effects of fasting and exogenous glucose delivery on cardiac tolerance to low-flow ischemia in adult...

Mechanisms of Ageing and Development

116 (2000) 15–32

Effects of fasting and exogenous glucosedelivery on cardiac tolerance to low-flow

ischemia in adult and senescent rats

Francois Boucher *, Stephane Tanguy,Marie-Claire Toufektsian, Sophie Besse, Joel de LeirisLaboratoire Stress Cardio6asculaires et Pathologies Associees, Uni6ersite Joseph Fourier,

Batiment Jean Roget-Domaine de La Merci, 38700 La Tronche, France

Received 13 March 2000; received in revised form 15 April 2000; accepted 18 April 2000

Abstract

Metabolic disorders due to changes in cytosolic glucose utilisation are suspected to beinvolved in the increased sensitivity of the aged myocardium to ischemia. This study presentsthe first direct measurement of glucose utilisation in hearts from senescent rats duringlow-flow ischemia under different conditions of substrate delivery and glycogen stores.Isolated hearts from young adult (4-months-old) and senescent (24-months-old) rats weresubjected to 30 min coronary flow restriction (residual flow rate=2% of control flows).Experiments were performed using glucose-free or glucose-enriched (11 mmol/L) perfusionmedia. The effects of increased glycogen stores were assessed after 24-h fasting in both agegroups. Ischemic contracture was measured via a left-ventricular balloon. Ageing increasedischemic contracture under both conditions of substrate delivery in fed rat hearts. Theincrease in ischemic tolerance induced by fasting in senescent rat hearts was less than thatseen in young rat hearts. Moreover, fasting decreased glucose utilisation in hearts fromyoung rats, an effect which was not found in hearts from old rats. Furthermore, myocardialglycogen utilisation was increased in all groups of aged rats compared with that of youngadults, particularly under fasting conditions. It is concluded that fasting is less detrimental tothe aged myocardium during low-flow ischemia than to the young myocardium because itdoes not further reduce exogenous glucose utilisation, and it stimulates glycogen consump-tion. Moreover, a reduction in exogenous glucose utilisation, which is only partly compen-sated for by increased glycogenolytic flux could be, at least in part, responsible for the

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* Corresponding author. Tel.: +33-0476-637117; fax: +33-0476-637117.E-mail address: [email protected] (F. Boucher).

0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0047 -6374 (00 )00125 -1

F. Boucher et al. / Mechanisms of Ageing and De6elopment 116 (2000) 15–3216

increased ischemic contracture in hearts from old fed rats. Finally, our glucose-free experi-ments suggest that residual oxidative phosphorylation during low-flow ischemia might be lessrelevant in hearts from senescent rats than in those from young adults. © 2000 ElsevierScience Ireland Ltd. All rights reserved.

Keywords: Ageing; Fasting; Glycogenolysis; Glycolytic flux; Ischemic contracture

1. Introduction

Ageing is associated with extensive changes in myocardial function, biochemistryand structure (Folkow and Svanborg, 1993; Roffe, 1998). In addition, the suscepti-bility of the myocardium to ischemia and reperfusion has been shown to increasewith age (Ataka et al., 1992; Boucher et al., 1998). In humans, advanced age(\70-years-old) has been identified as a significant risk factor for the operativemortality during cardiac surgery in coronary artery bypass (Christakis et al., 1989).In animals, among the few experimental data available on this topic, Ataka et al.(1992) have shown that the aged rabbit heart accumulates more calcium duringischemia and exhibits less functional recovery upon reperfusion than does theyoung mature heart. Moreover, we have recently shown that the contracturedeveloped by the myocardial sarcomeres in response to total ischemia increasesprogressively with age in rats (Boucher et al., 1998).

It is now well established that ischemic injury is multifactorial in origin (Cooleyet al., 1972; Apstein et al., 1983; Steenbergen et al., 1990). The hypothesisimplicating disturbances in calcium homeostasis or intracellular buffering in agedischemic hearts has already been tested (Ataka et al., 1992), and the age-dependentdecrease in cardiac ability to maintain high energy phosphates under hypoxic (Baket al., 1998) or ischemic (Muscari et al., 1993) conditions has already been shown.However, the precise mechanisms of these phenomena still remain unknown.

Ample experimental evidence shows that the ability of the adult myocardium toincrease its glycolytic ATP production during ischemia is a crucial factor determin-ing cell survival (Owen et al., 1990; Eberli et al., 1991; King et al., 1995).

Thus, King et al. (1995) have shown that the severity of coronary flow restrictionduring experimental ischemia in young adult rat hearts plays a crucial role indetermining the rate of delivery of glucose and therefore its beneficial effects. Inaddition, these authors have reported that glycolysis was more cardioprotectiveduring ischemia when supplied with exogenous glucose than with glycogen (King etal., 1995).

In the present study, we compared the effects of 30 min of coronary flowreduction (with a residual flow rate of 2% of control flow) in young adult(4-months-old) and senescent (24-months-old) rat hearts under different conditionsof substrate delivery (glucose-free or glucose 11 mmol/L perfusion). Because of thepotential contribution of glycogen to the glycolytic pathway during ischemia, twomyocardial levels of glycogen where tested in each group of age. Thus, short termfasting (24 h) was used as a mean of increasing myocardial glycogen content.

F. Boucher et al. / Mechanisms of Ageing and De6elopment 116 (2000) 15–32 17

Finally, since fatty acids are the main substrate utilised by the normoxic heart,the delivery of an alternate non-carbohydrate substrate reflects a more physiologicalsystem. For this reason, we have used acetate, a homologue of fatty acids withoutdetergent effects, throughout all experiments.

2. Materials and methods

2.1. Animals

Adult male Wistar rats of 4- and 24-months of age were used. They were housedunder conditions of constant temperature, humidity and standard light–dark cycle(12 h/12 h). They had free access to tap water and standard food (A04, UAR,Villemoisson-sur-Orge, France). All animals were treated according to the guideli-nes of the Recommendations from the Declaration of Helsinki and the GuidingPrinciples in the Care and Use of Animals (L358-86/609/EEC).

2.2. High pre-ischemic le6el of glycogen

Myocardial glycogen content is known to be increased by short fasting in adultrats. In the present study, two groups of adult and two groups of old rats werefasted for the 24 h preceding the experiments.

2.3. Heart preparations

Rats were anesthetised with sodium pentobarbital (Sanofi, France; 60 mg/kgbody weight, i.p., reduced to 40 mg/kg body weight in senescent rats), and heparin(Sigma Chimie, France; 500 IU/kg body weight, i.v.) was injected via a femoralvein. Hearts were excised and placed in cold (+4°C) Krebs-Henseleit buffer.Within 1 min, aortic perfusion was initiated at a constant pressure of 9.81 kPa (1m H2O). A non-compliant water-filled ultra-thin balloon, connected to a pressuretransducer (P23ID, Gould Allco, Ballainvilliers, France) via rigid polyethylenetubing was introduced into the left ventricle through the mitral valve and inflatedto impose an end-diastolic pressure of 3–5 mmHg. The volume of the balloon(150–250 ml) was kept constant throughout the experiment. The sinus node wasremoved by cutting the right atrium, and the atrio-ventricular node was isolated bycutting the upper septum. The heart was paced at 5 Hz (300 bpm) via a monopolarsilver electrode placed on the left atrial wall and connected to a stimulator (Harvard6002, Edenbridge, UK).

After a 10 min control perfusion period, the hearts were made globally ischemicfor 30 min by imposing a residual coronary flow of 2% of the pre-ischemic valuewith a Gilson Minipuls pump.

During ischemia, the hearts were placed in a thermostated jacket (37°C), andtheir temperature was monitored by a thermocouple (Jenco Electronic, Taiwan)inserted into the right ventricle. Electrical pacing was stopped during ischemia.


2.4. Measurement of heart function and cardiac ischemic contracture

Left-ventricular diastolic and systolic pressures, the maximal rate of rise (+dP/dt) and the maximal rate of decrease (−dP/dt) in left-ventricular pressure weremeasured via the pressure transducer connected to a 2200 GOULD recorder(Gould Allco, Ballainvilliers, France). Cardiac ischemic contracture was recorded asthe increase in resting pressure (in mmHg) above baseline as recorded by theintraventricular balloon following the start of ischemia (King et al., 1995).

2.5. Perfusion liquid

The Krebs-Henseleit crystalloid buffer was equilibrated with a gas-mixture ofO2/CO2 (95%/5%) at 37°C, pH 7.4, and contained (mmol/L): NaCl 118.5; NaHCO3

25.0; KCl 4.75; MgSO4·7H2O 1.19; KH2PO4 1.18; CaCl2·2H2O 1.36.The substrate used was sodium acetate (5 mmol/L) with or without glucose (11

mmol/L).

2.6. Glucose extraction and exogenous glucose utilisation measurements

Glycolytic flux from exogenous glucose (mmol/min/g prot) was determined bymeasuring the formation of tritiated water from D-[2-3H(N)]-glucose (DuPontNEM) according to Rovetto et al. (1975). A trace amount (8.7×10−2 mmol/L; 0.2mCi/L) of D-[2-3H(N)]-glucose was added to the perfusate. In the glycolyticreaction catalysed by isomerase, tritium in position 2 of D-glucose exchanges withH2O. The rate of 3H2O production was estimated by collecting the samples ofcoronary effluent at various times during the perfusion, separating the labelledglucose from 3H2O using DOWEX (1×4-200, Sigma Chimie, France) mini-columns, and counting the H2O samples in a scintillation cocktail (Ready Safe,Beckman) in a b-counter (TriCarb 2000CA, Packard). Glucose extraction wasexpressed as a percentage of glucose delivery.

2.7. Biochemical assays

At the end of the ischemic period, the hearts were freeze-clamped, pulverised atliquid nitrogen temperature and divided into two homogenous samples: one for theassessment of glycogen content and one for acidic extraction of ATP, ADP, AMPand lactate.

2.7.1. Glycogen assayThe frozen myocardial powder was placed at 100°C for 30 min in KOH 40% (1

mL/100 mg wet tissue). After precipitation in ethanol (3 mL /100 mg wet tissue) at4°C for 15 h, the pellet containing glycogen was first dried then hydrolysed in HCl2N (1 mL/100 mg wet tissue) at 100°C for 3 h. The resulting acid samples whereneutralised by NaOH 2N addition. Glycogen content was measured by assayingglucose content using a kit (Glucose TRINDER, Sigma Chimie, France).


Tissue levels of glycogen are expressed as micromol glucose equivalents per gramprotein (mmol/g prot).

Glycogen utilisation during the 30 min ischemia was estimated by cal-culating the difference between initial myocardial glycogen content (measured inhearts clamped after 5 min of normoxic perfusion) and post-ischemic myocardialglycogen content (measured in hearts clamped after 30 min of low-flowischemia).

2.7.2. Acidic extractionThe frozen myocardial powder was homogenised in perchloric acid (0.6 N) in a

glass tube. Samples were kept at 4°C for 15 min before being centrifuged (1000×g,8 min, 4°C). The pellet was resuspended in NaOH (1N) for protein assay. Thesupernatant was neutralised with KOH (6 N) and placed on ice for 30 min beforebeing centrifuged (1000×g, 8 min, 4°C). The acid-soluble supernatant was storedat −80°C until assay.

2.7.3. Energy charge determinationThe adenyl nucleotides (ATP, ADP, AMP) were assessed by high-performance

liquid chromatography (HPLC). The equipment used was a spectro-photometer (Waters Millipore model 150; Molsheim, France) linked to aUV-detector (Waters Millipore model 481 LC; 254 nm). Eluent (sodium pyrophos-phate (0.01 M) 95%, methanol 5%) was used over a reverse-phase column(C18 Lichrosphere; Merck, Darmstadt, Germany). The peak identification wasbased on the retention times, which were checked with a mixture of syntheticstandards.

The energy charge (EC) was calculated as follows:

EC=[ATP]+0.5[ADP]

[ATP]+ [ADP]+ [AMP]

2.7.4. Lactate assayLactate tissue content was assessed on the acid-soluble extracts after neutralisa-

tion according to the enzymatic method of Gutmann and Wahlefeld (1974).

2.7.5. Protein assay and edema quantificationThe protein content was assayed by the modified method of Lowry et al., (1951),

using bovine serum albumin as standard. Tissue edema was expressed as tissue wetweight (g) per protein (g).

2.8. Statistics

Results are expressed as mean9standard error (S.E.). Analysis of variance(ANOVA) followed by the t-test with Bonferroni correction were used to examinedifferences between groups.

A difference was considered statistically significant when PB0.05.


3. Results

3.1. General aspects

As expected, ageing was associated with a significant increase in body and heartweights (Table 1).

3.2. Normoxic conditions of perfusion

3.2.1. Effect of age on fed rat heartsAgeing induced a marked decrease in cardiac function in terms of left ventricular

developed pressure and 9dP/dt without significantly modifying coronary flow(Table 2).

Ageing did not affect cardiac energy charge, exogenous glucose extraction andutilisation or myocardial lactate content under normoxic conditions of perfusion

Table 1Body weight and heart weight

24-months-old4-months-old

Fasted Fed FastedFed

493912* 495921*Body weight (g) 41295 399910(n=27) (n=14)(n=24) (n=14)

1.8490.10*1.3090.09Heart weight (g) 1.8090.06*1.2790.04(n=14)(n=24) (n=27) (n=14)

* Means9S.E. PB0.01 vs the corresponding 4-month-old rats group.

Table 2Pre-ischemic myocardial function and coronary flow

4-months-old 24-months-old

Fed FastedFed Fasted

104.293.23 70.893.32*106.296.7LVDevP (mmHg/g wwt)a 70.595.19*(n=6)(n=6)(n=12) (n=14)

+dP/dt (mmHg/s/g wwt)b 28469196*30829157*5641939055869199(n=14) (n=6)(n=12) (n=6)

40689258 22259108*35789127−dP/dt (mmHg/s/g wwt)b 21169149*(n=6)(n=14)(n=6)(n=12)

15.890.7 11.990.613.490.4CF (mL/min/g wwt)c 13.390.6(n=6)(n=6)(n=12) (n=14)

a LVDevP: Left ventricular developed pressure.b 9dP/dt: Positive and negative maximal first derivative of left ventricular pressure.c CF: Coronary flow.* Means9S.E. PB0.01 vs the corresponding 4-month-old rats group.


Table 3Exogenous glucose extraction and utilisation, energy charge and lactate myocardial content in isolatedperfused rat hearts after 5 min of normoxic perfusion with glucose (11 mmol/L) and acetate (5mmol/L) as substrates

Exogenous glucoseGlucose extraction Energy chargeaGroup Lactate(mmol/g prot)(% of delivery) utilisation

(mmol/min/g wwt)

0.8190.04 0.7790.03 9.995.84 months fed 0.4890.04(n=6)

0.3890.05†† 0.7690.02 ns0.2090.03†† 7.594.8 ns4 months fasted(n=6)

0.6990.09 ns 0.7990.01 ns24 months fed 4.991.5 ns0.3490.08 ns(n=6)

0.4190.06† 0.8090.01 ns0.1790.03† 1.290.2*,†24 monthsfasted (n=5)

a Energy charge: EC= ([ATP]+0.5[ADP])/([ATP]+[ADP]+[AMP]).† Means9S.E. PB0.05 vs the corresponding fed group.†† Means9S.E. PB0.01 vs the corresponding fed group.* Means9S.E. PB0.05 vs the corresponding 4 months group.

(Table 3). However, a significant increase in myocardial glycogen content wasobserved in the group of aged rats compared with young adults (24-month-old fedrats: 214.5915.9 mmol/g prot vs 4-month-old fed rats: 162.397.4 mmol/g prot,PB0.05; Table 4).

3.2.2. Effect of fastingA mild non-significant increase in coronary flow was observed in fasted rat hearts

from both age groups, without any modification in cardiac function (Table 2). Asexpected, fasting significantly increased myocardial glycogen content (20–35%) inboth young adults (pB0.05) and aged rats (pB0.05) (Table 4). Moreover, itdecreased glucose extraction (−60% in 4-month-old rats, PB0.01; −50% in24-month-old rats, PB0.05) and utilisation (−53% in 4-month-old rats, PB0.01;−41% in 24-month-old rats, PB0.05) and lowered cardiac lactate content (−25%in 4-month-old rats, ns; −75% in 24-month-old rats, PB0.05) in hearts of bothgroups without affecting cardiac energy charge (Table 3).

3.3. Indices of ischemic damage

Ageing significantly increased ischemic contracture in fed rat hearts under bothconditions of substrate delivery (glucose 11 mmol/L or glucose-free perfusion).However, it did not significantly modify ischemic contracture in fasted rat hearts(Fig. 1).

No tissue edema was detected in young rat hearts of any group. However, anincrease in wet weight over protein content ratio was measured in all groups ofischemic aged rat hearts. This phenomenon was less severe in the fasted group (Fig.2).


Tab

le4

Eva

luat

ion

ofgl

ycog

enco

nsum

ptio

nin

isol

ated

perf

used

rat

hear

tsdu

ring

30m

inlo

wflo

wis

chem

iaa

4m

onth

s24

mon

ths

Fed

Fas

ted

Ace

tate

Fas

ted

Fed

Ace

tate

214.

59

15.9

*18

9.89

9.0†

273.

79

8.7*

*,†

158.

99

8.7

203.

69

15.9

*16

2.39

7.4

Pre

-isc

hem

icgl

ycog

en(m

mol

/gpr

ot)

(n=

6)(n

=6)

(n=

5)(n

=6)

(n=

6)(n

=6)

28.69

3.3†

†11

4.19

15.7

70.29

5.2†

†30

.99

4.1†

†55

.99

10.9

†P

ost-

isch

emic

glyc

ogen

(mm

ol/g

prot

)83

.69

6.3

(n=

8)(n

=9)

(n=

8)(n

=6)

(n=

6)(n

=7)

85.6

%G

lyco

gen

cons

umpt

ion

48.5

%70

.5%

80.6

%46

.8%

74.4

%

aB

oth

fed

and

fast

edgr

oups

wer

epe

rfus

edw

ith

am

ixed

subs

trat

e(g

luco

se11

mm

ol/L

+A

ceta

te5

mm

ol/L

).T

heac

etat

egr

oups

wer

efe

dan

dhe

arts

perf

used

wit

ha

gluc

ose-

free

solu

tion

(ace

tate

5m

mol

/L).

Gly

coge

nva

lues

are

expr

esse

din

gluc

ose

equi

vale

nt(m

mol

)pe

rpr

otei

n(g

);m

eans9

S.E

.Gly

coge

nco

nsum

ptio

ndu

ring

the

30m

inis

chem

iaw

asca

lcul

ated

asth

edi

ffer

ence

betw

een

init

ial

(mea

sure

daf

ter

5m

inof

norm

oxic

perf

usio

n)an

dre

mai

ning

(mea

sure

daf

ter

30m

inof

isch

emia

)gl

ycog

enco

nten

t(e

xpre

ssed

as%

ofpr

e-is

chem

icva

lues

).†

PB

0.05

vsth

eco

rres

pond

ing

fed

grou

p.†

†PB

0.01

vsth

eco

rres

pond

ing

fed

grou

p.*

PB

0.05

vsth

eco

rres

pond

ing

4m

onth

sgr

oup.

**PB

0.01

vsth

eco

rres

pond

ing

4m

onth

sgr

oup.


3.4. Exogenous glucose utilisation

Ageing induced a dramatic decrease in both ischemic glucose extraction (Table 5)and consumption (Fig. 3) in fed rat hearts. However, under our experimentalconditions of myocardial ischemia, fasting did not affect glucose extraction orutilisation in aged rats, while it markedly decreased these parameters in hearts fromyoung adults (Table 5 and Fig. 3).

3.5. Glycogen utilisation and total glycolytic flux

Fasting and glucose-free perfusion increased glycogen consumption in both agegroups (Table 4). Moreover, ageing increased the initial glycogen content in allgroups of rats (Table 4).

Figure 4 gives an estimation of total cardiac glycolytic flux (i.e. includingexogenous glucose and glycogen utilisation) in the different experimental groups.Ageing induced a 30% (PB0.05) decrease in exogenous glucose utilisation over the30 min of ischemia, a phenomenon which was partly compensated for by increasedglycogenolysis (+30%). Fasting significantly decreased (−35%, PB0.05) exoge-

Fig. 1. Maximal ischemic contracture measured in isolated perfused rat hearts during low-flow ischemia.Glc: glucose 11 mmol/L; A: sodium acetate 5 mmol/L. Means9S.E. †PB0.05; ††PB0.01 vs thecorresponding fed group. *PB0.05; **PB0.01 vs the corresponding 4-month-old rats group. ns: nonsignificant.


Tab

le5

Glu

cose

extr

acti

on,

ener

gych

arge

and

lact

ate

cont

ent

ofis

olat

edpe

rfus

edra

the

arts

subm

itte

dto

30m

inlo

wflo

wis

chem

iaa

24m

onth

s4

mon

ths

Ace

tate

Fed

Fas

ted

Ace

tate

Fas

ted

Fed

(Glc

+A

)

6.209

0.65

**8.

649

4.03

†6.

809

0.62

Glu

cose

extr

acti

on(%

/gw

wt)

00

13.4

99

0.82

(n=

9)(n

=8)

(n=

6)(n

=8)

0.289

0.04

††

0.579

0.03

0.579

0.04

0.259

0.02

††

Ene

rgy

char

ge0.

499

0.05

†0.

629

0.01

(n=

7)(n

=8)

n=

8)(n

=6)

(n=

9)(n

=6)

85.6

19

16.3

8*91

.189

16.4

9†85

.569

8.99

43.8

69

3.43

61.3

39

4.56

Lac

tate

(mm

ol/g

prot

)67

.049

6.33

††

(n=

8)(n

=9)

(n=

7)(n

=6)

(n=

8)(n

=6)

aB

oth

fed

and

fast

edgr

oups

wer

epe

rfus

edw

ith

am

ixed

subs

trat

e(g

luco

se11

mm

ol/L

+A

ceta

te5

mm

ol/L

).T

heac

etat

egr

oups

wer

efe

dan

dhe

arts

perf

used

wit

ha

gluc

ose-

free

solu

tion

(ace

tate

5m

mol

/L).

Glu

cose

extr

acti

on(p

erg

wet

tiss

ue)

isex

pres

sed

aspe

rcen

tof

the

gluc

ose

deliv

ered

over

the

30m

inis

chem

ia;

ener

gych

arge

(EC

=([

AT

P]+

0.5[

AD

P])/(

[AT

P]+

[AD

P]+

[AM

P])

)an

dla

ctat

eco

nten

tw

ere

asse

ssed

onm

yoca

rdia

lsa

mpl

efr

ozen

atth

een

dof

isch

emia

;m

eans9

S.E

.†

PB

0.05

vsth

corr

espo

ndin

gfe

dgr

oup.

††

PB

0.01

vsth

corr

espo

ndin

gfe

dgr

oup.

*PB

0.05

24m

onth

svs

the

corr

espo

ndin

g4

mon

ths

grou

p.**

PB

0.01

24m

onth

svs

the

corr

espo

ndin

g4

mon

ths

grou

p.


Fig. 2. Myocardial edema calculated as wet tissue weight (g) per protein (g). Means9S.E. Glc: glucose11 mmol/L; A: sodium acetate 5 mmol/L. †PB0.05; ††PB0.01 vs the corresponding fed group.*PB0.05; **PB0.01 vs the corresponding 4-month-old rats group. ns: non significant.

nous glucose utilisation in the group of 4-month-old rats. Finally, ischemic glyco-gen consumption under fasting conditions was stimulated to a greater extent inhearts from senescent rats than in young rat hearts (+106% and +70%,respectively).

3.6. Post-ischemic energy charge and lactate content

Ageing increased tissue lactate accumulation in fed rat hearts perfused duringischemia with a mixture of glucose (11 mmol/L) and acetate (5 mmol/L). Fastingand glucose-free perfusion increased lactate accumulation in hearts from 4-month-old rats, an effect which was not found in hearts from aged rats (Table 5).

Finally, both fasting and glucose-free perfusion decreased ischemic cardiacenergy charge in young rat hearts while only glucose free perfusion exerted thisdetrimental effect in old rat hearts (Table 5).

4. Discussion

4.1. Myocardial ageing

Numerous experimental studies on cardiovascular ageing have been performed inthe rat on the basis that the modifications to its cardiovascular system during the


Fig

.3.

Exo

geno

usgl

ucos

eco

nsum

ptio

nof

isol

ated

rat

hear

tsdu

ring

30m

inlo

w-fl

owis

chem

ia.

Mea

ns9

S.E

.G

lc:

gluc

ose

11m

mol

/L;

A:

sodi

umac

etat

e5

mm

ol/L

.†PB

0.05

;††

PB

0.01

vsth

eco

rres

pond

ing

fed

grou

p.*PB

0.05

;**

PB

0.01

vsth

eco

rres

pond

ing

4-m

onth

-old

rats

grou

p.


30–36 months of its life span are comparable to the effects of cardiovascular ageingin human. Four-month-old male Wistar rats are considered as adults or mature ratsand 24-month-old rats are senescent rats. The spontaneous mortality rate at24-months of age in this population is 50% (Folkow and Svanborg, 1993). Thepresent study was designed to investigate the effects of ageing, rather than develop-ment or maturation, on cardiac metabolic adaptation to ischemia. For this reason,only two groups of age were considered: 4-month-old (adults) and 24-month-old(senescent) rats.

Biological and morphological studies have shown that marked changes occur inthe myocardium between these two groups of ages (Lakatta, 1993). Global myocar-dial mass increases without modifying metabolic mass, under the effect of thedevelopment of a tissular fibrosis (Klima et al., 1990; Boucher et al., 1998) and theloss of cardiomyocytes (Klima et al., 1990) which is almost compensated for by acellular hypertrophy (Fraticelli et al., 1989).

4.2. Iso6olumic performance and bioenergetics under normoxic conditions ofperfusion

The results presented here confirm that isolated perfused senescent rat heartshave reduced mechanical activity compared with young rat hearts. Under condi-tions of constant perfusion pressure and with identical rate of electrical pacing,

Fig. 4. Glucose utilisation of isolated rat hearts during 30 min low-flow ischemia. Hatched bars:exogenous glucose utilisation (Means9S.E.). Open bars: glycogen utilisation (difference betweennormoxic and post-ischemic glycogen contents). The bold frame of each bar represents total glycolyticflux. Glc: glucose 11 mmol/L; A: sodium acetate 5 mmol/L. †PB0.05; ††PB0.01 vs the correspondingfed group. *PB0.05; **PB0.01 vs the corresponding 4-month-old rats group.


LVdevP and 9dP/dt were significantly lower in the group of senescent rat hearts(−30% and −40%, respectively).

It has been shown previously that ageing induces profound changes in myocar-dial energy balance (Muscari et al., 1992). Despite the lack of direct glycolytic fluxmeasurements in the studies available in the literature, some authors have suggestedthat exogenous glucose utilisation could be increased in old rat hearts as acompensatory response to ATP depletion (Barry et al., 1987). Our study demon-strates for the first time that under normoxic conditions of perfusion, neitherglycolytic flux from exogenous glucose nor glucose extraction by the myocardiumare significantly modified by ageing in either fed and fasted rats. Moreover, cardiacenergy charge was found to remain constant during ageing under our experimentalconditions of normoxic perfusion, a result which is consistent with a recent31P-NMR study (Bak et al., 1998). Finally, our results confirm previous findingsthat myocardial glycogen stores are increased during ageing (Jullien et al., 1989).

The present study describes a major finding regarding the consequences of fastingin both the young and the senescent myocardium. Exogenous glucose extractionand utilisation during normoxic perfusion were decreased by �50% in fasted rathearts from both age groups and tissue lactate accumulation was reduced. Thisinhibition of glycolytic flux might be the consequence of a stimulation of acetateutilisation by the mitochondria under fasting conditions. Indeed, in our experi-ments, the direct supply of acetate to the myocardium might maintain an inhibitionof phosphofructokinase due to the high rate of production of high energy phos-phates from acetate, and to the inhibition of the pyruvate dehydrogenase complexby acetyl-CoA produced directly in the mitochondria from acetate. We believe thatthis consequence of fasting on glucose utilisation, which might be relevant in 6i6o,has never been described in isolated perfused hearts to date because all previousstudies that were carried out in this model used glucose as sole substrate (Depre andHue, 1997).

4.3. Low-flow ischemia with glucose in fed rat hearts

Ageing increased ischemic contracture in hearts from fed rats perfused withglucose (11 mmol/L). This phenomenon was accompanied by a decrease in exoge-nous glucose extraction and consumption which was only partly compensated forby an increase in glycogen utilisation. Several studies have been devoted to theinvestigation of cellular calcium accumulation or diastolic cardiac dysfunction inthe aged myocardium under conditions of hypoxia (Bak et al., 1998), low-flowischemia (Assayag et al., 1998) or total ischemia (Ataka et al., 1992; Snoeckx et al.,1993; Boucher et al., 1998). Despite important differences between the models used,all these studies have concluded that calcium homeostasis is less well maintained inthe aged myocardium than in the young myocardium under conditions of oxygensupply or coronary flow reduction. According to the literature (Bak et al., 1998),these alterations in calcium handling in the aged myocardium could result fromdecreased density and activity of the sarcoplasmic reticulum calcium ATPase(Maciel et al., 1990), decreased activity of the sodium calcium exchange (Heyliger


et al., 1988) and/or decreased activity of the sodium potassium ATPase (Carre et al.,1993). Our results suggest that the decrease in exogenous glucose extraction andutilisation in the aged ischemic myocardium may contribute to this phenomenoneven if it is partly compensated for by an increase in glycogen utilisation due to theactivation of glycogen phosphorylase by calcium overload. Indeed, it has beenshown that if the stimulation of glycolytic flux in ischemia is beneficial, glycogenutilisation is less cardioprotective than exogenous glucose consumption in youngadult rats (King et al., 1995).

4.4. Low-flow ischemia with glucose in fasted rat hearts

Fasting induced an increase in ischemic contracture which was significant in thegroup of young rats. However, the ischemic contracture that developed in heartsfrom aged rats under fasting conditions was equivalent to that measured in youngrat hearts under the same conditions. Moreover, fasting induced a significantdecrease in exogenous glucose extraction and utilisation in the group of 4-month-oldrats whereas it had no effect on these parameters in 24-month-old rats. Finally,ischemic glycogen consumption was substantially increased under fasting conditionsin old rat hearts.

It is well established that overnight fasting increases myocardial glycogen content.The results presented here show an increase of �20% in myocardial glycogencontent in both young and senescent rat hearts after 24 h fasting. Nevertheless, sinceglycogen stores were already higher under basal conditions of normal feeding in oldrat hearts, the absolute increase in myocardial glycogen content under fastingconditions was greater in this group compared with young adults. The consequenceof increased glycogen stores in young rat hearts was an increased glycogenolytic fluxduring ischemia which promoted lactate accumulation and inhibited, in turn,exogenous glucose utilisation. The resulting global glycolytic flux under suchconditions was comparable to that of normally fed hearts, with a greater utilisationof glycogen and a decreased utilisation of exogenous glucose. Conversely, theincreased glycogenolytic flux in fasted senescent rat hearts did not induce any furtherreduction in exogenous glucose utilisation. As a consequence, global glycolytic flux,resulting from both glycogen and exogenous glucose utilisation, was increased in thisgroup of hearts leading to a better preservation of cellular energy charge.

The metabolic consequences of glycogen breakdown during ischemia is still amatter of debate and has been discussed elsewhere (King et al., 1995; Depre andHue, 1997). Several studies point to deleterious effects of glycogenolysis on cellularintegrity (Neely and Grotyohann, 1984; King et al., 1995; Schaefer and Ramasamy,1997), whereas others underline the beneficial effects of glycogen breakdown(McElroy et al., 1989; Doenst et al., 1996; Depre and Hue, 1997). One reason forthis discrepancy could be the nature of the parameters chosen as end points ofcardiac ischemic damage. Nevertheless, we propose that the effect of fasting onischemic contracture in hearts from senescent rats could be the result of thecombination of a deleterious action of increased glycogen breakdown and abeneficial effect of preserved cardiac energy charge.


4.5. Glucose-free low-flow ischemia in fed rat hearts: possible role for residualoxidati6e phosphorylation

Surprisingly, under glucose-free conditions of perfusion, whereas glycogen con-sumption was higher in aged rat hearts, ischemic contracture was more severe in thegroup of 24-month-old rats than in the group of 4-month-old rats (young adults).Previous studies by King et al. (1995) have shown that residual oxidative phospho-rylation under comparable conditions of ischemia contributes to �25% of totalATP production in 2–3-month-old rats. In hearts of senescent rats, oxidativephosphorylation was found to be markedly reduced compared with youngeranimals mainly due to a reduction in complex I activity or to an age-dependentdecrease in cytochrome oxidase (Roffe, 1998). On this basis, we suggest thatischemic ATP production may be more affected in hearts from senescent rats thanin young adults, the stimulation of glycogenolytic flux being unable to compensatefully for the decrease in energy charge.

4.6. Limitations

‘‘Stone heart‘‘ is the clinical equivalent of ischemic contracture. It is an importantcomplication of severe ischemia which might be the consequence of decreased ATPavailability and/or cytosolic calcium accumulation (Kleber and Oetliker, 1992).Ischemic contracture may, by mechanical compression of coronary arteries, furtherreduce residual blood flow, thereby precipitating irreversible injury. Ischemic con-tracture is therefore widely used as an index of ischemic damage in isolatedperfused rat hearts (McElroy et al., 1989; King et al., 1995; Boucher et al., 1998).

Our experiments were performed on isovolumic heart preparations, and hence,the development of ischemic contracture might induce a transmural redistributionof coronary flow which could contribute to the metabolic disorders observed.

While this study has focused on the role of cytosolic utilisation of glucose, wehave not measured oxygen consumption in our heart preparations. However, theresults presented here suggest that residual oxidative phosphorylation could play acrucial role, which remains to be determined.

4.7. Conclusions

It is concluded that fasting is less detrimental to the aged myocardium duringlow-flow ischemia than to the young myocardium because it does not further reduceexogenous glucose utilisation, and it stimulates glycogen consumption. Moreover,the increased ischemic contracture in hearts from old fed rats can be attributed, atleast in part, to a reduction in exogenous glucose extraction and utilisation, whichis only partly compensated for by increased glycogenolytic flux. Finally, ourglucose-free experiments suggest that residual oxidative phosphorylation duringlow-flow ischemia might be less relevant in hearts from senescent rats than in heartsfrom young adults.


Acknowledgements

This work was supported by the ‘‘Conseil Regional Rhone-Alpes‘‘ (grant97.021.219). The authors wish to thank Mr J.-P. Matthieu for his technicalassistance and Pr C. Seban, from Centre d’Explorations Fonctionnelles, HopitalCharles Foix, Ivry-sur Seine, for having provided the senescent animals.

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