The primary cardiac event in MI is cell death. Genetic studies in mice have established that most cardiomyocyte death during reperfused MI (MI/R) occurs through regulated cell death programs. This suggests that it should be possible in concept to limit cardiomyocyte death during MI/R to obtain the full benefit of reperfusion in maintaining cardiac function. A significant obstacle in achieving this goal, however, is that cardiomyocyte death in MI/R is mediated by several regulated apoptosis and necrosis programs, and the mechanisms that link these programs to produce an integrated cell death response remain poorly understood. This gap in knowledge has been a critical impediment for the rational design of therapies to limit cardiac damage during MI/R. Accordingly, a goal of our lab has been to understand the molecular framework that connects cell death programs in MI/R. Our preliminary studies have identified caspase-9 as an important link between apoptosis and necrosis programs during MI/R. The canonical role of caspase-9 in apoptosis is to activate downstream procaspases-3 and -7. However, we have identified a new pathway in which caspase-9 mediates cardiomyocyte necrosis during MI/R. In contrast to caspase-9-mediated apoptosis, caspases-3/7 are dispensable for caspase-9-mediated necrosis indicating the distinctness of the two pathways. However, as in apoptosis, caspase-9 enzymatic activity is required. To further dissect the pathway, we performed a proteomics-based screen for procaspase-9 interacting proteins in the heart during MI/R, which revealed proteins known to be involved in various aspects of necrosis. Thus far, we have focused on one procaspase-9 interactor, SERCA2a, which we hypothesize functions downstream of caspase-9 to mediate cell-intrinsic killing. Our data suggest that caspase-9 cleaves SERCA2a during MI/R disabling its function consistent with prior genetic studies showing that SERCA2a loss exacerbates cardiomyocyte necrosis and infarct size. We believe, however, that the relationship between procaspase-9 and SERCA2a is more complex. In addition to caspase-9 inducing SERC2a loss, our data suggest that SERCA2a loss may contribute to caspase-9 activation through Ca2+ overload. Thus, we hypothesize that a bidirectional mutually-reinforcing relationship exists between caspase-9 and SERCA2a and contributes to cardiomyocyte necrosis and infarct generation in MI/R. This project investigates critical aspects of the caspase-9-mediated necrosis pathway including activation mechanisms, downstream signaling, the relationship of the pathway to cell death programs thought to be involved in MI/R, and whether this pathway can be therapeutically inhibited to limit infarct size. Aim 1. To genetically dissect the caspase-9 necrosis axis during MI/R in vivo. Aim 2. To delineate the bidirectional regulation between caspase-9 and SERCA2A in cardiomyocyte necrosis during MI/R. Aim 3. To assess the therapeutic benefit of specifically inhibiting procas...