Possible Mechanism of Rottlerin Induced Modulation of Ischemia Reperfusion Injury in Isolated Rat Hearts

Abstract

Coronary artery diseases represent a global burden on health care resources and poised to become the leading cause of morbidity and mortality in the world by 2020. Early reperfusion is essential to salvage ischemic myocardium; however reperfusion of ischemic region is not devoid of deleterious effects. Reperfusion of ischemic myocardium is noted to produce detrimental morphological and functional changes in the form of reperfusion injury manifested as reperfusion arrhythmias, myocardial stunning, micro-vascular and endothelial dysfunction leading to impairment of reflow. 1,2)Myocardial ischemia and reperfusion lead to activation of two novel PKC iso-forms (PKCd and PKCe) which are reported to play opposing roles during myocardial ischemia and reperfusion (I/R).3,4) PKCd has been reported to mediate I/R induced myocardial injury through activation of mitochondrial pathway, 5,6) whereas PKCe has been reported to provide cardioprotection through preconditioning. 7) PKCd has been implicated in I/R induced myocardial injury by producing apoptotic and necrotic cell death. 4,5) A selective peptide inhibitor of PKCd i.e. dV1-1, has been reported to reduce I/R induced myocardial injury by inhibiting apoptotic as well as necrotic cell death. 6)Ischemia and reperfusion has also been reported to activate protein kinases such as p-38 mitogen activated protein kinase (p-38 MAPK); which further increases transcription of pro-inflammatory cytokines such as tumor necrosis factora (TNF-a).8,9) Administration of SB 203580, a selective p-38 MAPK inhibitor, has been reported to attenuate the apoptotic and necrotic cell death of cardiomyocytes.10) Rottlerin is a specific non-peptide inhibitor of PKCd,11,12) which is also reported to activate p-38 MAPK.13) Therefore, the present study was designed to investigate the modulatory effects of rottlerin, an inhibitor of PKCd and activator of p-38 MAPK on ischemia reperfusion induced myocardial injury. MATERIAL AND METHODSWistar albino, rats of either sex weighing 150-200 g maintained at standard animal diet (Kisan Feeds Ltd., Chandigarh, India) and tap water ad libitum were employed in the present study. The animals were procured from 'disease free small animal house' Chaudary Charn Singh, Haryana Agriculture University, Hisar (Haryana), India. They were exposed to 12 h cycle of light and dark. The protocol of the study was duly approved by Institutional Animal Ethics Committee and care of the animals was taken as per guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India (Reg. No. 107/CPCSEA 1999).Drugs and Chemicals Rottlerin (Sigma Aldrich, St. Louis, U.S.A.) and FR-167653 (Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) were dissolved in 1 : 5 mixtures of ethanol with normal saline and saline respectively. All other reagents used in the study were of analytic grade.Isolated Rat Heart Preparation Rats were heparinised (500 I.U., i.p.) about 20 min before sacrificing the animal by cerv...