The cardiac ATP sensitive potassium channel (KATP) in surface sarcolemmal membrane and in mitochondria have both been implicated in the pathogenesis of cardiac ischemia, particularly in an important protective phenomenon called ischemic preconditioning (IPC). Proposed mechanisms for IPC are complex and the role and relative importance of KATP for IPC remains controversial, in part because of heavy reliance on KATP pharmacology. The sulfonylurea receptor subunit SUR2 regulates pharmacology and nucleotide interactions for surface KATP, but the molecular nature of the subunit for mitochondria is unknown. In the last period of this project we described novel SUR2 splice variants in heart involving exons 14 and 17. Experiments in Aim 1 will address the hypotheses that 1A) the pharmacology of SUR2 splice variants accounts for the pharmacology of mitochondrial KATP, and 1B) a splice variant of SUR2 is targeted to mitochondria and accounts for mitochondrial KATP activity. These hypotheses are based upon our characterization of an SUR2 knockout mouse developed during the last period that lacks cardiac KATP activity at both the surface and in mitochondria. This mouse provides a novel non-pharmacological model to assess the role of KATP in IPC. Experiments in Aim 2 will address the hypotheses that 2A) KATP is required for early IPC and 2B) KATP openers and blockers and other triggers of IPC such as adenosine affect IPC by non-KATP mechanisms. Experiments in Aim 3 will address the hypotheses that 3A) KATP is required for a type of protection called delayed IPC, and 3B) KATP underlies late but not early ST elevation in ischemia. Aim 4 will develop novel transgenic mice that "rescue" SUR2 in a tissue and splice variant specific manner. These mice will serve as models to further elucidate the relative role of mitochondrial versus sarcolemmal SUR2 and KATP in the mechanism of lPC. This proposal utilizing transgenic animals represents a novel approach to elucidating the mechanism of IPC, a means of cardioprotection with important clinical implications, and it will also suggest more specific therapeutic targets in the SUR2 variants to be studied. It will also provide important new information on mitochondrial structure and function in heart.