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Abstract

Background:

Ischemic heart disease is the leading cause of death in postmenopausal women. The expression of caveolin, a membrane protein and a negative regulator of nitric oxide (NO), increases after menopause. The present study was designed to determine the effect of daidzein (DDZ), a phytoestrogen in attenuated cardioprotective effect of ischemic preconditioning (IPC) in ovariectomized rat heart.

Methods:

Heart was isolated from ovariectomized rat and mounted on Langendorff’s apparatus, subjected to 30 min ischemia and 120 min reperfusion. IPC was mediated by four cycles of 5 min ischemia and 5 min reperfusion. The infarct size was estimated using triphenyltetrazolium chloride stain, and coronary effluent was analyzed for lactate dehydrogenase and creatine kinase MB (CK-MB) release to assess the degree of myocardial injury. The release of NO was estimated indirectly by measuring the release of nitrite in coronary effluent.

Results:

IPC-induced cardioprotection was significantly attenuated in ovariectomized rats as compared to normal rats, which was restored by treatment of DDZ, a caveolin inhibitor (0.2 mg/kg subcutaneously) for 1 week. However, this observed cardioprotection was significantly attenuated by perfusion of l-nitroarginine methyl ester, an endothelial nitric oxide synthase (eNOS) inhibitor (100 µM/L) and glibenclamide, an adenosine triphosphate-sensitive potassium ion channel blocker (10 µM/L) alone or in combination, noted in terms of increase in myocardial infarct size, release of LDH and CK-MB, and also decrease in the release of NO.

Conclusion:

Thus, it is suggested that DDZ restores the attenuated cardioprotective effect in ovariectomized rat heart, which may be due to downregulation of caveolin and subsequent increase in the activity of eNOS.

Keywords

Ovariectomy daidzein nitric oxide caveolin ischemic preconditioning

Introduction

In the reproductive years, men are more susceptible to the risk of cardiovascular diseases than women.¹ However after menopause, the risk of cardiovascular disease in women reaches to the same level as in men of same age.^1,2 Ischemic heart diseases have been remarked as a major cause of morbidity and mortality.³ Reperfusion of an ischemic heart is necessary to regain the normal functioning of heart.⁴ However, abrupt reperfusion of an ischemic heart elicits a cascade of adverse events that lead to injury of myocardium, that is, ischemia–reperfusion (I/R) injury.^5,6 The endogenous powerful strategy to protect the ischemic heart is ischemic preconditioning (IPC), in which the myocardium is subjected to short periods of sublethal I/R before the prolonged ischemic insult.⁷ The IPC induces cardioprotection by activation of phosphoinositide-3-kinase/Akt pathway,^8,9 generation of nitric oxide (NO), and opening of mitochondrial adenosine triphosphate-sensitive potassium ion (K_ATP) channels.^10,11 However, the cardioprotective effect of IPC gets attenuated in certain pathological conditions such as hypertension,^12,13 diabetes,^14,15 hyperlipidemia,¹⁶ aging,¹⁷ and heart failure.¹⁸

Caveolae are the morphologically distinct plasma membrane of endothelial cells.¹⁹ Caveolin, the caveolar membrane protein is a negative regulator of endothelial nitric oxide synthase (eNOS), as its interaction and binding suppress the activity of eNOS by making caveolin–eNOS complex.^20
–22 Caveolin maintains eNOS in inactivated state, which leads to decrease in NO production.^23
–25 IPC produces cardioprotection by disrupting the caveolin–eNOS complex and subsequent activation of eNOS.²² Moreover, it has been documented that during IPC, NO produces cardioprotection by opening K_ATP channel, and at the same time, caveolin facilitates the interaction of NO with K_ATP channel by forming a suitable signaling platform.²⁶

Ovariectomy has been reported to upregulate caveolin 1 expression.^27,28 Estrogen upregulates eNOS and downregulates its inhibitory protein caveolin 1.^29,30 Moreover, protein kinase B (Akt) can be also activated by estrogen, which further activates eNOS by phosphorylating it at serine 1177 residue.^31
–33 This phosphorylation not only activates eNOS but also increases the efficiency of activation by calcium ion/calmodulin.³⁴ Thus, estrogen increases bioavailability of NO and decreases myocardial injury. Therefore, the present study has been designed to investigate the role of daidzein (DDZ), a caveolin inhibitor in modulation of cardioprotective effect of IPC in ovariectomized rat heart.

Materials and methods

For the purpose of this study, female Wistar rats weighing about 180–250 g were used. The rats were housed in animal cages and provided with 12-h light/12-h dark cycle. They were fed on 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/2013/P.Col) in accordance with the national guidelines on the use of laboratory animals.

Drugs and chemicals

DDZ (0.2 mg/kg/day, subcutaneously (s.c.); Sigma Aldrich [P] Ltd, Bangalore, Karnataka, India) was used as caveolin inhibitor. l-Nitroarginine methyl ester (l-NAME); 100 µM/L, Sigma Aldrich [P] Ltd) and glibenclamide (10 µM/L; Medirose Drugs and Pharmaceuticals Pvt. Ltd, Ghaziabad, Uttar Pradesh, India) were added to Krebs–Henseleit (K-H) buffer for perfusion. All other reagents used in this study were of analytical grade and always freshly prepared before use.

Induction of experimental ovariectomy

Rats were divided into nine groups with six rats in each group. The rats were anesthetized with pentobarbitone (45 mg/kg intraperitoneally (i.p.)). Ovariectomy was performed by making peritoneal incision of 0.4–0.6 cm in the middle part of the abdomen slightly toward right. Ovary and associated fat were easily located and exteriorized by gentle retraction. Ovaries along with uterus were pulled out, and suture was applied at the end of uterus and beginning of ovary. Ovaries were removed and the uterus was pushed back, and incisions were sutured in layers. Neomycin antibiotic powder was applied twice daily on wounds for 1 week, and animals were allowed to recover for 4 weeks.

Isolated rat heart preparation

Rats were administered heparin (500 IU/L, i.p.; Gland Pharma Ltd, Hyderabad, Telangana, India) 20 min prior to killing by cervical dislocation. Heart was rapidly excised and was immediately mounted on Langendorff’s apparatus.³⁵ The heart was enclosed in double-walled jacket, and temperature was maintained at 37°C by circulating warm water. Isolated heart was retrogradely perfused at constant pressure of 80 mmHg and coronary flow rate of 7–9 mL/min with K-H buffer (sodium chloride, 118 mM; potassium chloride, 4.7 mM; calcium chloride, 2.5 mM; magnesium sulfate hexahydrate, 1.2 mM; potassium dihydrogen phosphate, 1.2 mM; and glucose, 11 mM), pH 7.4, maintained at 37°C, bubbled with 95% oxygen and 5% carbon dioxide. Global ischemia was produced for 30 min by blocking the inflow of K-H solution, followed by 120 min of reperfusion. Coronary effluent was collected before ischemia, immediately, 5 min after reperfusion, and 30 min after reperfusion for the estimation of lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), and nitrite release.^15,36

Assessment of myocardial injury

The myocardial infarction was assessed by the estimation of LDH and CK-MB in the coronary effluent and measurement of infarct size, which determines the extent of myocardial injury. The assessment of myocardial infarct size was done using triphenyltetrazolium chloride (TTC) staining method, while LDH and CK-MB were estimated using commercial kits.^14,15 Values of LDH and CK-MB were expressed in international units per liter.

Estimation of LDH and CK-MB

LDH was estimated in samples of coronary effluent collected before stabilization, immediately, and 30 min after reperfusion using commercially available kit (Span Diagnostics Ltd., Surat, Gujarat, India) spectrophotometrically at 340 nm, while CK-MB release was estimated before stabilization, immediately, and 5 min after reperfusion using commercially available kit (Coral Clinical Systems, Goa, India) spectrophotometrically at 340 nm.

Myocardial infarct size measurement

Heart was removed from the Langendorff’s apparatus. Both the auricles and root of aorta were excised and ventricles were kept overnight at −4°C temperature. Frozen ventricles were sliced into uniform sections of about 2–3 mm thickness and incubated at 37°C for 30 min in 1% w/v TTC stain in 0.2 M Tris–chloride buffer, pH 7.4.³⁷ The viable cells were stained brick red due to the conversion of TTC to red formazone pigment by nicotinamide adenine dinucleotide reduced and dehydrogenase enzyme.³⁸ The infarcted cells lost the enzyme and cofactor and thus remained dull yellow or unstained. Infarct size was measured macroscopically and expressed as percentage of average infarcted ventricular volume.^39,40

Nitrite estimation

Unlike NO, nitrite can be measured easily, and nitrite concentrations can be used to infer the levels of NO production.⁴¹ Nitrite release in coronary effluent was measured.^42,43 Greiss reagent (0.5 mL, 1:1 solution of 1% sulfanilamide 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 spectrophotometer. Nitrite concentration was calculated by comparing the spectrophotometer readings of standard solution of sodium nitrite prepared in K-H buffer.¹⁵ Results were expressed as micromoles per liter.

Experimental protocol

The present study was conducted on nine groups, with six rats in each group. The diagrammatic representation of experimental protocol is shown in Figure 1.

Figure 1.

Diagrammatic representation of experimental protocol. S: stabilization; P: perfusion; I: ischemia; R: reperfusion.

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

Group IV: (IPC 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 IPC as described earlier in group III.

Group V: (IPC in dimethyl sulfoxide (DMSO; vehicle)-treated ovariectomized rat heart; n = 6): Isolated rat heart preparation from DMSO-treated ovariectomized rat was allowed to stabilize for 10 min and then subjected to four cycles of IPC as described earlier in group III.

Group VI: (IPC in DDZ-pretreated ovariectomized rat heart; n = 6): Isolated rat heart preparation from DDZ (0.2 mg/kg/day, s.c. in DMSO, 1 week)-treated ovariectomized rat was allowed to stabilize for 10 min and then subjected to four cycles of IPC as described earlier in group III.

Group VII: (IPC in DDZ-pretreated, l-NAME-perfused ovariectomized rat heart; n = 6): Isolated rat heart preparation from DDZ (0.2 mg/kg/day, s.c. in DMSO, 1 week)-treated ovariectomized rat was allowed to stabilize for 10 min and perfused with l-NAME (100 µM/L K-H buffer) for 30 min and subjected to four cycles of IPC as described earlier in group III.

Group VIII: (IPC in DDZ-pretreated, glibenclamide-perfused ovariectomized rat heart; n = 6): Isolated rat heart preparation from DDZ (0.2 mg/kg/day, s.c. in DMSO, 1 week)-treated ovariectomized rat was allowed to stabilize for 10 min and perfused with glibenclamide (10 µM/L K-H buffer) for 30 min and subjected to four cycles of IPC as described earlier in group III.

Group IX: (IPC in DDZ-pretreated glibenclamide- and l-NAME-perfused ovariectomized rat heart; n = 6): Isolated rat heart preparation from DDZ (0.2 mg/kg/day, s.c. in DMSO, 1 week)-treated ovariectomized rat was allowed to stabilize for 10 min and perfused with glibenclamide (10 µM/L K-H buffer) and l-NAME (100 µM/L K-H buffer) for 30 min and subjected to four cycles of IPC as described earlier in group III.

Statistical analysis

All values were expressed as mean ± standard deviation. Statistical analysis was performed using Sigmastat software (Version-Sigmastat 3.5; Aspire software international, California, USA). The data obtained from various groups were statistically analyzed using one-way analysis of variance (ANOVA) and two-way ANOVA, followed by Tukey’s multiple comparison test. The values of p < 0.05 was considered to be statistically significant.

Results

Effect of IPC and pharmacological interventions on myocardial injury

Global ischemia of 30 min, followed by reperfusion of 120 min significantly increased the myocardial infarct size and the release of LDH and CK-MB in coronary effluents as compared to the sham control group. Four cycles of 5 min ischemia and 5 min reperfusion (IPC) were sufficient to markedly prevent the ischemia reperfusion-induced increase in infarct size and the release of LDH and CK-MB in normal rat heart but not in ovariectomized rat heart. However, DDZ pretreatment significantly restored the IPC-induced reduction of myocardial injury in ovariectomized rat heart. Perfusion of l-NAME (100 µM/L) or glibenclamide (10 µM/L) for 30 min in DDZ-pretreated ovariectomized rat heart significantly abolished the restoration of DDZ pretreatment-induced decrease of infarct size and the release of LDH and CK-MB. However, the perfusion of glibenclamide (10 µM/L) with l-NAME (100 µM/L) for 30 min in DDZ-pretreated ovariectomized rat heart was unable to produce any additional effect on infarct size and the release of LDH and CK-MB (Figures 2 to 4).

Figure 2.

Effect of I/R on myocardial infarct size, effect of IPCon myocardial infarct size in normal and ovariectomized rat heart, effect of DDZ pretreatment, effect of l-NAME perfusion in DDZ-pretreated rats, effect of glibenclamide perfusion in DDZ-pretreated rats, effect of l-NAME and glibenclamide perfusion in DDZ-pretreated rats on myocardial infarct size in ovariectomized rat heart. Values are expressed as mean ± SD. ^a p < 0.05: versus sham control; ^b p < 0.05: versus I/R control; ^c p < 0.05: versus IPC in normal rat heart; ^d p < 0.05: versus IPC in OVX rat heart; ^e p < 0.05: versus IPC in OVX rat heart with pretreated DDZ. I/R: ischemia–reperfusion; IPC: ischemic preconditioning; DDZ: daidzein; l-NAME: N-nitro-l-arginine methyl ester; OVX: ovariectomy.

Figure 3.

Effect of I/R on the release of LDH, effect of IPC on the release of LDH in normal and ovariectomized rat heart, effect of DDZ pretreatment, effect of l-NAME perfusion in DDZ-pretreated rats, effect of glibenclamide perfusion in DDZ-pretreated rats, effect of l-NAME and glibenclamide perfusion in DDZ-pretreated rats on the release of LDH in ovariectomized rat heart. Values are expressed as mean ± SD. ^a p < 0.05: versus sham control; ^b p < 0.05: versus I/R control; ^c p < 0.05: versus IPC in normal rat heart; ^d p < 0.05: versus IPC in OVX rat heart; ^e p < 0.05: versus IPC in OVX rat heart with pretreated DDZ. I/R: ischemia–reperfusion; IPC: ischemic preconditioning; DDZ: daidzein; l-NAME: N-nitro-l-arginine methyl ester; OVX: ovariectomy; LDH: lactate dehydrogenase.

Figure 4.

Effect of I/R on the release of CK-MB, effect of IPC on the release of CK-MB in normal and ovariectomized rat heart, effect of DDZ pretreatment, effect of l-NAME perfusion in DDZ-pretreated rats, effect of glibenclamide perfusion in DDZ-pretreated rats, effect of l-NAME and glibenclamide perfusion in DDZ-pretreated rats on the release of CK-MB in ovariectomized rat heart. Values are expressed as mean ± SD. ^a p < 0.05: versus sham control; ^b p < 0.05: versus I/R control; ^c p < 0.05: versus IPC in normal rat heart; ^d p < 0.05: versus IPC in OVX rat heart; ^e p < 0.05: versus IPC in OVX rat heart with pretreated DDZ. I/R: ischemia–reperfusion; IPC: ischemic preconditioning; DDZ: daidzein; l-NAME: N-nitro-l-arginine methyl ester; OVX: ovariectomy; CK-MB: creatine kinase MB.

Effect of IPC and pharmacological interventions on the release of nitrite

The release of nitrite in coronary effluent was noted to be significantly reduced in ovariectomized rat heart when compared with the normal rat heart. Treatment with DDZ, a caveolin inhibitor (0.2 mg/kg/day s.c., 1 week) significantly increased the release of nitrite in coronary effluent of ovariectomized rat heart when compared with untreated ovareictomized rat heart. Moreover, DDZ-induced restoration of the release of nitrite in coronary effluent of ovariectomized rat heart was significantly suppressed by perfusion of l-NAME. However, DDZ-induced release of nitrite in coronary effluent of glibenclamide-perfused ovariectomized rat heart was not affected. Perfusion of glibenclamide (10 µM/L) with l-NAME (100 µM/L) in combination was unable to further increase the release of nitrite in coronary effluent of DDZ-pretreated ovariectomized rat heart (Figure 5).

Figure 5.

Effect of I/R on the release of NO, effect of IPC on the release of NO in normal and ovariectomized rat heart, effect of DDZ pretreatment, effect of l-NAME perfusion in DDZ-pretreated rats, effect of glibenclamide perfusion in DDZ-pretreated rats, effect of l-NAME and glibenclamide perfusion in DDZ-pretreated rats on the release of NO in ovariectomized rat heart. Values are expressed as mean ± SD. ^a p < 0.05: versus sham control; ^b p < 0.05: versus I/R control; ^c p < 0.05: versus IPC in normal rat heart; ^d p < 0.05: versus IPC in OVX rat heart; ^e p < 0.05: versus IPC in OVX rat heart with pretreated DDZ. I/R: ischemia–reperfusion; IPC: ischemic preconditioning; DDZ: daidzein; l-NAME: N-nitro-l-arginine methyl ester; OVX: ovariectomy; NO: nitric oxide.

Discussion

Myocardial injury was noted in terms of increased myocardial infarct size, release of LDH and CK-MB, and decreased release of nitrite in coronary effluent. LDH and CK-MB are good indicators of myocardial injury and nitrite, a potent vasodilator of stable nitrogen intermediate formed from the spontaneous degradation of NO.

In our 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. Moreover, four cycles of 5 min of ischemia and 5 min of reperfusion, preceding 30 min of ischemia and 120 min of reperfusion, were sufficient to significantly attenuate the I/R-induced increased myocardial infarct size, release of LDH and CK-MB, and decreased release of nitrite in coronary effluent of normal rat heart. These results are in accordance with earlier published reports from our laboratory.^14
–16

Caveolae are 50–100 nm plasma membrane invaginations on the surface of endothelial cells, which form lipid raft with caveolin proteins.¹⁹ These proteins act as signaling platform^44
–46 for G-protein-coupled receptor and other molecules such as Src-like kinases and NOS.^47
–49 IPC can modulate microenvironment of caveolin and promotes the signaling involved in the protection of myocardium against I/R-induced injury.²² It has been reported that expression of caveolin is unregulated in ovariectomized rat heart.²⁷ Increased expression of caveolin leads to increased interaction with eNOS, it maintains eNOS in inactive state by making caveolin–eNOS complex and subsequently decreases the generation of NO.^25,50 In our study, IPC-induced cardioprotection and the release of nitrite in ovariectomized rats was significantly reduced as compared to normal rat heart, which is supported by the findings of different laboratories.⁵¹ It may be due to increased expression of caveolin and subsequent decrease in eNOS activity.

DDZ, a phytoestrogen, has been noted to decrease the caveolin expression^52,53 and facilitate the IPC-induced release of NO by decreasing its binding with eNOS.^15,28 In the present study, 1 week treatment of ovariectomized rats with DDZ significantly restored the cardioprotective effect of IPC and increased the release of NO in the ovariectomized rat heart. Perfusion of DDZ-pretreated ovariectomized rat heart with l-NAME, an NOS inhibitor, restricted the DDZ-induced increase in release of NO, decrease in myocardial infarct size, and release of LDH and CK-MB in the coronary effluent of ovariectomized rat heart, which indicates that some defect in caveolin–eNOS complex may be involved in this process as indicated in our study by a decrease in the release of nitrite in coronary effluent in the ovariectomized rat heart.

Opening of mitochondrial K_ATP channels protects the myocardium from I/R-induced injury.⁵⁴ Various mediators, namely, adenosine, bradykinin, angiotensin, prostaglandins and NO, which are released by the stimuli of IPC, produce cardioprotection by the opening of mitochondrial K_ATP channel.^55,56 Further, perfusion of glibenclamide, a K_ATP channel blocker in DDZ-pretreated ovariectomized rat heart significantly abolished the cardioprotective effect of IPC without affecting the IPC-mediated release of NO. It is suggested that the observed cardioprotective effect of IPC in normal rat and DDZ-pretreated rat may be due to the opening of mitochondrial K_ATP channel. Our results are in accordance with previous reports from our laboratory.¹⁵

Moreover, perfusion of l-NAME with glibenclamide in DDZ-pretreated ovariectomized rat heart was unable to produce any additional effect in comparison with individual drugs. These findings reflect that NO generated due to IPC in DDZ-pretreated ovariectomized rat heart produce cardioprotection by the opening of K_ATP channels.

Conclusion

On the basis of the above discussion, it may be concluded that DDZ restores the attenuated cardioprotective effect of IPC in ovariectomized rat heart, which may be due to downregulation of caveolin that leads to increased availability of NO and consequent increase in the activation of mitochondrial K_ATP channels. This contention is supported by the fact that perfusion of l-NAME, an eNOS inhibitor, and glibenclamide, a K_ATP channel blocker, significantly attenuated the DDZ-induced cardioprotective effect of IPC in ovariectomized rat heart.

Limitation of the present study

Ideally, the proposed caveolin–eNOS interaction should have been assessed by coimmunoprecipitation study or caveolin isolation.

Footnotes

Acknowledgments

We are grateful to Shri. Narayan Das Agrawal Ji, Chancellor, GLA University; Prof. DS Chauhan, Vice Chancellor, GLA University; Prof. Pradeep Mishra, Director, Institute of Pharmaceutical Research, GLA University; and Prof. Anoop Kumar Gupta, Director, Institute of Applied Science and Humanities, GLA University, Mathura, Uttar Pradesh, India, for their praiseworthy inspiration and constant support for this study.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart

Abstract

Background:

Methods:

Results:

Conclusion:

Keywords

Introduction

Materials and methods

Drugs and chemicals

Induction of experimental ovariectomy

Isolated rat heart preparation

Assessment of myocardial injury

Estimation of LDH and CK-MB

Myocardial infarct size measurement

Nitrite estimation

Experimental protocol

Statistical analysis

Results

Effect of IPC and pharmacological interventions on myocardial injury

Effect of IPC and pharmacological interventions on the release of nitrite

Discussion

Conclusion

Limitation of the present study

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Funding

References