Harlokesh Narayan Yadav

Modulation of the cardioprotective effect of ischemic preconditioning in hyperlipidaemic rat heart.

Ischemic preconditioning (IPC) produces cardioprotection by phosphorylation of glycogen synthaes kinase-3beta (GSK-3beta) that inhibits the opening of mitochondrial permeability transition pore (MPTP), and this cardioprotective action of IPC is attenuated by hyperlipidaemia. The present study investigated the role of GSK-3beta in attenuation of cardioprotective effect of IPC, by hyperlipidaemia in the rat heart. Hyperlipidaemia was produced in rat by feeding high fat diet for six weeks. Isolated perfused rat heart was subjected to 30 min of ischemia followed by 120 min of reperfusion. Myocardial infarct size was estimated by triphenyltetrazolium chloride (TTC) staining and lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) was analyzed from coronary effluent. IPC significantly decreased the myocardial infarct size and the release of LDH and CK-MB from normal rat heart. IPC induced myocardial protection was attenuated in hyperlipidaemic rat heart. However, cardioprotective effect of pharmacological preconditioning with GSK-3beta inhibitors i.e. Lithium Chloride (LiCl) (20mM), Indirubin - 3 Monooxime (1 microM) and 3-(2, 4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2, 5-dione (SB216763) (3 microM), was not attenuated. This differential attenuation by hyperlipidaemia, of IPC and pharmacological preconditioning induced cardioprotection is a new finding in our study. GSK-3beta inhibition is reported to increase the threshold of opening for MPTP during reperfusion. Administration of atractyloside (20 microM), an opener of MPTP, significantly attenuated the cardioprotective effect of IPC in normal heart, and pharmacological preconditioning in the hyperlipidaemic rat heart. Thus, the attenuation of cardioprotective effect of IPC in hyperlipidaemic heart may be due to inhibition of protective signaling pathways upstream of GSK-3beta and inhibition of opening of MPTP.

Possible involvement of caveolin in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart.

BackgroundNitric oxide (NO) has been noted to produce ischemic preconditioning (IPC)-mediated cardioprotection. Caveolin is a negative regulator of NO, which inhibits endothelial nitric oxide synthase (eNOS) by making caveolin-eNOS complex. The expression of caveolin is increased during diabetes mellitus (DM). The present study was designed to investigate the involvement of caveolin in attenuation of the cardioprotective effect of IPC during DM in rat.MethodsExperimental DM was induced by single dose of streptozotocin (50 mg/Kg, i.p,) and animals were used for experiments four weeks later. Isolated heart was mounted on Langendorffs apparatus, and was subjected to 30 min of global ischemia and 120 min of reperfusion. IPC was given by four cycles of 5 min of ischemia and 5 min of reperfusion with Krebs-Henseleit solution (K-H). Extent of injury was measured in terms of infarct size by triphenyltetrazolium chloride (TTC) staining, and release of lactate dehydrogenase (LDH) and creatin kinase-MB (CK-MB) in coronary effluent. The cardiac release of NO was noted by measuring the level of nitrite in coronary effluent.ResultsIPC- induced cardioprotection and release of NO was significantly decreased in diabetic rat heart. Pre-treatment of diabetic rat with daidzein (DDZ) a caveolin inhibitor (0.2 mg/Kg/s.c), for one week, significantly increased the release of NO and restored the attenuated cardioprotective effect of IPC. Also perfusion of sodium nitrite (10 μM/L), a precursor of NO, significantly restored the lost effect of IPC, similar to daidzein in diabetic rat. Administration of 5-hydroxy deaconate (5-HD), a mito KATP channel blocker, significantly abolished the observed IPC-induced cardioprotection in normal rat or daidzein and sodium nitrite perfused diabetic rat heart alone or in combination.ConclusionsThus, it is suggested that attenuation of the cardioprotection in diabetic heart may be due to decrease the IPC mediated release of NO in the diabetic myocardium, which may be due to up -regulation of caveolin and subsequently decreased activity of eNOS.

Possible involvement of α1-adrenergic receptor and KATP channels in cardioprotective effect of remote aortic preconditioning in isolated rat heart

Background: Remote preconditioning is a phenomenon in which brief episodes of ischemia and reperfusion to remote organs protect the target organ against sustained ischemia/reperfusion (I/R)-induced injury. Protective effects of remote aortic preconditioning (RAPC) are well established in the heart, but their mechanisms still remain to be elucidated. Objective: This study has been designed to investigate the possible involvement of α-1-adrenergic receptor (AR) and KATP channels in cardio-protective effect of RAPC in isolated rat heart. Materials and Methods: Four episodes of ischemia and reperfusion, each comprising of 5 min occlusion and 5 min reperfusion, were used to produce RAPC. Isolated perfused rat heart was subjected to global ischemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analyzed for LDH and CK-MB release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Results: Phenylephrine (20 μ/kg i.p.), as α-1-AR agonist, was noted to produce RAPC-like cardio-protection. However, administration of glibenclamide concomitantly or prior to phenylephrine abolished cardioprotection. Moreover, prazocin (1 mg/kg. i.p), as α-1-AR antagonist and glibenclamide (1 mg/kg i.p), a KATP channel blocker, abolished the cardioprotective effect of RAPC. Conclusion: These data provide the evidence that α-1-AR activation involved in cardioprotective effect of RAPC-mediated trough opening of KATP channels.

Mechanism of cardioprotective effect of erythropoietin-induced preconditioning in rat heart.

Objective: The cardioprotective potential of human recombinant erythropoietin (alpha) (Epo) against ischemia-reperfusion-induced injury is well known. But, the underlying mechanisms are not well elucidated. The aim of this study was to characterize the mechanism involved in the cardioprotective effect of Epo-induced preconditioning in isolated rat heart. Materials and Methods: The heart was mounted on a Langendorff apparatus. After 10 min of stabilization, four cycles of ischemic preconditioning (IPC) were given followed by 30 min of global ischemia and 120 min of reperfusion. Epo preconditioning was induced by four cycles of 5-min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution. Myocardial infarct size was estimated macroscopically using the triphenyltetrazolium chloride staining technique. The extent of myocardial injury was measured by release of lactate dehydrogenase and creatine kinase-MB in the coronary effluent. Results: The present study demonstrates that Epo preconditioning was almost as effective as IPC. Administration of Wortmannin (100 nM), a PI-3K inhibitor, or Chelerythrine (1 µM), a protein kinase-C (PKC) inhibitor, or AG490 (5 µM), a JAK-2 inhibitor, significantly attenuated the cardioprotective effects of Epo-induced preconditioning. Conclusion: Our result suggest that the cardioprotective potential of Epo-induced preconditioning in isolated rat heart was due to an interplay of the JAK-2, PI-3K and PKC pathways. Inhibition of any one of the three pathways was sufficient to block the cardioprotective effect of Epo-induced preconditioning in isolated rat heart.

Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart

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.

Role of atrial natriuretic peptide in ischemic preconditioning–induced cardioprotection in the diabetic rat heart

BACKGROUND It has been noted that nitric oxide (NO) is involved in the ischemic preconditioning (IPC)-mediated cardioprotection. Diabetes is a downregulator of atrial natriuretic peptide (ANP), resulting in low expression of endothelial nitric oxide synthase (eNOS) by which NO level get reduced. The purpose of the present study was to investigate the role of ANP in attenuated cardioprotective effect of IPC in the diabetic rat heart. METHODS The heart was isolated from the diabetic rat and mounted on Langendorffs 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 creatinine kinase-MB release to assess the degree of myocardial injury. The cardiac release of NO was estimated indirectly by measuring the release of nitrite in coronary effluent. RESULTS IPC-mediated cardioprotection was significantly attenuated in the diabetic rat as compared with the normal rat. Perfusion of ANP (0.1 μM/L) in the diabetic rat heart significantly restored the attenuated cardioprotective effect of IPC and also increased the release of NO. However, this observed cardioprotection was significantly attenuated by perfusion of N-nitro L-arginine methyl ester, an eNOS inhibitor (100 μM/L) noted in terms of increase in myocardial infarct size, release of lactate dehydrogenase and creatinine kinase-MB, and also decreases in release of NO. CONCLUSIONS Thus, it is suggested that ANP restores the attenuated cardioprotective effect in the diabetic heart which may be due to increase in the expression of eNOS and subsequent increase in the activity of NO.

Effect of pioglitazone on the abrogated cardioprotective effect of ischemic preconditioning in hyperlipidemic rat heart.

Objectives: The signaling pathways upstream of glycogen synthase kinase-3β (GSK-3β) get reduced during ischemic preconditioning (IPC) in hyperlipidemic rat heart. Pioglitazone, an insulin sensitizer, exerts cardioprotection through GSK-3β. The objective of the study is to investigate the role of pioglitazone on the attenuated cardioprotective effect of IPC in hyperlipidemic rat heart. Materials and Methods: The rats were administered high-fat diet for 8 weeks to induce experimental hyperlipidemia (HL). After mounting on a Langendorff apparatus, isolated perfused hearts were given four cycles of IPC; each consists of 5 min of both ischemia and reperfusion followed by 30 min of ischemia and 120 min of reperfusion. Insulin (50 mU/ml) was perfused alone and in combination with pioglitazone (2 μM), while in other groups, this combination was repeated with wortmannin (100 nM), a selective PI3K inhibitor and rapamycin (1 nM), a selective mammalian target of rapamycin (mTOR) inhibitor, separately, and in combination. Myocardial injury was assessed by measuring infarct size and the levels of creatinine kinase-myocardial band (CK-MB) and lactate dehydrogenase (LDH) in the coronary effluent. Results: IPC significantly decreased the infarct size and levels of LDH and CK-MB in normal but not in HL rat heart. Perfusion of insulin along with pioglitazone significantly reduced the infarct size and release of CK-MB and LDH in IPC-treated HL rat hearts. Perfusion of wortmannin or rapamycin alone significantly and in combination almost completely abolished the pioglitazone-induced restored cardioprotection (P < 0.05). Conclusion: Cardioprotective effect of IPC gets lost in hyperlipidemic rat heart. The results suggest that perfusion of pioglitazone restored the cardioprotective effect of IPC in hyperlipidemic rat heart, an effect that may be via PI3K and mTOR.

Hemin, a heme oxygenase-1 inducer, restores the attenuated cardioprotective effect of ischemic preconditioning in isolated diabetic rat heart

Background: Attenuated cardioprotective effect of ischemic preconditioning (IPC) by reduced nitric oxide (NO) is a hallmark during diabetes mellitus (DM). Recently, we reported that the formation of caveolin–endothelial nitric oxide synthase (eNOS) complex decreases the release of NO, which is responsible for attenuation of IPC-induced cardioprotection in DM rat heart. Heme oxygenase-1 (HO-1) facilitates release of NO by disrupting caveolin–eNOS complex. The activity of HO-1 is decreased during DM. This study was designed to investigate the role of hemin (HO-1 inducer) in attenuated cardioprotective effect of IPC in isolated diabetic rat heart. Methods: DM was induced in male Wistar rat by single dose of streptozotocin. Cardioprotective effect was assessed in terms of myocardial infarct size and release of lactate dehydrogenase and creatine kinase in coronary effluent. The release of NO was estimated indirectly by measuring the release of nitrite in coronary effluent. Perfusion of sodium nitrite, a precursor of NO, was used as a positive control. Result: IPC-induced cardioprotection and increased release of nitrite were significantly attenuated in a diabetic rat as compared to a normal rat. Pretreatment with hemin and daidzein, a caveolin inhibitor, alone or in combination significantly restored the attenuated cardioprotection and increased the release of nitrite in diabetic rat heart. Zinc protoporphyrin, a HO-1 inhibitor, significantly abolished the observed cardioprotection and decreased the release of nitrite in hemin pretreated DM rat heart. Conclusion: Thus, it is suggested that hemin restores the attenuated cardioprotective effect in diabetic rat heart by increasing the activity of HO-1 and subsequently release of NO.

Involvement of atrial natriuretic peptide in abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart

Background: Nitric oxide (NO) is an effective mediator of ischemic preconditioning (IPC)-induced cardioprotection. Atrial natriuretic peptide (ANP) is downregulated after ovariectomy, which results in reduction in the level of NO. The present study deals with the investigation of the role of ANP in abrogated cardioprotective effect of IPC in the ovariectomized rat heart. Methods: Heart was isolated from ovariectomized rat and mounted on Langendorff’s apparatus, subjected to 30 min of ischemia and 120 min of reperfusion. IPC was given by four cycles of 5 min of ischemia and 5 min of reperfusion with Krebs–Henseleit solution. The myocardial infract size was estimated employing triphenyltetrazolium chloride stain, and coronary effluent was analyzed for creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) release to consider the degree of myocardial injury. The cardiac release of NO was estimated by measuring the level of nitrite in coronary effluent. Results: IPC-mediated cardioprotection was significantly attenuated in ovariectomized rat as compared to normal rat, which was restored by perfusion with ANP. However, this observed cardioprotection was significantly attenuated by perfusion with L-NAME, an endothelial nitric oxide synthase inhibitor, and Glibenclamide, a KATP channel blocker, alone or in combination noted in terms of increase in myocardial infract size, release of CK-MB and LDH, and also decrease in release of NO. Conclusion: Thus, it is suggested that ANP restores the attenuated cardioprotective effect of IPC in the ovariectomized rat heart which may be due to increase in the availability of NO and consequent increase activation of mitochondrial KATP channels.