Free Radicals, PKC Delta Signal ACh Preconditioning

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DESCRIPTION (provided by applicant): Preconditioning single or multiple periods of brief ischemia and reperfusion before a prolonged period of coronary.artery occlusion, reduces myocardial infarct size and apoptosis. Acetylcholine (ACh) mimics preconditioning effects in vitro and in vivo. Apoptosis has been identified in many cardiac conditions associated with ischemia-reperfusion injury. The goal of this proposal is to elucidate the intracellular signaling pathways by which ACh blocks cardiocyte apoptosis and preserves cardiac function. Protein kinase C (PKC) and mitochondrial K ATP channels are signaling components of preconditioning and ACh. ACh generates oxygen radicals that correlate with cardioprotection in ischemia-reperfusion. ACh affects coronary endothelium, circulating blood elements, and activates a series of signal transduction cascades in cardiocytes such as activating PKCdelta isoform. Which effect accounts for cardioprotection remains unclear. We hypothesize that opening mitochondrial K ATP channels releases oxygen radicals that activate PKCdelta isoform. Through this signaling cascade, ACh blocks cardioeyte apoptosis and preserves cardiac function in ischemia-reperfusion. To test this hypothesis, we will measure oxygen radical production directly in neonatal rat cardiomyocytes. We will use recombinant adenoviral vectors to overexpress in cardiocytes an active PKC6 isoform carrying its full length of cDNA (AdCMVPKCepsilon-FL) or a dominant negative mutant inactive PKCS (AdCMVPKC delta-DN). The effects of these recombinant constructs on cardiocyte function, apoptosis, infarction, and oxidant stress will be examined in both in vitro and in vivo rat models of ischemia-reperfusion. Understanding of the role of specific signaling pathways by which ACh blocks cardiocyte apoptosis and development of local modulation of the pathways through somatic gene transfer may provide novel therapies for the management of many clinical disorders.