The long-term goal of this project is to characterize how Second Mitochondria-derived Activator of Caspase (Smac) promotes apoptosis in the heart. More specifically, we will address how Smac interacts with xIAP and caspases during apoptosis of cardiomyocytes. Smac is a pro-apoptotic protein that inhibits the caspase inhibitor, MAP. Since Smac is expressed at high levels in the heart, inhibition of Smac could inhibit cardiomyocyte apoptosis. Apoptosis causes myocyte loss and heart failure with ischemia reperfusion injury, as happens with therapeutic revascularization of the heart. Therefore, attenuating Smac could decrease apoptosis and potentially have therapeutic benefit after cardiac revascularization. To fully inhibit Smac, we must explore all of its actions. Smac may be involved in xIAP and caspase-independent apoptosis. ? ? Our hypothesis is that Smac has an xIAP-independent function that ultimately promotes apoptos is in the cardiomyocyte. To test this hypothesis, our specific aims are 1.) To test if overexpress ion of Smac with deleted xIAP-binding site promotes caspase activation and apoptosis. Smac with deleted xIAP-binding site will be compared to Smac with an active xIAP-binding site. Each will be transfected into human embryo kidney 293 cells and exposed to apoptotic stimuli. Caspase activity and apoptosis will be measured. To test if Smac has a caspase-independent function in 293 cells, exposure to apoptotic stimuli will be repeated with a broad caspase inhibitor. 2.) To test if overexpression of Smac with a deleted xIAP-binding site in an adenovirus promotes caspase activation and apoptosis in the cardiomyocyte. Cultured cardiomyocytes will be infected with adenovirus containing either of the two mutants described above and exposed to hypoxia-reoxygenation. Caspase activity and apoptosis will be measured. To test if Smac has a caspase-independent function in cardiomyocytes, exposure to apoptotic stimuli will be repeated with a broad caspase inhibitor. ? ?
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