression of left ventricular dysfunction38. Accordingly, we showed that withdrawal of chronic ACE inhibition in rats with myocardial infarction was not only associated with accelerated development of left ventricular hypertrophy and reduced cardiac contractility, but most notably, a rapid development of marked endothelial dysfunction (chapters 6 and 7). This is accordance with previous observations in patients with myocardial infarction, where ACE-I

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withdrawal resulted in a high incidence of cardiac ischemic events39,40. Endothelial dysfunction with increased tendency for coronary vasospasm and acute coronary thrombotic processes is associated with an increased risk for ischemic events. Thus endothelial dysfunction, rather than progression of heart failure41 appears a matter of concern during the early phase after abrupt cessation of ACE inhibition.

Conclusion

As far as animal studies can be extrapolated to man, it seems improbable that on the long-term dietary sodium restriction will substantially improve prognosis of patients with myocardial infarction treated with an optimal dose of ACE-I. Although addition of sodium restriction to ACE-I therapy did improve intermediate parameters in rats with myocardial infarction, treatment outcome was not altered. In contrast, diuretic treatment improved early chronic survival and cardiac function. The underlying mechanism was not RAAS activation, but either potentiation of ACE inhibition via a pharmacokinetic interaction, or a direct effect of HCTZ, independent of sodium status or ACE inhibition. However, on the long term, these beneficial effects of combining ACE inhibition with diuretic therapy were abolished, presumably as a result of adverse renal effects of chronic combination therapy. Based on our results, patients with LV dysfunction after MI may benefit from addition of a diuretic to ACE inhibition therapy, but loss of renal function may be a drawback during prolonged treatment. Combination treatment for a limited period of time (weeks to months) after MI is a strategy deserves consideration. ACE inhibition after MI remains effective during extended follow-up, and abrupt cessation - even for brief periods - should be avoided. During the early phase after withdrawing ACE inhibition, endothelial dysfunction and a subsequent risk for cardiac ischemia, rather than cardiac remodeling, appears a matter of concern.

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Summary_samenvatting_dankwoord

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Summary Left ventricular (LV) dysfunction is characterized by a progressive loss of cardiac pump function, which eventually leads to the clinical syndrome of chronic heart failure (CHF). Heart failure is associated with high morbidity and mortality. The incidence of heart failure has increased during the last decades, mainly because improved survival after acute myocardial infarction. Renin angiotensin aldosterone system (RAAS) activation plays a major role in the progression of left ventricular dysfunction towards chronic heart failure. Angiotensin-converting enzyme (ACE) inhibition therapy improves cardiac function and reduces morbidity and mortality. Although ACE-inhibition slows the gradual progression of myocardial dysfunction towards overt CHF, it does not prevent it. In order to reach maximal therapy response to ACE inhibition a low sodium intake may be required. The expected underlying mechanism was that an activated RAAS – i.e. elevated renin production – induced by sodium restriction is required for an optimal therapeutic response to ACE inhibition. Hence, the first main subject of this thesis was to study intervention with the sodium balance in order to optimize ACE-I therapy. In chapter 2 we describe the effects of dietary salt restriction on ACE inhibitor treatment in rats with myocardial infarction, after 10 weeks of follow-up. Whereas dietary salt restriction alone did not worsen or improve any of the studied parameters, it augmented left ventricular –but not renal or plasma- ACE inhibition with zofenopril. This further reduction of ACE activity was associated with a more potent anti-hypertrophic effect of the drug. Although LV hypertrophy reduction with zofenopril was improved by dietary salt restriction, we did not find a further improvement of in vitro cardiac function. We subsequently studied whether on the long term dietary salt restriction may improve the outcome of long term ACE inhibition therapy. Therefore, we studied mortality during a period of 14 months, and in vivo cardiac function. The results are described in chapter 3, and show that during 14 months of treatment with the ACE inhibitor quinapril dietary sodium restriction did not improve survival or cardiac function in rats with myocardial infarction. Based on these results, we question the benefit of dietary sodium restriction during long-term ACE inhibition for post-infarct left ventricular dysfunction. More encouraging results were obtained with addition of a diuretic (hydrochlorothiazide) to quinapril treatment. This pharmacological strategy to induce a negative sodium balance was also studied, as it is easier to maintain for the patient, and already commonly used in the cardiologic practice to reduce symptoms of fluid retention. We showed an improvement in survival and cardiac function during the first 8 months of follow-up. However, towards the end of follow-up period (14 months), the beneficial effects of hydrochlorothiazide waned off. This discrepancy between early beneficial effects and loss of benefit towards the end of long-term treatment is further discussed below, but first we focused on short-term effects of hydrochlorothiazide on ACE-I therapy. In chapter 4 we investigated whether the (early) benefits of adding hydrochlorothiazide to ACE inhibition could be explained by a pharmacokinetic interaction. We observed


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that indeed the diuretic caused accumulation of two different ACE inhibitors. The hydrophilic ACE inhibitor lisinopril accumulated in plasma, whereas the lipophilic ACE inhibitor zofenopril accumulated mainly in heart and kidney tissue. We showed that the most likely mechanism is a hydrochlorothiazide-induced volume depletion, either directly or via a GFR decrease leading to reduction in renal clearance of the ACE inhibitors. The potential risk of this loss of kidney function is the main issue in chapter

5. Here we show that combining hydrochlorothiazide with quinapril caused an increase in plasma creatinine, which indicates decreased renal function. Furthermore we showed that increased plasma creatinine concentrations measured after 4 months of treatment were significantly associated with increased mortality in rats with myocardial infarction towards 14 months of follow-up. Furthermore, histopathology showed that addition of hydrochlorothiazide to quinapril in rats with myocardial infarction caused tubular degeneration as well as inflammation and fibrotic lesions in renal proximal tubular interstitium. From this study we conclude that during long-term treatment post-MI combining ACE inhibition with a diuretic can causes renal abnormalities, which could cancel out the early beneficial effects of addition of a diuretic to ACE inhibitor therapy. Second main topic of this thesis was to study the consequences of ACE inhibitor withdrawal on cardiac function and neurohormones, and endothelial function. In contrast to the well-studied beneficial effects of ACE inhibition therapy itself, remarkably little has been published about the consequences of its withdrawal. In chapter 6 we report progressive left ventricular hypertrophy and activation of the renin-angiotensin-aldosterone system after 4 and 6 weeks of ACE inhibitor withdrawal. Secondly, we found that substantial endothelial dysfunction, which plays a central role in the pathophysiology of heart failure, developed within 4 weeks after cessation of therapy. In chapter 7, we studied the involvement of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in the vascular effects of the ACE-I quinapril and its withdrawal. We showed that long-term ACE inhibition in rats with myocardial infarction markedly improved non-NO/prostanoid-mediated (i.e. EDHF-mediated) endothelium-dependent vasorelaxation. Within four weeks after treatment withdrawal this beneficial effect completely disappeared. These findings highlight the potential importance of EDHF impairment in development of endothelial dysfunction after myocardial infarction, and the possibility to improve EDHF-mediated vasodilation with chronic ACE inhibitor therapy. The observed alterations after withdrawing long-term post-MI quinapril treatment in the present study may account for an increased risk for ischemic events. By that our findings highlight the potentially harmful effects associated with abrupt discontinuation of long-term post-MI ACE inhibition, and imply careful clinical consideration in this matter. Chapter 8 provides a final overview on the issue of sodium depletion to augment tissue ACE inhibition. Sodium restriction or diuretic therapy per se does not influence the progression of LV dysfunction into chronic heart failure, with the exception of aldosterone receptor antagonists. Furthermore, it seems improbable that on the long-


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term sodium depletion will substantially improve prognosis of patients with myocardial infarction treated with ACE inhibition in an optimal dose. Although addition of sodium restriction improved intermediate parameters in rats with myocardial infarction, treatment outcome was not altered. Diuretic treatment improved early chronic survival and cardiac function. Thus long-term treatment with this diuretic in combination with ACE inhibition after myocardial infarction may be safe, as far as animal experiments can be extrapolated to humans. As long-term survival was similar in rats treated with ACE-I alone and combined with hydrochlorothiazide, the balance between beneficial and adverse effects of adding diuretics to ACE inhibition therapy thus far ends up undecided.


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Samenvatting in het Nederlands Linkerventrikel disfunctie wordt gekenmerkt door een progressieve verslechtering van de pompfunctie van het hart, en kan uiteindelijk leiden tot hartfalen. De belangrijkste oorzaak voor linkerventrikel disfunctie is verlies van functioneel hartweefsel na een infarct. Activatie van het renine angiotensine aldosteron systeem (RAAS) speelt een belangrijke rol in het voortschrijden van linkerventrikel disfunctie. Hoewel dit systeem acuut de conditie van de patiënt verbetert, leidt langdurige activatie van het RAAS tot veranderingen aan hart en vaten die juist verslechtering van de hartfunctie tot gevolg hebben, waardoor een neerwaartse spiraal ontstaat. Blokkade van het RAAS met angiotensin-converting enzyme (ACE)-remmers geeft derhalve een duidelijke verbetering van de prognose bij linkerventrikel disfunctie. Echter, ACE remmers verminderen, maar voorkómen niet het voortschrijden van linkerventrikel disfunctie. Bovendien vertonen niet alle patiënten een goede therapie respons op ACE remmers. Er zijn aanwijzingen dat een optimale therapie respons op ACE remmers wordt verkregen bij een lage zoutinname. ACE remmers worden niet alleen gebruikt bij de behandeling van linkerventrikel disfunctie, maar ook bij nierfalen en hypertensie. Bij deze laatste aandoeningen is een grote hoeveelheid bewijs voorhanden dat een negatieve zoutbalans zorgt voor optimale therapie respons op ACE remmers. Het onderliggende mechanisme is onduidelijk, maar waarschijnlijk resulteert activatie van het RAAS als gevolg van de verlaagde zoutinname in een groter behandelingseffect van de ACE remmer. In het onderzoek beschreven in het eerste deel van dit proefschrift werd derhalve gekeken naar de effecten van interventie met de zoutbalans ter verbetering van ACE remmer therapie bij linkerventrikel disfunctie. In hoofdstuk 2 beschrijven we de effecten van een laag zout dieet op ACE remmer behandeling in ratten waarin een hartinfarct geïnduceerd is door ligatie van de kransslagader die een groot deel van het linkerventrikel van bloed voorziet. Het geven van een laag zout dieet alleen had geen effect. Echter, in ratten behandeld met een ACE remmer resulteerde zoutbeperking in een verdere reductie van de ACE enzym activiteit in het hartweefsel. Dit effect op de ACE activiteit werd niet gevonden in het plasma en de nieren. Deze verbetering van de remming van cardiaal ACE was gecorreleerd aan een sterkere reductie van linkerventrikel hypertrofie in de ratten die werden behandeld met zoutbeperking en ACE remmer. Het meten van de hartfunctie in een perfusie- opstelling voor geïsoleerde rattenharten liet echter geen verbetering van de hartfunctie zien. Vervolgens werd gekeken of zoutbeperking op de lange termijn zorgt voor een verbetering van ACE remmertherapie op harde eindpunten, namelijk sterfte en in vivo hartfunctie in ratten met een hartinfarct. In hoofdstuk 3 is beschreven dat een laag zout dieet geen verbetering gaf van hartfunctie of overleving in ratten met een hartinfarct die 14 maanden behandeld werden met een hoge dosis ACE remmer. Meer bemoedigende resultaten werden gevonden met het toevoegen van het diureticum hydrochloorthiazide aan de ACE remmer therapie. Dit is een alternatieve strategie om een negatieve zoutbalans te creëren die klinisch zeer relevant is. Diuretica worden namelijk vaak voorgeschreven aan patiënten met linkerventrikel disfunctie om


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vochtretentie tegen te gaan. Deze studie liet zien dat het toevoegen van het diureticum hydrochloorthiazide aan ACE remmer therapie geen verbetering gaf van overleving en hartfunctie in ratten met een hartinfarct op de complete vervolgperiode van 14 maanden, maar wel gedurende de eerste helft van deze periode. De discrepantie tussen de vroege gunstige effecten en het verdwijnen ervan in de late fase kwamen in hoofdstuk 5 van dit proefschrift aan bod, maar eerst werden de effecten van hydrochloorthiazide op korte termijn verder uitgediept. In hoofdstuk 4 werd onderzocht of de gunstige effecten van hydrochloorthiazide op ACE remmer therapie na een hartinfarct kunnen worden verklaard door een farmacokinetische interactie. Inderdaad veroorzaakte dit diureticum accumulatie van 2 verschillende ACE remmers. De hydrofiele ACE remmer lisinopril accumuleerde in het plasma, terwijl de concentratie van de lipofiele ACE remmer zofenopril juist was verhoogd in nieren hartweefsel. De meest waarschijnlijke oorzaak van deze bevinding volumedepletie door de combinatie van hydrochloorthiazide en ACE remmer, hetzij direct, of via verminderde nierklaring van de ACE remmers als gevolg van een nierfunctie daling. Het potentiële risico van deze verminderde nierfunctie is het onderwerp van hoofdstuk 5. Daarin werd wederom een duidelijke daling van de nierfunctie gevonden in ratten met een hartinfarct die hydrochloorthiazide naast de ACE remmer kregen. Verder werd gedemonstreerd dat verhoogde plasma creatinine concentraties waren geassocieerd met verhoogde mortaliteit na 14 maanden. Afgezien van nierfunctie werd ook naar de morfologie van de nieren gekeken. Hydrochloorthiazide veroorzaakte degeneratie van de tubuli en interstitiële fibrose. Uit deze bevindingen werd geconcludeerd dat het toevoegen van het diureticum hydrochlorothiazide aan ACE remmer therapie ongunstige effecten op de nieren heeft, die op lange termijn de gunstige effecten van de combinatietherapie bij linkerventrikel disfunctie teniet zouden kunnen doen. In het tweede deel van dit proefschrift werden de gevolgen van het stopzetten van ACE remmer therapie in kaart gebracht, met name in relatie tot vaatfunctie. Hierover is opvallend weinig bekend, hoewel de effecten van ACE remmer therapie uitgebreid bestudeerd zijn. In hoofdstuk 6 wordt beschreven dat het stopzetten van ACE remmer behandeling in ratten met een hartinfarct na 4 weken resulteerde in uitgesproken linkerventrikel hypertrofie en activatie van het RAAS. Verder werd al na 4 weken sterke endotheel disfunctie gevonden, hetgeen van belang is in de pathofysiologie van linkerventrikel disfunctie na een hartinfarct. Er zijn verschillende mediatoren bekend waarmee de endotheelcellen relaxatie van de gladde spiercellen in de bloedvatwand kunnen veroorzaken, maar er is tegenstrijdige literatuur over welke van deze mediatoren verslechteren gedurende het proces van voortgang van linkerventrikel disfunctie, en over de effecten van ACE remmers hierop. In hoofdstuk 7 werd gekeken naar de relatieve bijdrages van deze mediatoren aan endotheelafhankelijke vasodilatie op lange termijn na een hartinfarct, en vervolgens naar de effecten van ACE remmer therapie en het stopzetten ervan op deze componenten. Het bleek dat de bijdrage van endothelium-derived hyperpolarizing factor (EDHF) aan endotheelafhankelijke vasodilatatie was verslechterd in ratten met een hartinfarct, en dat ACE remmer


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therapie een sterke verbetering van EDHF gaf. Deze verbetering steeg zelfs boven het niveau van gezonde ratten, waarschijnlijk door het tegengaan van het normale vasculaire verouderingsproces en verlies van endotheelfunctie met het toenemen van de leeftijd af te remmen. Vier weken na het stopzetten van de ACE remmer behandeling was dit effect op EDHF-gemedieerde vasorelaxatie volledig verdwenen. Onze bevindingen bevestigen dat EDHF-gemedieerde vasodilatatie een belangrijke rol speelt in het optreden van endotheeldisfunctie bij linkerventrikel disfunctie, en dat verbetering van EDHF mogelijk is met ACE remmer behandeling. De combinatie van sterke linkerventrikelhypertrofie en endotheeldisfunctie kort na het stopzetten van chronische ACE remmer behandeling veroorzaakt een hoog risico op ischemie-gerelateerde complicaties. Het stopzetten van ACE remmer therapie dient derhalve in patienten met linkerventrikel disfunctie na een hartinfarct zeer zorgvuldig overwogen te worden. De algemene discussie over het in dit proefschrift beschreven onderzoek is te vinden in hoofdstuk 8. Zoutrestrictie of diureticum behandeling op zich geven geen verbetering van de prognose bij linkerventrikel disfunctie na een hartinfarct. De enige uitzondering hierop is de klasse van aldosteron receptor antagonisten. Verder is het, op basis van onze dierexperimentele resultaten, onwaarschijnlijk dat een laagzout dieet op lange termijn de werking van ACE remmer zo verbetert, dat dit leidt substantiële verbetering van de prognose van patiënten met LV disfunctie. Behandeling met het diureticum hydrochloorthiazide toegevoegd aan ACE remmer therapie verbetert mogelijk de overleving en hartfunctie in de vroege chronische fase na het hartinfarct. Echter, verslechtering van de nierfunctie en beschadiging van het interstitiële nierweefsel als gevolg van het toevoegen van een diureticum dragen er mogelijk toe bij dat dit gunstige effect na een lange vervolgperiode verdwenen was. Dit suggereert dat de in veel gevallen noodzakelijke chronische diureticum therapie ter bestrijding van vochtretentie veilig gegeven kan worden aan patiënten met linkerventrikel disfunctie na een hartinfarct. De balans van gunstige en ongunstige effecten van het toevoegen van een diureticum aan ACE remmer behandeling eindigt vooralsnog onbeslist.


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Dankwoord ‘Science seldom proceeds in the straightforward logical manner imagined by outsiders. Instead, its

steps forward (and sometimes backward) are often very human events in which personalities and

traditions play major roles.’

James Watson in “The Double Helix”

Bovenstaand citaat gaat perfect op voor het doen van onderzoek bij de Klinische Farmacologie, met medewerkers van diverse pluimage, en samenwerkingsverbanden met de afdelingen Cardiologie, Nefrologie, en Pathologie. Velen waren betrokken bij de totstandkoming van dit proefschrift, en slechts een aantal hiervan kan ik hier vermelden. Ten eerste wil ik mijn promotoren, professor Wiek van Gilst en professor Dirk Jan van Veldhuisen, bedanken. Wiek, bedankt voor je vertrouwen, vanaf het sollicitatiegesprek tot aan het afronden van mijn boekje. Verder bewaar ik erg goede herinneringen aan de door jou georganiseerde “Groningse avonden” als we weer eens op congres waren. Hopelijk kruisen onze onderzoekswegen elkaar nog eens. Dirk Jan, ik heb veel geleerd van je zakelijke aanpak en je klinische kijk op onze studies. Mijn copromotoren Regien Schoemaker en Hendrik Buikema wil ik bedanken voor de begeleiding, en waar nodig de morele steun. Hendrik, jij bent een van degenen die me direct op mijn gemak deden voelen toen ik hier in het hoge noorden kwam wonen. Ik waardeer je relativerende en bespiegelende kijk op de wetenschap én andere zaken; geen wonder dat de refereeravonden bij jou thuis altijd nét even langer duurden... Regien, toen ik na ongeveer anderhalf jaar met een gigantische berg data zat, kwam jij als geroepen. Je kennis en praktische aanpak waren van grote waarde. Ik zie uit naar mijn nieuwe avontuur in Canada (dank je wel!), en ik houd je natuurlijk op de hoogte. Ik dank professor Danser, professor Navis, en professor Zijlstra voor het snelle beoordelen van mijn proefschrift. Wat was er voor vier jaar onderzoek een hoop (bio)technische ondersteuning nodig! Ik bedank Alex Kluppel, Allard Wagenaar, Azuwerus van Buiten, Egbert Scholtens, Jacko Duker, Jan Roggeveld, Janneke van der Wal, Kristien Boddeus, Marjolein Hensens, Marry Duin, en Martin Houwertjes voor de ondersteuning en gezelligheid. Bianca, bedankt voor het vele biotechnische werk dat jij hebt verricht. Ik wens jou en je kindjes een mooie toekomst. Egbert Scholtens, ik dank je voor het werk (300 hartinfarctoperaties in vier weken is echt gekkenwerk!), maar vooral zal ik je grillen en je mooie verhalen nooit vergeten. Voor de vaatexperimenten was de hulp van Azuwerus en Marjolein (zonder jou is het stil op het vaatlab) onmisbaar. Azuwerus, zoals jij al rennend ongeveer alle opstellingen op de 5e verdieping tegelijk draaiend weet te houden: respect!


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Voor de secretariële ondersteuning ben ik dank verschuldigd aan Alexandra Doeglas, Alma Guikema, Ardy Kuperus, Ellen la Bastide, en Olga Klompstra. Wessel Sloof, dank voor het oplossen van computerellende. Van grote waarde was het contact met de andere promovendi. During the 4 years I worked as a PhD student, I have witnessed the start of a new era at the department: the increasing participation of Ubbo Emmius bursaries. I have greatly appreciated the presence of the PhD students from all over the world; Cheng, Erik, Haang, Heidrun, Larissa, Maria, Peter Ochodnicky, Sascha, Simone Gschwend, Simone Vetoretti, Ying, you made me really feel a world citizen! Peter & Erik, I consider it inappropriate to thank you in English, as your progress in mastering the Dutch language was really impressive. Therefore: som vám veľmi vďačný za všetky spoločné spoločenské/športové/krčmové stretnutia a ak sa mi podarí prispôsobiť sa v Kanade aspoň spolovice tak dobre ako sa podarilo vám v Groningene, tak to budem považovať za svoj úspech. Ste tam vítaní ma navštíviť! Dan natuurlijk de aboriginals: Annemarieke, Bas, Bernadet, Els (mijn wandelende niervraagbaak), Hiddo, Hisko, Jacoba, Jacoline, Menno, Peter Mol, Willeke en Rik, bedankt voor jullie gezelligheid en kennis. Lieve Willemijn en Mirjam, jullie lieten voor mij het zonnetje schijnen op de KF. Bedankt voor veel te veel om op te schrijven. Mirjam, met jou als paranimf móet het gewoon wel een mooie promotie worden!

Verder dank ik alle overige medewerkers van de afdeling Klinische Farmacologie. Een aantal stafleden wil ik nog in het bijzonder noemen. Dick de Zeeuw, je bent dan niet mijn promotor, jouw ideeën liggen wel ten grondslag aan dit proefschrift. Dank voor je enthousiasme en kritische kijk. Anton Roks, ouwe gitaargod van me, dank je voor alle gezelligheid. Rob Henning, ondanks dat je niet direct bij mijn onderzoek betrokken was, heb ik veel opgestoken van je enthousiasme, kennis, en creativiteit. Stefano Evangelista, thank you for the support and help in the zofenopril studies. Frans Boomsma, je plasma bepalingen waren van groot belang in de totstandkoming van dit proefschrift. Aan het Kidney Center heb ik goede herinneringen; allen bedankt voor de interesse en behulpzaamheid. Gerjan Navis, je enthousiasme en kennis zijn inspirerend. Harry van Goor en Inge Hamming, dank voor de vruchtbare samenwerking, ik vind het jammer dat die pas zo laat in mijn aanstelling tot stand kwam. De (ex-)promovendi van de afdeling Cardiologie: Daan, Folkert, Martin, Michiel, Peter van der Meer (moeten we vaker doen, samen door nachtelijk Bratislava zwerven…), Pim, Rudolf, Tom, Vincent, en Wim bedank ik voor de gezelligheid, zowel op de KF en in het ziekenhuis als tijdens de buitenlandse congressen. Op echte vrienden moet je zuinig zijn, en dat lukt niet altijd even goed als je in het verre Groningen met je proefschrift bezig bent. Jan Peter, ik mis Utrecht nog steeds,


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niet in de laatste plaats door jou. Tof dat je mijn paranimf wilt zijn. Michiel (ja ik weet het, te weinig daglicht) & Karin, Dries Jan (Bromtol-man van het eerste uur), Wijnand, Martijn & Amanda: op een mooie dag komen we weer eens wat dichter bij jullie wonen! Lieve papa, mama en Inge, ontzettend bedankt voor jullie onvoorwaardelijke steun. Door jullie ben ik wie ik ben, en ligt dit proefschrift nu hier. De weekendjes Heerde waren altijd een oase van rust in het hoofd (maar niet aan de oren). Jaap en Riet van de Bovenkamp, Henk en Marie, Ruud en Martine, Bart en Sylvia, ik ben een rijk man met zo’n schoonfamilie! Liefste Marja, hoe zou ik in vredesnaam op deze plek kunnen uitdrukken hoe belangrijk jij was, bent en blijft? Volgens mij kan jij het proefschrift net zo goed verdedigen als ik. En verder: “smile and just say no more”...


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Publications

Full papers (English) Westendorp B; Schoemaker RG; Buikema H; de Zeeuw D; van Veldhuisen DJ; van Gilst WH. Dietary sodium restriction specifically potentiates left ventricular ACE inhibition by zofenopril, and is associated with attenuated hypertrophic response in rats with myocardial infarction. J.Renin Angiotensin Aldosterone Syst. 2004; 5(1): 27-31 Westendorp B; Schoemaker RG; Buikema H; Boomsma F; van Gilst WH; van Veldhuisen DJ. Beneficial effects of add-on hydrochlorothiazide in rats with myocardial infarction optimally treated with quinapril. Eur. J. Heart Fail. 2005; in press. Westendorp B; Schoemaker RG; Buikema H; Boomsma F; van Veldhuisen DJ; van Gilst WH. Progressive left ventricular hypertrophy after withdrawal of long-term ACE inhibition following experimental myocardial infarction. Eur. J. Heart Fail. 2005; in press. Westendorp B; Schoemaker RG; van Gilst WH; Buikema H. Improvement of EDHF by chronic ACE inhibition declines rapidly after withdrawal in rats with myocardial infarction. J. Cardiovasc. Pharmacol. 2005; accepted for publication. Full papers (Dutch) Westendorp B, Buikema H. Vroege ACE-remmer therapie bij AMI: een focus op zofenopril. Klinische Cardiologie 2002: 2; 14-21 Abstracts Rook DW; Westendorp B; Van het Schip AD; Nijsen; JFW. Efficacy of holmium-166 loaded microspheres in the treatment of liver tumours in rabbits. J. Nucl. Med. 2001; 42: Suppl. S273-274 Westendorp B; Buikema H; Schoemaker RG; van Veldhuisen DJ; van Gilst WH. Rebound after withdrawing chronic angiotensin-converting enzyme inhibitor treatment post-myocardial infarction. Eur. Heart. J. 2003; 24: Suppl. S 526 Westendorp B; Schoemaker RG; Buikema H; de Zeeuw D; van Veldhuisen DJ; van Gilst WH. Dietary sodium restriction enhances cardiac ACE inhibition after experimental myocardial infarction. Circulation 2003; 108 (17): Suppl. S 429


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Curriculum vitae Bart Westendorp werd geboren op 7 mei 1977 in Heerde. In 1996 behaalde hij het VWO diploma aan de Chr. Scholengemeenschap “de Heertganck” te Heerde. In datzelfde jaar werd begonnen met de opleiding Biomedische Wetenschappen aan de Universiteit van Utrecht. Tijdens de doctoraalfase werden 2 stages gelopen. Een bijvakstage werd gedaan bij het NMR laboratorium Experimentele Cardiologie van het Universitair Medisch Centrum Utrecht onder leiding van professor C.J.A. van Echteld. Onderzocht werd wat de rol was van de natrium/bicarbonaat cotransporter bij natrium-ophoping bij ischemie en reperfusie in geïsoleerde rattenharten. De hoofdvakstage werd gelopen bij de afdeling Nucleaire Geneeskunde en Biofarmacie onder leiding van professor W. Hennink en dr. F. Nijsen. Doel was de effectiviteit en biodistributie van radioactieve polymelkzuur-microsferen bij behandeling van levermetastasen te onderzoeken. In 2001 werd het doctoraalexamen behaald. Van 2001 tot en met 2005 was hij als onderzoeker in dienst van de afdeling Klinische Farmacologie aan de Universiteit van Groningen. In deze functie werd het onderzoek dat in dit proefschrift beschreven is verricht.


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Westendorp B; Schoemaker RG; Buikema H; van Gilst WH; van Veldhuisen DJ. Hydrochlorothiazide further improves survival during angiotensin-converting enzyme inhibitor therapy after experimental myocardial infarction. J. Am. Coll. Cardiol. 2004; 43 (5): Suppl. A 457 Westendorp B; Buikema H; Schoemaker RG; van Veldhuisen DJ; van Gilst, WH. Hydrochlorothiazide increases tissue concentrations of the ACE inhibitor zofenoprilat in rats with myocardial infarction. Eur. Heart J. 2004; 25: Suppl S506 Westendorp B; Schoemaker RG; Buikema H; van Gilst WH. Withdrawing chronic quinapril treatment causes rebound volume overload and endothelial dysfunction in rats with myocardial infarction. Circulation 2004; 110: Suppl. III 297 Westendorp B; Schoemaker RG; Buikema, H; van Veldhuisen DJ; van Gilst WH. Rebound after withdrawing chronic quinapril treatment in rats with myocardial infarction. Naunyn-Schmiedebergs Arch. Pharmacol. 2005; 371: Supp. R11

university of groningen tissue ace inhibition and sodium status … · (ace) is first-line therapy...

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