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Role of ACE and ACE-2 in abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart

Abstract

Ischemic heart disease is the leading cause of death in postmenopausal women. The activity of heart ACE increases whereas the activity of ACE-2 decreases after menopause. The present study was designed to investigate the role of ACE and ACE-2 in the abrogated cardioprotective effect of IPC in OVX rat heart. The heart was isolated from OVX rat and mounted on Langendorff’s apparatus for giving intermittent cycles of IPC. The infarct size was estimated using TTC stain, and coronary effluent was analyzed for LDH, CK-MB, and nitrite release. IPC induced cardioprotection was significantly attenuated in the ovariectomized rat heart as compared to the normal rat heart. However, this attenuated cardioprotection was significantly restored by perfusion of DIZE, an ACE-2 activator, and captopril, an ACE inhibitor, alone or in combination noted in terms of decrease in myocardial infarct size, the release of LDH and CK-MB, and also increase in the release of NO as compared to untreated OVX rat heart. Thus, it is suggested that DIZE and captopril, alone or in combination restore the attenuated cardioprotective effect of IPC in OVX rat heart which is due to an increase in ACE-2 activity and decrease in ACE activity after treatment.

Keywords:
Ovariectomy; Captopril; Diminazene aceturate; Nitric oxide; Ischemic Preconditioning

INTRODUCTION

Ischemic heart disease (IHD) has been identified as the world's leading cause of mortality and morbidity (Murray, Lopez, 1997Murray CJ, Lopez AD. Alternative projections of mortality and disability by cause 1990-2020: Global burden of disease study. Lancet. 1997;349(9064):1498-1504.). Reperfusion of the ischemic myocardium is mandatory for the restoration of the normal functioning of the myocardium (Topol, Califf, Vandormael, 1992Topol EJ, Califf RM, Vandormael MA. Randomized trial of late reperfusion therapy for acute myocardial infarction. Thrombolysis and angioplasty in myocardial infarction-6 study group. Circulation. 1992;85(6):2090-9.). However, abrupt reperfusion of an ischemic heart produces further damage of the myocardium, described as ischemic/reperfusion (I/R) injury (Baxter, Ebrahim, 2002Baxter GF, Ebrahim Z. Role of bradykinin in preconditioning and protection of the ischaemic myocardium. Br J Pharmacol. 2002;135(4):843-854.; Piper, Abdullah, Schafer, 2004Piper HM, Abdullah Y, Schafer C. The first minutes of reperfusion: a window of opportunity for cardioprotection. Cardiovasc Res. 2004;61(3):365-371.). Ischemic preconditioning (IPC) is a powerful endogenous protective phenomenon that is used to protect the myocardium from ischemic insults, and it comprises short intermittent cycles of sublethal ischemia and reperfusion before the subsequently prolonged ischemic insult (Murray, Jenning, Reimer, 1986Murray CE, Jenning JB, Reimer KA. Preconditioning with ischemic: a delay of lethal injury in ischemic myocardium. Circulation. 1986;74(5):1124-36.). IPC produces cardioprotection by various mechanisms (Stokoe et al., 1997Stokoe D, Stephens LR, Copeland T, Gaffiney PR, Reese CB, Painter GF, et al. Dual role of phosphatidylinositol3,4,5 trisphosphate in the activation of protein kinase B. Science. 1997;277(5325):567-570. ; Ferdinandy, Schulz, Baxter, 2007Ferdinandy P, Schulz R, Baxter GF. Interaction of cardiovascular risk factors with myocardial ischemia/reperfusion injury, preconditioning and postconditioning. Pharmacol Rev. 2007;59(4):418-458.; Prendes et al., 2007Prendes MGM, Gonzalez M, Savino EA, Varela A. Role of endogenous nitric oxide in classic preconditioning in rat hearts. Regul Pept. 2007;139(1-3):141-145.; Garg et al., 2010Garg K, Yadav HN, Singh M, Sharma PL. Mechanism of cardioprotective effect of erythropoietin-induced preconditioning in rat heart. Indian J Pharmacol. 2010;42(4):219-223. Goyal et al., 2016Goyal A, Semwal BC, Yadav HN. Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart. Hum Exp Toxicol. 2016;35(6):644-653.; Charan et al., 2016Charan K, Goyal A, Gupta JK, Yadav HN. Role of atrial natriuretic peptide in ischemia preconditioning induced cardioprotection in the diabetic rat heart. J Surg Res. 2016;201(2):272-278. ). However, the cardioprotective effect of IPC gets attenuated in certain pathological conditions such as diabetes mellitus (Ajmani et al., 2011Ajmani P, Yadav HN, Singh M, Sharma PL. Possible involvement of caveolin in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart. BMC Cardiovasc Disord. 2011;11:43.; Charan et al., 2016Charan K, Goyal A, Gupta JK, Yadav HN. Role of atrial natriuretic peptide in ischemia preconditioning induced cardioprotection in the diabetic rat heart. J Surg Res. 2016;201(2):272-278. ), hyperlipidaemia (Yadav, Singh, Sharma, 2010Yadav HN, Singh M, Sharma PL. Involvement of GSK-3β in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart. Mol Cell Biochem . 2010;343(1-2):75-81.), hypertension (Snoeckx et al., 1986Snoeckx LH, Vander Vusse GJ, Coumans WA, Willemsen PH, van der Nagel T, Reneman RS. Myocardial function in normal and spontaneously hypertensive rats during reperfusion after a period of global ischemia. Cardiovasc Res . 1986;20(1):67-75.; Snoeckx et al., 1996Snoeckx LH, Vander Vuesse GJ, Coumans WA, Willemsen PH, Reneman RS. Differences in ischaemia tolerance between hypertrophied hearts of adult and aged spontaneously hypertensive rats. Cardiovasc Res . 1996;27(5):874-881.), aging (Abete et al., 1996Abete P, Ferrara N, Cioppa A, Ferrara P, Bianco S, Calabrese C, et al. Preconditioning does not prevent postischemic dysfunction in aging heart. J Am Coll Cardiol. 1996;27(7):1777-1786.; Liu et al., 2004Liu J, Kam KWL, Zhou JJ, Yan WY, Chen M, Wu S, et al. Effects of heat shock protein 70 activation by metabolic inhibition preconditioning or к- opioid receptor stimulation on Ca2+ homeostasis in rat ventricular myocytes subjected to ischemic insults. J Pharmacol Exp Ther. 2004;310(2):606-613.), hypertrophy (Singh et al., 2008Singh VP, Le B, Khode R, Baker KM, Kumar R. Intracellular Angiotensin II production in diabetic rats is correlated with cardiomyocyte apoptosis, oxidative stress, and cardiac fibrosis. Diabetes. 2008;57(12):3297-3306.), obesity (Sasaki et al., 2007Sasaki H, Ogawa K, Shimizu M, Mori C, Takatsuka H, Okazaki F, et al. The insulin sensitizer pioglitazone improves the deterioration of ischemic preconditioning in Type 2 diabetes mellitus rats. Int Heart J. 2007;48(5):623-635.), and estrogen deficiency (Goyal, Semwal, Yadav, 2016Goyal A, Semwal BC, Yadav HN. Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart. Hum Exp Toxicol. 2016;35(6):644-653.). Estrogen deficiency is one of the major risk factors of ischemic heart disease (Shinmura, Nagai, Tamaki, 2008Shinmura K, Nagai M, Tamaki K. Loss of ischemic preconditioning in ovariectomized rat hearts: possible involvement of impaired protein kinase C€ phosphorylation. Cardiovasc Res . 2008;79(3):387-394.). It is well documented that men are more susceptible to the risk of IHD than women (Barrett-Connor, 1997Barrett-Connor E. Sex differences in coronary heart disease: why are women so superior? The 1995 ancel keys lecture. Circulation. 1997;95(1):252-264.). But after menopause, the risk of IHD in women reaches the same level as in men of the same age (Barrett-Connor, 1997Barrett-Connor E. Sex differences in coronary heart disease: why are women so superior? The 1995 ancel keys lecture. Circulation. 1997;95(1):252-264.; Clarkson, Cline, Williams, 1997Clarkson TB, Cline JM, Williams JK. Gonadal hormone substitutes: effects on cardiovascular system. Osteoporosis Int. 1997;7 (Suppl. 1):43-51.). Hence there is an urge to detect a possible mechanism involved in the abrogated cardioprotective effect of IPC in ovariectomized (OVX) rat heart.

The heart renin-angiotensin system (HRAS) plays an important role in the homeostasis of the cardiovascular system (David, Kenneth, 1999David ED, Kenneth MB. The cardiac renin-angiotensin system conceptual, or a regulator of cardiac function? Circ Res. 1999;85(7):643-650. ). It has been demonstrated that an increase in activity of heart angiotensin-converting enzyme (ACE) stimulates cardiomyocytes hypertrophy and fibroblast proliferation that lead to left ventricular hypertrophy, arrhythmia, heart failure, increased myocardial infarct size, and ultimately cardiac death (Alderman, 2004Alderman MH. Renin Angiotensin system and the heart. Circulation. 2004;110:496-497.). Further, it has been well suggested that angiotensin-converting enzyme-2 (ACE-2), a new component of RAS exhibit an opposing function to the ACE (Ferreira, Santos, Almeida, 2001Ferreira AJ, Santos RA, Almeida AP. Angiotensin-(1-7): cardioprotective effect in myocardial ischemic/reperfusion. Hypertension. 2001;38(3 Pt 2):665-668.). The equilibrium of both enzymes is necessary to maintain the homeostasis of the cardiovascular system. This contention is supported by the other laboratories that the administration of ACE inhibitors and ACE-2 activators decrease the myocardial infarct size as well as decrease the release of markers of myocardial injury (LDH and CK-MB) (Martinez, Molina, 2003Martinez LA, Molina RV. Early and chronic captopril or losarten therapy reduce infarct size and avoids congestive heart failure after myocardial infarction in rats. Arch Med Res. 2003;34(5):357-361. ; Katovich, Raizada, 2013Katovich MJ, Raizada MK. Diminazene aceturate enhances angiotensin-converting Enzyme 2 activity and attenuates ischemia-induced cardiac pathophysiology. Hypertension. 2013;62(4):746-752.; Fraga-Silva et al., 2015Fraga-Silva RA., Costa-Fraga FP, Montecucco F, Sturny M, Faye Y, Mach F, et al. Diminazene protect corpus cavernosum against hypercholesterolemia-induced injury. J Sex Med. 2015;12(2):289-302.). However, it has also been documented that the pretreatment of ACE inhibitors and ACE-2 activators produce cardioprotection by facilitating the release of nitric oxide (NO) (Comini et al., 2007Comini L, Bachetti T, Cargnoni A, Bastianon D. Therapeutic modulation of the nitric oxide: all ace inhibitors are not equivalent. Pharmacol Res. 2007;56(1);42-48.; Brancaleone, Bucci, 2008Brancaleone V, Bucci M. ACE­inhibition ameliorate vascular reactivity and delays diabetes outcome in NOD mice. Vascul Pharmacol. 2008;49(2-3):84-90.; Fraga-Silva et al., 2015Fraga-Silva RA., Costa-Fraga FP, Montecucco F, Sturny M, Faye Y, Mach F, et al. Diminazene protect corpus cavernosum against hypercholesterolemia-induced injury. J Sex Med. 2015;12(2):289-302.). Further, it has been well known that NO produces IPC mediated cardioprotection, and their down-regulation abrogates the cardioprotective effect of IPC (Ajmani et al., 2011Ajmani P, Yadav HN, Singh M, Sharma PL. Possible involvement of caveolin in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart. BMC Cardiovasc Disord. 2011;11:43.; Goyal, Semwal, Yadav, 2016Goyal A, Semwal BC, Yadav HN. Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart. Hum Exp Toxicol. 2016;35(6):644-653.).

The cardioprotective effect of IPC gets attenuated in estrogen-deficient or OVX rat hearts (Goyal, Semwal, Yadav, 2016Goyal A, Semwal BC, Yadav HN. Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart. Hum Exp Toxicol. 2016;35(6):644-653.). It has been documented that the level of ACE gets upregulated whereas the level of ACE-2 gets downregulated during estrogen deficiency which further decreases the release of NO which is known to produce IPC mediated cardioprotection (Lindsey et al., 2009Lindsey SH, Cohen JA, Brosnihan KB, Gallagher PE, Chappell MC. Chronic treatment with the G protein-coupled receptor 30 agonist G-1 decreases blood pressure in ovariectomized mRen2.Lewis rats. Endocrinology. 2009;150(8):3753-3758.; Goyal, Semwal, Yadav, 2016Goyal A, Semwal BC, Yadav HN. Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart. Hum Exp Toxicol. 2016;35(6):644-653.). Therefore, the present study has been designed to investigate the role of ACE and ACE-2 in the modulation of the cardioprotective effect of ischemic preconditioning in the ovariectomized rat heart.

MATERIAL AND METHODS

Female Wistar rats weighing about 180-250 g were kept in the animal house and provided 12h light, and the 12h dark cycle was employed in this study. They were fed on a standard chow diet (wheat flour 22.5%, roasted Bengal gram powder 60%, skimmed milk powder 5%, casein 4%, refined oil 4%, salt mixture with starch 4% and vitamin and choline mixture 0.5%) and provided water ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee (GLAIPR/CPCSEA/IAEC/2016/P.Col/R15) in accordance with the national guidelines on the use of laboratory animals.

Drugs and chemicals

Captopril (ACE inhibitor) (100μM/L) and Diminazene aceturate (DIZE) (ACE-2 activator) (26.35mg/L) (Sigma Aldrich [P] Ltd., Bangalore, India) were dissolved in Kreb’s-Henseleit (K-H) buffer and then perfused to the isolated heart in four cycles of reperfusion. All other reagents used in this study were of analytical grade and always freshly prepared before use.

Induction of experimental ovariectomy

Total seven groups have been used in the present study; each group consists of six female Wistar rats. Female rats were anesthetized with pentobarbitone (45 mg/Kg i.p.). Ovariectomy was performed by making a peritoneal incision of 0.4-0.6 cm on the middle part of the abdomen slightly towards the right. Ovary and associated fat were easily located and exteriorized by gentle retraction. Ovaries along with the uterus were pulled out, and the suture was applied at the end of the uterus and beginning of the ovary. Ovaries were removed, the uterus was pushed back, and incisions were sutured in layers. Neomycin antibiotic powder was applied twice daily on wounds for one week, and animals were allowed to recover for four weeks (Goyal, Semwal, Yadav, 2016Goyal A, Semwal BC, Yadav HN. Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart. Hum Exp Toxicol. 2016;35(6):644-653.).

Isolated rat heart preparation

Rats were administered heparin (500 IU/L, i.p.) (Gland Pharma Ltd., Hyderabad, India) 20 min prior to sacrifice by cervical dislocation, and the Heart was rapidly excised and was immediately mounted on Langendorff’s apparatus (Langendorff, 1895Langendorff O. Untersuchungen am uberlebenden saugethierherzen. Pflugers Arch Ges Physiol Mensch Tiere. 1895;61(6):291-332.). The heart was enclosed in a double-walled jacket, and the temperature was maintained at 37 ℃ by circulating warm water. The isolated heart was retrogradely perfused at a constant pressure of 80 mmHg and coronary flow rate of 7-9 mL/min with Kreb’s Henseleit (K-H) buffer (NaCl 118 mM; KCl 4.7 mM; CaCl2 2.5 mM; MgSO4.7H 1.2 mM; KH2PO4 1.2 mM; C6H12O6 11 mM), pH 7.4, maintained at 37 ℃ bubbled with 95% O2 and 5% CO2. Global ischemia was produced for 30 min by blocking the inflow of Kreb’s Henseleit solution, which was followed by 120 min of reperfusion. Coronary effluent was collected before ischemia, immediately, 5 min, and 30 min after reperfusion for estimation of lactate dehydrogenase (LDH), creatine kinase (CK-MB), and nitrite release (Skrzypiec-Spring, Grotthus, Szela, 2007Skrzypiec-Spring M, Grotthus B, Szela A. Isolated heart perfusion according to langendorff-still viable in the new millennium. J Pharmacol Toxicol Methods. 2007;55(2):113-126.; Ajmani et al., 2011Ajmani P, Yadav HN, Singh M, Sharma PL. Possible involvement of caveolin in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart. BMC Cardiovasc Disord. 2011;11:43.).

Assessment of myocardial injury

The myocardial injury was assessed by the estimation of lactate dehydrogenase (LDH) and creatinine kinase-MB (CK-MB) in the coronary effluent (Skrzypiec-Spring, Grotthus, Szela, 2007Skrzypiec-Spring M, Grotthus B, Szela A. Isolated heart perfusion according to langendorff-still viable in the new millennium. J Pharmacol Toxicol Methods. 2007;55(2):113-126.; Yadav, Singh, Sharma, 2010Yadav HN, Singh M, Sharma PL. Involvement of GSK-3β in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart. Mol Cell Biochem . 2010;343(1-2):75-81.) by using a commercially available kit (Coral clinical system, Goa, India). Values are expressed in international unit IU per liter (IU/L).

Myocardial infarct size measurement

The heart was removed from Langendorff’s apparatus. Both the auricles and root of the aorta were excised, and ventricles were kept overnight at -4 ℃ temperature. Frozen ventricles were sliced into uniform sections of about 2-3 mm thickness and incubated at 37 ℃ for 30 min in 1% w/v triphenyltetrazolium chloride stain (TTC stain) in 0.2 M Tris-chloride buffer, pH 7.4 (Fishbein, Meerbaum, Rit, 1981Fishbein MC, Meerbaum S, Rit J. Early phase acute myocardial infarct size quantification: validation of the triphenyltetrazolium chloride tissue enzyme staining technique. Am Heart J. 1981;101(5):593-600.). The viable cells were stained brick red due to the conversion of TTC to red formazone pigment by NADH and dehydrogenase enzyme (Nachlas, Schnitka, 1963Nachlas M and Schnitka C. Macroscopic identification of early myocardial infarcts by alterations in dehydrogenase activity. Am J Pathol. 1963;42(4):379-406.). While the infarcted cells have lost the enzyme and co-factor and thus remained dull yellow or unstained. Infarct size was measured macroscopically and expressed as a percentage of average infracted ventricular volume (Klein, Pushman, Schaper, 1981Klein HH, Pushman S, Schaper J. The mechanism of the tetrazolium reaction in identifying experimental infarction. Eur Soc Pathol, Virchows Archiv. 1981;393(3):287-297.; Chopra, Singh, Kaul, 1992Chopra K, Singh M, Kaul N. Decrease of myocardial infarct size with desferroxamine: possible role of oxygen free radicals in its ameliorative effect. Mol Cell Biochem. 1992;13(1):71-76.).

Nitrite Estimation

Unlike NO, nitrite can be measured easily, and nitrite concentrations can be used to infer levels of NO production (Marletta, Yoon, Iyenger, 1988Marletta MA, Yoon PS, Iyenger R. Macrophageoxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry. 1988;27(24):8706-8711.). Nitrite release in coronary effluent was measured (Szabo, Thiemermann, Vane, 1993Szabo C, Thiemermann C, and Vane JR. Dihydropyridine modulators of calcium channel inhibit the induction of nitric oxide synthase by endotoxin in cultured J774.2 cells. Biochem Biophys Res Commun. 1993;196(2):825-830. ; Szabo, Wu, Mitchell, 1993Szabo C, Wu CC, Mitchell JA. Platelet activating factor contributes to the induction of nitric oxide synthase by bacterial lipopolysaccharide. Circ Res . 1993;73(6):991-999.). Greiss reagent 0.5 ml (1:1 solution of 1% sulphanilamide in 5% phosphoric acid and 0.1% N-(1-Naphthyl) ethylenediamine dihydrochloride in water) was added to 0.5 ml of coronary effluent. The optical density at 550 nm was measured using a spectrophotometer. Nitrite concentration was calculated by comparison with spectrophotometer readings of the standard solution of sodium nitrite prepared in K-H buffer (Ajmani et al., 2011Ajmani P, Yadav HN, Singh M, Sharma PL. Possible involvement of caveolin in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart. BMC Cardiovasc Disord. 2011;11:43.). Results were expressed as micromoles per litre (µM/L).

Experimental Protocol

The present study was conducted on seven groups, and each group comprised of six rats. The diagrammatic representation of the experimental protocol is shown in Figure 1.

FIGURE 1
Diagrammatic representation of experimental protocol. S, P, I, R, DIZE, Cap denote stabilization, perfusion, ischemia, reperfusion, diminazene aceturate, captopril.

Group I - (Sham control; n=6): Isolated rat heart preparation was stabilized for 10 min and then perfused continuously with K-H buffer solution for 190 min without subjecting them to global ischemia.

Group II - (Ischemia/reperfusion Control; n=6): Isolated rat heart preparation was allowed to stabilize for 10 min then it was subjected to 30 min global ischemia followed by 120 min of reperfusion.

Group III - (Ischemic preconditioning control; n=6): Isolated rat heart preparation was allowed to stabilize for 10 min and subjected to four cycles of ischemic preconditioning, each cycle comprised of 5 min global ischemia followed by 5 min reperfusion with K-H buffer solution. Then the preparation was subjected to 30 min global ischemia followed by 120 min of reperfusion.

Group IV - (Ischemic preconditioning in ovariectomized rat heart; n=6): Isolated heart preparation from ovariectomized rat was allowed to stabilize for 10 min and subjected to four cycles of ischemic preconditioning as described earlier in group III.

Group V - (Ischemic preconditioning in DIZE (26.35 mg/L) perfused ovariectomized rat heart; n=6): Isolated rat heart preparation was allowed to stabilize for 10 min and perfused with DIZE (26.35 mg/L) in each cycle of 5 min reperfusion. The rest of the protocol was the same as described in group III.

Group VI - (Ischemic preconditioning in Captopril (100μM/L) perfused ovariectomized rat heart; n=6): Isolated rat heart preparation was allowed to stabilize for 10 min and perfused with captopril (100μM/L) in each cycle of 5 min reperfusion. The rest of the protocol was the same as described in group III.

Group VII - (Ischemic preconditioning in DIZE (26.35 mg/L) and Captopril (100μM/L) perfused ovariectomized rat heart; n=6): Isolated rat heart preparation was allowed to stabilize for 10 min and perfused with DIZE (26.35 mg/L) and captopril (100μM/L) in each cycle of 5 min reperfusion. The rest of the protocol was the same as described in group III.

Statistical Analysis

All values were expressed as mean ± S.D (standard deviation). Statistical analysis was performed using Sigmastat Software. The data obtained from the various groups were statistically analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. p-value of less than 0.05 was considered to be statistically significant.

RESULTS

Effect of IPC and pharmacological intervention on myocardial infarct size

Global ischemia for 30 min, followed by reperfusion of 120 min significantly increased the myocardial infarct size as compared to sham control. Four cycles of 5 min ischemia and 5 min reperfusion (IPC) were sufficient to markedly prevent I/R induced increase in myocardial infarct size in normal rat heart but not in ovariectomized rat heart. However, perfusion of DIZE (ACE-2 Activator) (26.35mg/L) and Captopril (ACE Inhibitor) (100µM/L) alone or in combination in each cycle of 5 min of reperfusion significantly restored the IPC induced decrease in myocardial infarct size in ovariectomized rat heart (Figure 2).

FIGURE 2
Myocardial infarct size with Images of the TTC-stained sections of the heart (1-7); Effect of I/R on myocardial infarct size, the effect of ischemic preconditioning (IPC) on myocardial infarct size in normal and OVX rat heart, the effect of DIZE perfusion on myocardial infarct size in ovariectomized rat heart and effect of Cap. alone or in combination with DIZE on myocardial infarct size in OVX rat heart. I/R denotes ischemia-reperfusion; IPC denotes ischemic preconditioning; OVX denotes ovariectomy; DIZE denotes diminazene aceturate; Cap. denotes captopril. Values are expressed as mean ± S.D, a = p< 0.05 vs. Sham control; b = p< 0.05 vs. I/R control; c = P< 0.05 vs. IPC in normal rat heart; d = p< 0.05 vs. IPC in OVX rat heart.

Effect of IPC and pharmacological intervention on the release of LDH and CK-MB

Global ischemia for 30 min, followed by reperfusion of 120 min significantly increased the release of LDH and CK-MB as compared to sham control. Four cycles of 5 min ischemia and 5 min reperfusion were sufficient to markedly prevent I/R induced increase in the release of LDH and CK-MB in normal rat heart but not in ovariectomized rat heart. However, perfusion of DIZE (ACE-2 Activator) (26.35mg/L) and Captopril (ACE Inhibitor) (100µM/L) alone or in combination in each cycle of 5 min of reperfusion significantly restored the IPC induced decrease in the release of LDH and CK-MB in ovariectomized rat heart (Figure 3, 4).

FIGURE 3
Effect of I/R on the release of LDH, effect of ischemic preconditioning (IPC) on the release of LDH in normal and OVX rat heart, effect of DIZE perfusion on the release of LDH in ovariectomized rat heart and effect of Cap. alone or in combination with DIZE on the release of LDH in OVX rat heart. I/R denotes ischemia reperfusion; IPC denotes ischemic preconditioning; OVX denotes ovariectomy; DIZE denotes diminazene aceturate; Cap. denotes captopril. Values are expressed as mean ± S.D, a = p< 0.05 vs. Sham control; b = p< 0.05 vs. I/R control; c = P< 0.05 vs. IPC in normal rat heart; d = p< 0.05 vs. IPC in OVX rat heart.

FIGURE 4
Effect of I/R on the release of CK-MB, effect of ischemic preconditioning (IPC) on the release of CK-MB in normal and OVX rat heart, effect of DIZE perfusion on the release of CK-MB in ovariectomized rat heart and effect of Cap. alone or in combination with DIZE on the release of CK-MB in OVX rat heart. I/R denotes ischemia reperfusion; IPC denotes ischemic preconditioning; OVX denotes ovariectomy; DIZE denotes diminazene aceturate; Cap. denotes captopril. Values are expressed as mean ± S.D, a = p< 0.05 vs. Sham control; b = p< 0.05 vs. I/R control; c = P< 0.05 vs. IPC in normal rat heart; d = p< 0.05 vs. IPC in OVX rat heart.

Effect of IPC and pharmacological intervention on the release of nitrite

The release of nitrite in coronary effluent was noted to be significantly reduced in ovariectomized rat heart when compared to normal rat heart. Perfusion of DIZE (ACE-2 Activator) (26.35mg/L) and Captopril (ACE Inhibitor) (100µM/L) alone or in combination in each cycle of 5 min of reperfusion significantly increased the release of nitrite in ovariectomized rat heart when compared to untreated ovariectomized rat heart (Figure 5).

FIGURE 5
Effect of I/R on the release of nitrite, effect of ischemic preconditioning (IPC) on the release of nitrite in normal and OVX rat heart, effect of DIZE perfusion on the release of nitrite in ovariectomized rat heart and effect of Cap. alone or in combination with DIZE on the release of nitrite in OVX rat heart. I/R denotes ischemia reperfusion; IPC denotes ischemic preconditioning; OVX denotes ovariectomy; DIZE denotes diminazene aceturate; Cap. denotes captopril. Values are expressed as mean ± S.D, a = p< 0.05 vs. Sham control; b = p< 0.05 vs. I/R control; c = P< 0.05 vs. IPC in normal rat heart; d = p< 0.05 vs. IPC in OVX rat heart.

DISCUSSION

The extent of release of LDH and CK-MB is directly correlated with the degree of damage of myocardium during ischemia and reperfusion (I/R) (Wang, Cherednichenko, Hernandez, 2001Wang L, Cherednichenko G, Hernandez L. Preconditioning limits mitochondrial Ca 2+ during ischemia in rat hearts: Role of KATP channels. Am J Physiol Heart Circ Physiol. 2001;280(5):2321-2328.). An increase in myocardial infarct size elevated levels of LDH and CK-MB and decreased level of NO in coronary effluent are the indicators of ischemia/reperfusion-induced myocardial injury (Yadav, Singh, Sharma, 2010Yadav HN, Singh M, Sharma PL. Involvement of GSK-3β in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart. Mol Cell Biochem . 2010;343(1-2):75-81.; Kaur, Parikh, Sharma, 1997Kaur H, Parikh V, Sharma A. Effect of amiloride a Na+/H+ exchange inhibitor on cardioprotective effect of ischemic preconditioning: Possible involvement of resident cardiac mast cells. Pharmacol Res . 1997;36(2):95-102.). In the present study, 30 min of global ischemia and 120 min of reperfusion increased the myocardial infarct size, release of LDH and CK-MB, and decreased the release of nitrite in coronary effluent of normal rat heart which is consistent with our earlier finding (Goyal, Semwal, Yadav, 2016Goyal A, Semwal BC, Yadav HN. Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart. Hum Exp Toxicol. 2016;35(6):644-653.; Charan et al., 2016Charan K, Goyal A, Gupta JK, Yadav HN. Role of atrial natriuretic peptide in ischemia preconditioning induced cardioprotection in the diabetic rat heart. J Surg Res. 2016;201(2):272-278. ). However, four episodes of 5 min of ischemia and 5 min of reperfusion were sufficient to significantly attenuate the I/R induced increased myocardial infarct size, the release of LDH and CK-MB and decreased the level of NO in coronary effluent of normal rat heart. This observation is consistent with our previous reports (Goyal, Semwal, Yadav, 2016Goyal A, Semwal BC, Yadav HN. Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart. Hum Exp Toxicol. 2016;35(6):644-653.).

The HRAS is an important system for cardiac functions, and it exerts many actions through its components to regulate cardiac physiology (David, Kenneth, 1999David ED, Kenneth MB. The cardiac renin-angiotensin system conceptual, or a regulator of cardiac function? Circ Res. 1999;85(7):643-650. ). It has been documented that ACE and ACE-2 both enzymes maintain the homeostasis in the cardiovascular system and further it has been well suggested that an imbalance between ACE and ACE-2 for a longer duration cause several cardiovascular complications (Ferreira, Santos, Almeida, 2001Ferreira AJ, Santos RA, Almeida AP. Angiotensin-(1-7): cardioprotective effect in myocardial ischemic/reperfusion. Hypertension. 2001;38(3 Pt 2):665-668.; Sakima, Averill, Gallagher, 2005Sakima A, Averill DB, Gallagher PE. Impaired heart rate baroreflex in older rats: role of endogenous angiotensin-(1-7) at the nucleus tractus solitarii. Hypertension. 2005;46(2):333-340. ; Diz, Garcia-Espinosa, Gallagher, 2008Diz DI, Garcia-Espinosa MA, Gallagher PE. Angiotensin-(1-7) and baroreflex function in nucleus tractus solitarii of (mRen2)27 transgenic rats. J Cardiovasc Pharmacol. 2008;51(6):542-548. ). An increase in ACE activity and decrease in ACE-2 activity exert deleterious effects on cardiovascular function in ischemia/reperfusion challenged heart (Degraeff et al., 1988Degraeff PA, Delangen CDJ, Van Gikst WH, Bel K. Protective effect of captopril against ischemia/reperfusion- induced ventricular arrhythmias in vitro and in vivo. Am J Med. 1988;84(3):67-74.; Qi et al., 2013Qi YF, Zhang J, Cole-Jeffrey CT, Shenoy V, Espejo A, Hanna M, et al. Diminazene aceturate enhances angiotensin-converting enzyme 2 activity and attenuates ischemia-induced cardiac pathophysiology. Hypertension. 2013;62(4):746-752.). A galaxy of experimental studies reported the cardioprotective activity of ACE inhibitors and ACE-2 activators against I/R injury (Degraeff et al., 1988Degraeff PA, Delangen CDJ, Van Gikst WH, Bel K. Protective effect of captopril against ischemia/reperfusion- induced ventricular arrhythmias in vitro and in vivo. Am J Med. 1988;84(3):67-74.; Pfeffer, Braunwald, Moy, 1992Pfeffer MA, Braunwald E, Moy LA. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction: results of the survival and ventricular enlargement study. N Engl J Med. 1992;327(10):669-677.; Qi et al., 2013Qi YF, Zhang J, Cole-Jeffrey CT, Shenoy V, Espejo A, Hanna M, et al. Diminazene aceturate enhances angiotensin-converting enzyme 2 activity and attenuates ischemia-induced cardiac pathophysiology. Hypertension. 2013;62(4):746-752.). This contention is supported by the fact that I/R injury after regional or global ischemia involves damage to the cardiomyocytes, vascular smooth muscles, and endothelial cells and the administration of ACE inhibitors and ACE-2 activators protect the myocardium from I/R injury and limit the infarct size of the myocardium (Pfeffer, Braunwald, Moy, 1992Pfeffer MA, Braunwald E, Moy LA. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction: results of the survival and ventricular enlargement study. N Engl J Med. 1992;327(10):669-677.; Martinez, Molina, 2003Martinez LA, Molina RV. Early and chronic captopril or losarten therapy reduce infarct size and avoids congestive heart failure after myocardial infarction in rats. Arch Med Res. 2003;34(5):357-361. ; Qi et al., 2013Qi YF, Zhang J, Cole-Jeffrey CT, Shenoy V, Espejo A, Hanna M, et al. Diminazene aceturate enhances angiotensin-converting enzyme 2 activity and attenuates ischemia-induced cardiac pathophysiology. Hypertension. 2013;62(4):746-752.). Further, it has been reported that ACE inhibitors and ACE-2 activators increase the release of NO and limit the infarct size (Zhang et al., 1997Zhang X, Xie YW, Nasjletti A, Bastianon D. ACE inhibitor promote nitric oxide accumulation to modulate myocardial oxygen consumption. Circulation. 1997;95(1):176-182.; Fraga-Silva et al., 2015Fraga-Silva RA., Costa-Fraga FP, Montecucco F, Sturny M, Faye Y, Mach F, et al. Diminazene protect corpus cavernosum against hypercholesterolemia-induced injury. J Sex Med. 2015;12(2):289-302.). The activity of ACE is upregulated while the activity of ACE-2 is downregulated in estrogen deficiency which further decreases the level of nitric oxide (Lindsey et al., 2009Lindsey SH, Cohen JA, Brosnihan KB, Gallagher PE, Chappell MC. Chronic treatment with the G protein-coupled receptor 30 agonist G-1 decreases blood pressure in ovariectomized mRen2.Lewis rats. Endocrinology. 2009;150(8):3753-3758.; Pereira, Bertolami, Faludi, 2013Pereira IRO, Bertolami MC, and Faludi AA. Lipid peroxidation and nitric oxide inactivation in postmenopausal woman. Arq Bras Cardiol. 2013;80(4):406-423.).

In our study, IPC induced cardioprotection and the release of nitrite in OVX rats was significantly reduced as compared to normal rat heart, which is supported by the finding of our laboratory (Goyal, Semwal, Yadav, 2016Goyal A, Semwal BC, Yadav HN. Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart. Hum Exp Toxicol. 2016;35(6):644-653.). This may be due to increased activity of ACE and decreased activity of ACE-2.

Captopril, an ACE inhibitor has been noted to inhibit the ACE activity (Brown, aughan, 1998Brown NJ, Vaughan DE. Angiotensin-converting enzyme inhibitors. Circulation. 1998;97(14):1411-1420.) and facilitates the IPC induced release of NO (Zhang et al., 1997Zhang X, Xie YW, Nasjletti A, Bastianon D. ACE inhibitor promote nitric oxide accumulation to modulate myocardial oxygen consumption. Circulation. 1997;95(1):176-182.; Tian et al., 2015Tian Y, Li H, Liu P and Irwin MG. Captopril pretreatment produce an additive cardioprotective to isoflurane preconditioning in attenuating myocardial ischemic reperfusion injury in rabbits and in humans. Mediators Inflammation. 2015;1-12.). In the present study, perfusion of captopril restored the cardioprotective effect of IPC and increased the release of NO in the OVX rat heart. Further, the perfusion of diminazene aceturate (DIZE), an ACE-2 activator also decreased the myocardial infarct size, release of LDH and CK-MB in the coronary effluent of OVX rat heart as compared to untreated OVX rat heart. These findings also support that the infarct size-limiting effect of captopril and DIZE is mediated through NO. Furthermore, co-perfusion of captopril and DIZE was unable to produce any significant cardioprotective effect of IPC as compared to individual perfusion of both the drugs in OVX rat heart noted in terms of infarct size, the release of LDH, CK-MB, and nitrite in coronary effluent.

Our data indicate that individual treatment of captopril and DIZE with IPC protect the myocardium from estrogen deficiency-induced injury. So, the treatment of hypoestrogenism related patients undergoing cardiopulmonary bypass with captopril or DIZE could be a beneficial adjunctive for myocardial protection during open heart surgery.

CONCLUSION

On the basis of the above discussion, it is concluded that the perfusion of ACE inhibitor and ACE-2 activator restore the attenuated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart. This observed cardioprotective effect is due to the decreased ACE activity or increased ACE-2 activity.

ACKNOWLEDGMENT

We are grateful to Shri. Narayan Das Agrawal Ji, Chancellor, GLA University, Prof. D.S. Chauhan, Vice-Chancellor, GLA University, Prof. Pradeep Mishra, Director, Institute of Pharmaceutical Research, GLA University, India for their praiseworthy inspiration and constant support for this study.

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  • FINANCIAL/SOURCE OF FUNDS STATEMENT

    There are no funding sources.

Publication Dates

  • Publication in this collection
    08 July 2022
  • Date of issue
    2022

History

  • Received
    18 Apr 2019
  • Accepted
    27 Aug 2019
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br