The application proposes to continue examining the signal transduction processes transmitting death signal to the cardiomyocytes during myocardial ischemia and reperfusion. During the current years' funding, our laboratory locumented the invo'vement of 'MAP kinase signaling in the development of ischemic reperfusion injury, and more importantly in the process of myocardial adaptation to ischemic stress. The MAP kinase signaling was found to modulate the death signal by reprogramming of gene expression. A number of redox-regulated transcription factors and genes were found to be involved in the signaling cascade leading to cardiomyocyte death due to necrosis and apoptosis. The proposed research will further explore the regulation of death and survival signals of MAP kinase cascades by addressing the following Specific Aims: i) by studying Src kinase regulation of cardiomyocyte life and death and its relation to the downstream kinases p38MAPK, JNK and ERK as well as by determining the role of tyrosine phosphorylation of PLCy; ii) by examining PI-3-kinase-Akt signaling in transmitting survival signal during ischemia/reperfusion and ischemic adaptation; iii) by studying the significance of JAKJSTAT signaling in ischemic reperfusion injury and following the death signal through caspase activation; iv) by examining the regulation of the transcription factors and induction of gene expression that modulate the death and/or survival signal as well as by studying transcription regulation of downstream kinases, p38MAPK and ERK and JNK by some of these genes; and v) by examining the second messenger role of reactive oxygen species in MAP kinase signaling and their transcription regulation. The experimental models will involve both working and Langendorif rat and mouse hearts, genetically engineered mouse, isolated cardiomyocytes, antisense gene delivery and dominant negative cells. The studies will include determination of the key signaling members of MAP kinase pathway both at mRNA and protein levels, their post-translational modification and transcription regulation by transcription factors and/or genes responsible for transmitting death and/or survival signal, and evaluation of cardiomyocyte apoptosis by study its progression through cytochrome c release and caspase activation. The overall objective is to delineate the signal transduction processes responsible for transmitting death signal and to explore interventions that change the death signal into survival signal which would lead to developing therapeutic modalities to reduce cardiomyocyte death due to apoptosis and necrosis.
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