Vascular Pharmacology 53 (2010) 68–76

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Vascular Pharmacology

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Cardioprotective effect with carbon monoxide releasing molecule-2 (CORM-2) inisolated perfused rat heart: Role of coronary endothelium andunderlying mechanism

Hitesh Soni a, Praful Patel a, Akshyaya C Rath a, Mukul Jain a, Anita A Mehta b,⁎a Zydus Research Centre (ZRC communication no. # 302), Sarkhej-Bavla N.H 8A Moraiya, Ahmedabad-382210, Indiab Department of Pharmacology, L.M. College of Pharmacy, Navarangpura, Ahmedabad, Gujarat 380009, India

⁎ Corresponding author. Tel.: +91 9428418611(mobE-mail address: dranitalmcp@gmail.com (A.A. Meht

1537-1891/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.vph.2010.04.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 January 2010Received in revised form 12 March 2010Accepted 6 April 2010

Keywords:Carbon monoxide releasing molecule-2CardioprotectionCoronary endotheliumVascular smooth muscle cellKATP channel

Although the cardioprotective role of carbon monoxide (CO) has been studied against myocardial ischemia-reperfusion (I/R) injury, the role of coronary endothelium and underlying mechanism in carbon monoxide-induced cardioprotection is not well understood in isolated heart. The present study was designed todetermine the role of coronary endothelium in CORM-2-mediated cardioprotection during I/R injury inisolated rat heart. Preconditioning with 30 µM/l and 50 µM/l of CORM-2 for 10 min markedly reduced lactatedehydrogenase (LDH) and creatinin kinase (CK) levels in coronary effluent after global ischemia. There wasalso a significant improvement in coronary flow rate, heart rate, cardiodynamic parameters and markedattenuation in infarct size. However, protective effect was abolished when hearts were pretreated with100 µM CORM-2. We observed that pretreatment with L-NAME (100 µM/l), a nitric oxide synthase (NOS)inhibitor did not affect protection by CORM-2 (50 µM/l). On the other hand pretreatment with Triton X-100(0.05% for 20 s) to denude endothelium before CORM-2 treatment followed by I/R injury showed similarcardioprotection. Moreover, pretreatment with KATP channel inhibitor, glibenclamide almost completelyreversed the cardioprotective effect of CORM-2 in endothelium-denuded hearts. These results indicate thatcardioprotection by CORM-2 is highly concentration-dependent, independent of coronary endothelium andcardioprotective effect might be attributed to the activation of KATP channel present on vascular smoothmuscle cell (VSMC).

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1. Introduction

Carbon monoxide (CO) has been known as toxic gas formammalians since decades (Chance et al., 1970). In the last fewyears' research on CO in the regulation of various physiologicalprocesses has been emerged. CO is produced endogenously due to thebreakdown of hememoiety by the enzyme heme oxygenase (HO). HOexistsmainly in HO-1 (inducible) andHO-2 (constitutive) forms. HO-2is constitutively synthesized, and is expressed in many organs likebrain and testes. HO-3, recently identified, is similar to HO-2 but lessefficient heme catalyst (Perrella and Yet, 2003). Heme breakdown byHO-1 produces CO, bilirubin and iron (Fe++), inwhich a byproduct COis reported as cytoprotective in diseased conditionsmimicking the roleof HO-1 (Otterbein et al., 2003). Induction of HO-1 shows cardiopro-tection during myocardial ischemia-reperfusion (I/R) injury in wild-type (Clark et al., 2000; Hangaishi et al., 2000; Masini et al., 2003),transgenic (Yet et al., 2001; Vulapalli et al., 2002) and knockout

animals (Yoshida et al., 2001). Moreover, the pre-emptive delivery ofHO-1 inhibits postmyocardial infarct remodeling and restores ven-tricular function following I/R (Liu et al., 2006). HO-1 transduction byadeno-associated virus in ischemic heart promotes neovascularization(Lin et al., 2008). Delivery of CO in gas form has no control on releasepattern and concentration, which may lead to undesired effects.Emerging evidence suggests that exogenously applied CO could havebeneficial and therapeutic effect (Kim et al., 2006). This is conceivablytrue if delivery of CO can be probably controlled to convert toxic effectsin to beneficial signaling activities. Tricarbonyldichlororuthenium (II)dimmer known as CO-releasing molecule-2 (CORM-2), a lipid solublemolecule that was able to deliver CO in a controlled manner andsimulate the cytoprotective action of HO-1 derived CO in biologicalsystems (Józkowicz et al., 2003; Choi et al., 2003). Subsequentlytricarbonyldichloro (glycinato) ruthenium (II) (CORM-3), a water-soluble formhas been developed and demonstrated protection againstcardiac I/R injury (Motterlini et al., 2002; Clark et al., 2003; Guo et al.,2004; Stein et al., 2005; Fujimoto et al., 2004; Lavitrano et al., 2004;Akamatsu et al., 2004).

A recent report by Bak et al. (2005) showed that low concentrationof CO in perfusion buffer reduces the infarct size, improves

69H. Soni et al. / Vascular Pharmacology 53 (2010) 68–76

hemodynamic parameters and reduces ventricular fibrillation where-as higher concentration led to severe ventricular fibrillation inisolated perfused rat hearts. Therefore, our first objective was tofind out the concentration of CORM-2 required for the markedcardioporotection in isolated rat heart. CO has been shown to inhibitnitric oxide synthase (NOS) and it has also been reported that nitricoxide (NO) can inhibit human HO-1, leading to the suggestion thatinhibition of human HO-1 by NO can contribute to the signalinginterplay between NO and CO (Wang et al., 2003). These findingsdirected us to further investigate the cross talk between NO and CO inisolated rat heart. In a comparative study of the vasoactive effects ofNO and CO in isolated rabbit aorta, exogenous CO produced anendothelial-independent vasorelaxant response, albeit with a 1000-fold less potency than NO under the same conditions (Furchgott andJothianandan, 1991). In contrast, the vasodilation elicited by CORM-3required intact endothelium and an accessory role for endogenous NOproduction (Foresti et al., 2004). CORM-2 mediated cardioprotectionin intact and dysfunctional coronary endothelium has not beenstudied in isolated heart. Therefore, the major thrust of our work wasto observe the effect of CORM-2 in absence of coronary endotheliumand its underlying mechanism.

2. Materials and methods

2.1. Animals

Male wistar rats (250–300 g body weight) were used in this study.The animals were kept in individually ventilated cages in a roomwithcontrolled temperature (23±2 °C), lighting (12:12 h light–darkcycle) and relative humidity (55±10%). Animals had free access tostandard rat chow and water. The protocol for use of animals forconducting this study has been reviewed and approved by theInstitutional Animal Ethics Committee.

2.2. Chemicals

Glibenclamide was procured from Hi Media, India. Tricarbonyldi-chlororuthenium (II) dimmer (CORM-2), Ruthenium (III) chloridehydrate (RuCl3), Nω-nitro-L-arginine methyl ester (L-NAME), TritonX-100, Sodium nitroprusside (SNP), bradykinin, triphenyltetrazoliumchloride (TTC) and other chemicals purchased from Sigma Chemical,St. Louis, Mo., USA. CORM-2, RuCl3 (also termed as inactive carbonmonoxide releasing molecule–iCORM-2) and glibenclamide wereprepared freshly in DMSO before use.

2.3. Isolated perfused heart preparation

After heparinization (500 IU heparin/rat), rats were anaesthetizedand heartwas rapidly excised, placed in ice-cold Krebs–Henseleit (K–H)buffer, cannulated via aorta and perfused in the Langendorff's mode atconstant perfusion pressure 70 mmHg. Hearts were perfused withnon-recirculating K–H buffer containing (mmol/L): NaCl 118, KCl 3.2,MgSO41.2,NaHCO325,NaH2PO41.2, CaCl2 1.25 andglucose11 atpH7.4.The perfusatewas equilibratedwith 95%O2 and 5% CO2 andmaintainedat a temperature of 37 °C. The solution was filtered through a 5-µmporosity filter (Millipore, Bedford, MA, USA) to remove contaminants.Global ischemia was produced for 30 min by closing inflow of the K–Hsolution and it was followed by 120 min reperfusion by opening theinflow.

2.4. Procedure for removal of coronary endothelium

In Langendorff's hearts, coronary endotheliumwas disrupted with asingle bolus injection (0.2 ml, 20 s) of Triton X-100 (0.05% in K–Hsolution) in the aortic cannula. Concentration and time of Triton X-100to damage the coronary endotheliumwas established after several pilot

experiments. We have assessed endothelial denudation functionally bymeasurement of coronary flow with bradykinin (1 µm) and SNP(100 µM) alongwith the histopathological examinations. This approachhas been previously reported to selectively destroy the endothelium ofthe isolated rat heart,without affecting the smoothmuscle layer. (Stanglet al., 1997; Qi et al, 1998).

2.5. Determination of cardiac parameters

2.5.1. Assessment of myocardial injuryTo determine the extent of myocardial injury, levels of lactate

dehydrogenase (LDH) and creatinin kinase (CK) in coronary effluentwere measured before global ischemia (BGI) and immediately afterreperfusion (Imm Rep) by commercially available kits (RandoxLaboratories Ltd, UK). Imm Rep meaning that sampling wasperformed till 1 min of initiating reperfusion.

2.5.2. Assessment of heart rate (HR) and coronary flow (CF)An ECG was recorded by means of two silver electrode attached to

the aorta and apex of the heart using ECG machine (BPL MK801,Banglore, India) for monitoring HR. CF was measured by collection ofcoronary effluent BGI and 120 min after reperfusion (120 Rep) ingraduated measuring cylinder.

2.5.3. Measurement of cardiodynamicsA fluid-fillled latex balloon was inserted in to the left ventricle to

monitor cardiodynamic parameters. Balloon was connected to apressure transducer (Biopac-MP 100; Biopac, Santa Barbara, CA, USA)and inflated to achieve left vetricular end-diastolic pressure (LVEDP)of about 10 mm Hg. AcqKnowledge data acquisition software wasused to collect and process LVDP, dp/dtmax (indices of left ventricularcontraction) and dp/dt min (indices of left ventricular relaxation).

2.5.4. Measurement of infarct sizeHearts were removed at the end of the each experiment and

frozen. The frozen hearts were sliced into about 2–3 mm sections andincubated in 1% TTC in Phosphate buffer, pH 7.4 at 37 °C for 10 min,followed by a fixation with 10% formal saline for 30 min. Sectionswere scanned using HP scanner (HP ScanJet ADF, Colorado, USA),infarct size was measured by Image/J software version 1.37v andexpressed as percentage of total area. The normal myocardium wasstained brick red while the infarcted portion remained unstained.

2.6. Experimental protocol

Experiment was designed to determine the concentration ofCORM-2 required for cardioprotection and to observe the role ofL-NAME (NOS inhibitor) in CORM-2-mediated cardioprotection.Experimental protocol was illustrated in Fig. 1. The role of coronaryendothelium and underlying mechanism was also studied accord-ing to the protocol illustrated in Fig. 2.

In all experiments, 0.01–0.02% DMSO in K–H buffer was used toensure that effects were due to CO and not due to DMSO solvent. Alsoto dissociate the effects of CO from the donor molecule, RuCl3 whichhas the same basic structure as CORM-2 but do not liberate CO wasused as a negative control and termed as inactive carbon monoxidereleasing molecule (iCORM-2).

2.7. Statistical analysis

Results were expressed as mean±SEM. CK, LDH, HR and CF datawere analyzed by two-way ANOVA for comparison of different groupsand different time points followed by Bonferroni post-tests. One-wayANOVA followed by Dunnett's multiple comparison tests was used foranalysis of cardiodynamic parameters and % infarct size. All analysis

Fig. 1. After 10 min of stabilization, hearts were perfused for 20 min with K–H buffer, pretreated for 10 min with either vehicle or treatments as described followed by 30 min ofglobal ischemia and 120 min of reperfusion.

70 H. Soni et al. / Vascular Pharmacology 53 (2010) 68–76

was done using GraphPad Prism software version 4.0. Pb0.05 wasconsidered to be statistically significant.

3. Results

3.1. Concentration-dependent effect of CORM-2 againstischemia-reperfusion-induced myocardial injury and role of L-NAME

The first aim of the study was to identify the concentration ofCORM-2 required for cardioprotection. Pretreatment with 30 µM and50 µM CORM-2 showed significant reduction in CK and LDH levels(Fig. 3a, b) in coronary effluent immediately after reperfusion ascompared to the vehicle control group. There was also significantimprovement in HR and CF rate (Table 1) at 120 min of reperfusion.Hearts pretreatedwith 30 µMand 50 µMof CORM-2 showed significantpost-ischemic recovery in LVEDP, LVDP, dp/dt max and dp/dt min ascompared to the vehicle control group. LVEDP (% recovery) in post-ischemic isolated hearts, was significantly improved from 32.9±2.9(vehicle control) to53.3±2.1 (#pb0.01) and78.9±7.5 (#pb0.01)with30 µM and 50 µM CORM-2 respectively. Similar pattern of statisticalsignificance was observed for % recovery of LVDP, dp/dt max and dp/dtmin (Table 1). Percentage infarct size 53.33±2.28 in vehicle controlpost-ischemic rat hearts was dose dependently decreased by increasingconcentrations of CORM-2, 10 µM (46.8±1.0, pb0.05), 30 µM (35.7±1.1, pb0.01) and 50 µM (22.5±0.9, pb0.01) (Fig. 4a, b). Pretreatmentwith100 µMCORM-2 showednocardioprotection and50%of theheartsshowed arrhythmia during CORM-2 exposure, which indicated thatcardioprotection by CORM-2, is dependent on concentration. Interest-ingly it has been observed that there was significant reduction in heartrate (pb0.01)whenheartswerepretreatedwith100 µMCORM-2. Since

CORM-2 exhibited the maximum cardioprotection at 50 µM in isolatedrat heart preparation, this concentrationwas chosen for the subsequentexperiments.

CO and NO share many common signaling pathways as well asfunctions; still there are functional diversities in both the molecules.Hence we evaluated the cardioprotective effect of CORM-2 (50 µM) inthe presence of L-NAME. Interestingly we found that in presence ofL-NAME, CORM-2 showed similar patterns of recovery in cardiacparameters such as CK (Fig. 3a), LDH (Fig. 3b), HR, CF rate, LVEDP,LVDP, dp/dt max, dp/dt min (Table 1) and infarct size (Fig. 4a, b) ascompared to CORM-2 alone. However, tendency to increase ininfarct size was observed in L-NAME+CORM-2 treated group ascompared to CORM-2 alone.

3.2. Role of the coronary endothelium and underlying mechanism ofCORM-2 mediated cardioprotection

From the above observation we found that there was partialinvolvement of NO in CORM-2-mediated cardioprotection. Therefore,we initiated the experiment to evaluate the effect of CORM-2 inthe absence of coronary endothelium, which was the major focusof this study. Endothelium disruption was confirmed in somehearts by comparing the coronary flow changes using endotheli-um-dependent vasodilator bradykinin (1 µM, n=4) before TritonX-100 with its effects after treatment. In contrast, the coronaryflow change to endothelium-independent vasodilator sodiumnitroprusside (100 µM, n=4) was unaltered in the isolated hearts(Fig. 5). Endothelium disruption was also confirmed by histopa-thology (Fig. 6a, b).

Fig. 2. After 10 min of stabilization, some hearts as described above were treated with 0.2 ml of 0.05 % Triton X-100 for 20 s, followed by K–H buffer up to 5 min. Thereafter heartswere subjected to different treatments as stated and 30 min of global ischemia followed by 120 min of reperfusion.

71H. Soni et al. / Vascular Pharmacology 53 (2010) 68–76

To determine whether CORM-2-induced cardioprotection wasendothelium-dependent or independent, CORM-2 was adminis-tered to hearts with intact endothelium and to hearts in whichendothelium had been disrupted with Triton X-100. Pretreatmentwith CORM-2 subsequent to disruption of coronary endotheliumusing Triton X-100 showed significant reduction in CK and LDHlevels (Fig. 7a, b). There was also marked recovery in HR and CFrate (Table 2) in endothelium-damaged and intact hearts. Similarlypost-ischemic recovery of cardiodynamic parameters was observedin both conditions (Table 2). Post-ischemic rat hearts wereassociated with statistically significant reduction in % infarct sizefor CORM-2 pretreated heart with intact (28.7±1.6, pb0.01) anddenuded endothelium (37.7±1.5, pb0.01) as compared to vehiclecontrol (56.8±3.3) (Fig. 8). These results indicated that CORM-2mediated cardioprotection is independent of coronary endotheli-um. However, there was a trend for partial endothelium depen-dence but it was not statistically significant in our experimentaldesign. Furthermore, we explored the mechanism of cardioprotec-tion by CORM-2 in endothelium-denuded hearts using the KATP

channel blocker, glibenclamide. Pretreatment with glibenclamide(10 µM) followed by Triton X-100 and then CORM-2 significantlyprevented post-ischemic recovery in myocardial injury markers CK(Fig. 7a) and LDH (Fig. 7b). Addition of glibenclamide in

endothelium-denuded hearts significantly abolished the improve-ment in HR, CF, LVEDP, LVDP, dp/dt max and dp/dt min ascompared to Triton X-100+CORM-2 (50 µM) group (Table 2).Post-ischemic rat hearts showed 56.8±3.3 percentage infarct sizein vehicle control, which was significantly reduced to 37.7±1.5 byCORM-2 in endothelium disrupted heart. On the other hand, whenglibenclamide was used in endothelium-denuded heart to inhibitKATP channel, infarct size was found to be 46.3±1.2 whichindicates the significant prevention of cardioprotective effect ofCORM-2 (Fig. 8a, b). In the absence of Triton X-100, glibenclamideshowed cardiac injury which is similar to glibenclamide+TritonX-100+vehicle control group (data not shown).

4. Discussion

It is well known fact that CO is harmful when inhaled at high dosesor for prolonged period of time by living organisms. (Gorman et al.,2003). Tenhunen et al. (1968) showed that CO could be generatedendogenously in various tissues by degradation of heme protein.Mammalian cells endogenously generate CO via catalysis of heme byhemeoxygenase family of enzymes. Among the three reported HOisoforms (HO-1, -2, and -3), HO-1 is highly inducible and expressed inmany cell types in response to various stimuli. CO produced due to

Fig. 3. Effects of CORM-2 on ischemia-reperfusion-induced changes in (a) creatininkinase (CK) levels and (b) lactate dehydrogenase (LDH) levels from coronary effluent inisolated rat hearts. Values are expressed as mean±SEM (n=6). Levels determinedbefore global ischemia (BGI) and immediately after reperfusion (Imm Rep). @pb0.05,#pb0.01 and *pb0.001 vs vehicle control.

72 H. Soni et al. / Vascular Pharmacology 53 (2010) 68–76

induction of HO-1 appears to be cytoprotective to the cellularenvironment. (Otterbein et al., 2000; Brouard et al., 2000; Bak et al.,2002, 2003). It is now recognized that CO act as a key-signalingmolecule in biological system that regulates various cardiovascularfunctions (Durante et al., 2006). The cardioprotective effects of HO-1-derived CO as well CO applied exogenously have been previouslyreported (Vulapalli et al., 2002; Fujimoto et al., 2004), more recentlythe effect of CORM-3 in protection against myocardial infarction andgraft rejection has been published (Clark et al., 2003; Motterlini et al.,2005a, 2005b).

In vivo CO is almost immediately toxic at the concentration of 0.4%(4000 ppm) or more, but the concentration of 0.01% (100 ppm) is

tolerable and allowable for an exposure of several hours. CO at lowconcentrations (10–500 ppm) is well tolerated by cells, and therodents can be exposed to 500 ppm continuously for up to 2 yearswithout deleterious effects (Otterbein and Choi, 2000; Stupfel andBouley, 1970). These reports suggest that the concentration of CO tospecific tissue may be very important to distinguish betweentherapeutic effects and toxic effects. The results from our studyshowed that CORM-2 protects the myocardium, which was evidentfrom decreased CK, LDH, and reduced myocardial infarct size. Inaddition to that it improves cardiodynamic response, which wasreflected by the % recovery in dp/dt max, LVDP, LVEDP and dp/dt min.This protectionwas only observed at 30 µMand 50 µMconcentrations,however interestingly protective effect has been abolished at 100 µM.There was significant reduction in HR at this particular concentrationand some hearts showed arrhythmic behavior during CORM-2exposure. Similar observations were made by Patel et al. (2004) whohas reported that toxic effects of higher concentration of CO ismediated by the production of H2O2 and peroxynitrite (ONOO−). Ourdata suggests that the cardioprotective effect of CORM-2 is concen-tration-dependent, which supports the work previously undertakenby Bak et al. (2005).

CO and NO both bind to heme proteins and are gasotransmitters,which show many common downstream signaling pathways andfunctions. But the role of CO in the presence or absence of NO may beunpredictable and diverse depending on the concentration and tissuetype (Kajimura et al., 2003). Therefore, our next objective was toevaluate the effect of CORM-2 in the presence of L-NAME, an NOSinhibitor, in I/R-induced myocardial injury in isolated heart. Weobserved that CORM-2 mediated cardioprotection was not inhibitedby L-NAME treatment which suggests that CO showed cardioprotec-tion by NO-independent pathway. Moreover, we found that there wasa trend for some dependence on NO but further studies are required toconfirm the significant role of NO in CORM-2-mediated protectiveeffect.

Cardioprotection by CORM-2 in absence of coronary endotheliumand underlying mechanism by CORM-2 was the major focus of thisstudy. We observed that CORM-2 mediated cardioprotection wasindependent of coronary endothelium and it causes the heart to shiftto a preconditioned phenotype. Moreover, there was a tendency, atleast in part, for endothelium dependence but it was not statisticallysignificant. Therefore the protective effect of CORM-2 could be due tomodulation of different physiological processes that are involved inthe development of I/R injury. In I/R injury, mitochondrial KATP

channel was believed to be major end effectors of preconditioning(Garlid et al., 1997; Liu et al., 1998; Quayle and Standen, 1994; Taggartand Wray, 1998). The entire molecular structure of mitochondrialKATP channel has not been completely sequenced (Hanley and Daut,2005). Therefore, the finding solely restricts involvement of KATP

channel in the mechanism of I/R injury and preconditioning, certainpharmacological inhibitors of these channels can abolish thepreconditioning protective effect (Shojima et al., 2006; Gross andAuchampach, 1992). CO and signaling mediated by potassiumchannels is an interesting aspect of CO signaling, which was proposedseveral years ago but has been appreciated only recently (Wang andWu, 1997). This idea has always been received with skepticismbecause it was initially thought that potassium channels could notshow chemical reactivity towards CO, as they do not contain atransition metal. However, calcium-sensitive potassium channelshave been reported to bind covalently to iron protoporphyrin IX(heme) (Tang et al., 2003) and recent evidence suggests that heme isan important allosteric regulator of human maxi-K+ channels, wherethe binding of gaseous molecules including CO is likely to have aphysiological role (Williams et al., 2004; Horrigan et al., 2005). Forestiet al. (2004) have shown that CO released from tricarbonylchloro-(glycinato)ruthenium(II) (CORM-3) induces a concentration-depen-dent relaxation of aortic vessels pre-contracted with phenylephrine,

Table 1Effect of different concentrations of CORM-2 and L-NAME+CORM-2 on I/R-induced HR changes, CF changes and % recovery for LVEDP, LVDP, dp/dt max, dp/dt min using isolated ratheart. All values are expressed asmean±SEM (n=6). @pb0.05, #pb0.01, *pb0.01 vs vehicle control. BGI-values determined before global ischemia and 120 Rep-values determined120 min after reperfusion.

Srno.

Parameter Timepoint

Groups

Vehicle control(0.02% DMSO)

i CORM-2 CORM-2(10 μM)

CORM-2(30 μM)

CORM-2(50 μM)

CORM-2(100 μM)

L-NAME (100 μM)+iCORM-2

L-NAME (100 μM)+CORM-2 (50 μM)

1 Heart rate (beats/min) BGI 235.2±7.9 242.5±5.9 232.0±3.8 235.8±4.2 240.0±2.2 230.0±3.4 232.8±3.8 235.0±3.2120 Rep 125.5±4.8 128.2±5.2 126.7±4.4 153.0±2.9* 156.5±2* 92.5±3.8* 130.0±3.7 166.2±2.5*

2 Coronary flow (ml/min) BGI 7.3±0.2 7.2±0.2 7.2±0.1 7.2±0.1 7.2±0.1 7.2±0.1 7.2±0.1 7.3±0.1120 Rep 2.7±0.1 2.7±0.1 2.6±0.1 4.1±0.1* 4.1±0.1* 2.3±0.2 2.0±0.2# 4.5±0.2*

3 LVEDP (% recovery) 120 Rep 32.9±2.9 29.7±1.7 42.7±2.2@ 53.3±2.1# 78.9±3.1# 24.9±1.5 31.1±2.5 68.1±2.2#4 LVDP (% recovery) 120 Rep 40.7±3.5 36.9±1.8 44.4±2.9 66.9±3.9# 81.5±3.0# 28.7±2.2 28.7±2.6 70.7±6.2#5 dp/dt max (% recovery) 120 Rep 16.4±0.5 17.8±1.5 20.1±0.9 29.2±2.9# 42.0±1.5# 15.3±1.6 21.2±0.8 38.9±2.8#6 dp/dt min (% recovery) 120 Rep 22.5±0.7 25.3±2.6 27.8±2.6 43.8±1.2# 54.9±0.1# 20.2±1.5 22.9±1.7 51.2±2.3#

73H. Soni et al. / Vascular Pharmacology 53 (2010) 68–76

an effect that is partially reduced by inhibiting the activity ofpotassium channels. Furthermore, CORM-3 could also affect mito-chondrial function and consequently cardiac activity by interactingwith ATP dependent potassium channels (Stein et al., 2005).Stimulation of mitochondria KATP channel may provide a combinationof benefits including reduced cellular injury, reduced infarct size andimprovement in cardiodynamics. Therefore we further investigatedwhether CORM-2 is capable of generating a cardioprotective effect

Fig. 4. (a) Effect of CORM-2 on I/R-induced changes in % infarct size in isolated rat hearts. Valusing TTC staining. #pb0.01 vs vehicle control and (b) representative images of infarct size

when KATP channel inhibited using glibenclamide in endotheliumdisrupted isolated heart. We found that the inhibition of KATP channelsignificantly blocked the CORM-2-mediated cardioprotection suggest-ing that the activation of the KATP channel present on VSMC may beone of the important mechanisms for the cardioprotection. Stein et al.(2005) found similar observation related to KATP channel usingCORM-3. The difference between his study and ours is that they havenot observed the role of endothelium and vascular smooth muscle

ues are expressed as mean±SEM (n=6). Values determined 120 min after reperfusionafter ischemia/reperfusion injury.

Fig. 5. Effect of Triton X-100 on the coronary flow changes induced by endothelium-independent vasodilator (sodium nitropruside—SNP) and endothelium-dependentvasodilator (bradykinin) in isolated rat heart. Data are presented as mean±SEM.@pb0.05 and *pb0.001 vs before Triton X-100 values.

Fig. 6. Representative light micrograph of coronary artery of rat heart. (Magnification20× and stained with hematoxylin and eosin stain). Vehicle treated heart showsintact endothelium (a), where as Triton X-100 treated heart shows disruption ofendothelium (b).

Fig. 7. Effects of CORM-2 (50 µM) on ischemia-reperfusion-induced (a) creatine kinase(CK) and (b) LDH levels from coronary effluent in isolated rat heart in presence of intactand denuded coronary endothelium. Role of glibenclamide (Glib-K ATP channelinhibitor) in CORM-2 mediated CK and LDH changes in coronary endothelium-denudedisolated rat heart. Values are expressed asmean±SEM (n=6). CK and LDH determinedin coronary effluent before global ischemia (Basal) and immediately after reperfusion(Imm Rep). @pb0.05 and *pb0.001 vs vehicle control.

74 H. Soni et al. / Vascular Pharmacology 53 (2010) 68–76

cells whereas we have performed the study in absence of coronaryendothelium and concluded that the KATP channel present on vascularsmooth muscle cell plays a significant role in CORM-2-inducedcardioprotection in I/R injury model.

Table 2Effect of CORM-2 (50 µM) on I/R-induced changes in HR,CF,LVEDP,LVDP, dp/dt max, dp/dt min using isolated rat heart in presence of intact and disrupted coronary endothelium.Role of glibenclamide (Glib-K ATP channel inhibitor) in CORM-2 mediated changes in HR,CF,LVEDP,LVDP, dp/dt max, dp/dt min in coronary endothelium-denuded heart. All valuesare expressed as mean±SEM (n=6). @pb0.05, #pb0.01, *pb0.01 vs vehicle control. BGI-values determined before global ischemia and 120 Rep-values determined 120 min afterreperfusion.

Srno.

Parameter Timepoint

Groups

Vehicle control(0.02% DMSO)

TritonX-100+vehicle

TritonX-100+iCORM-2

Triton X-100+CORM-2 (50 μM)

CORM-2(50 μM)

Glib(10 μM)+Triton X-100+vehicle

Glib(10 μM)+Triton X-100+iCORM-2

Glib(10 μM)+TritonX-100+CORM-2(50 μM)

1 Heart Rate(beats/min)

BGI 226.6±4.9 236.7±2.8 229.5±3.3 235.8±4.7 224.2±4.0 216.7±4.0 219.2±3.3 215.8±3.8120Rep

126.2±2.5 132.5±5.3 123.3±4.2 156.2±2.9* 162.7±2.5* 117.0±3.7 120.0±3.4 139.2±2.4@

2 Coronary flow(ml/min)

BGI 7.2±0.1 7.2±0.2 7.2±0.1 7.2±0.1 7.3±0.2 7.0±0.0 7.2±0.1 7.2±0.1120Rep

2.1±0.3 2.1±0.1 2.1±0.1 4.1±0.2* 4.5±0.2* 1.8±0.2 1.8±0.2 2.6±0.1@

3 LVEDP (% recovery) 120Rep

31.3±2.5 33.8±4.0 37.0±3.7 71.8±4.6# 78.2±3.7 # 33.6±1.5 31.3±4.5 45.5±2.6

4 LVDP (% recovery) 120Rep

30.1±1.2 28.6±2.1 28.2±2.8 69.6±3.2# 79.4±3.7# 23.6±2.2 23.9±1.5 39.5±1.4@

5 dp/dt max (%recovery)

120Rep

26.9±1.8 21.7±0.7 23.8±2.6 44.7±3.9# 55.1±2.4# 28.1±1.4 21.7±1.0 36.8±1.7@

6 dp/dt min (%recovery)

120Rep

23.6±0.8 23.9±0.7 22.9±1.4 46.6±1.9# 52.9±2.2# 21.4±0.6 22.1±1.5 31.6±0.6

75H. Soni et al. / Vascular Pharmacology 53 (2010) 68–76

The present study demonstrates that pretreatment with CORM-2reduces I/R injury in Langendorff's perfused rat hearts which may beconcentration-dependent and endothelium-independent. Further,cardioprotective effect of CORM-2 may be due to the activation ofKATP channel present on VSMC.

Fig. 8. (a) Effect of CORM-2 (50 µM) on ischemia-reperfusion-induced myocardial infarct sizglibenclamide (Glib-K ATP channel inhibitor) in CORM-2 mediated changes in myocardireperfusion. All values are expressed as mean±SEM (n=6). @pb0.05 and #pb0.01 vs veh

Acknowledgements

Authors thank Dr Ajay Sharma for his valuable guidance for theseexperiments. Authors thank Dr Chitrang Trivedi and Mr JogeswarMohapatra for their assistance in improving the manuscript.

e in isolated rat heart in presence of intact and denuded coronary endothelium. Role ofal infarct size in coronary endothelium-denuded isolated rat heart after 120 min oficle control. (b) Representative images of infarct size after ischemia/reperfusion injury.

76 H. Soni et al. / Vascular Pharmacology 53 (2010) 68–76

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