Hydrogen sulfide (H2S) has recently been identified as a physiologically important endogenous gaseous signaling molecule with a diverse array of biological activities. H2S is produced in micromolar quantities by two endogenous enzymes, cystathionine 3lyase (CGL) and cystathionine 2 synthase (CBS) and is critical for the maintenance of cardiovascular homeostasis. H2S attenuates leukocyte adhesion, modulates mitochondrial respiration, and inhibits both apoptosis, and oxidative stress. These physiological actions are ideal for the treatment of myocardial ischemia-reperfusion (MI-R) injury. Preliminary data clearly demonstrate that physiological levels of H2S significantly ameliorate MI-R injury and preserve left ventricular function. Preliminary data also indicate that H2S therapy triggers both early and late myocardial preconditioning. We also demonstrate that mice with cardiac-restricted CGL overexpression exhibit significantly increased myocardial H2S bioavailability and protection against myocardial I-R injury. The central hypothesis for the proposed studies is that H2S triggers a cardioprotective signaling cascade that confers robust cardioprotection in the setting of MI-R injury. The proposed studies will evaluate the various cardioprotective signals induced by H2S therapy during both acute and chronic pharmacological preconditioning as well as the during acute H2S therapy at the time of reperfusion. Specific Aim 1: To investigate the contribution of ATP sensitive K+ channels (KATP channels) in H2S- mediated cardioprotection against myocardial ischemia-reperfusion injury. In vitro studies will evaluate the effects of H2S on KATP channel activation and mitochondrial function. In vivo Studies will be performed using gene-targeted mice with cardiac myocyte deletion of KATP (Sur 1, Kir 6.1, and Kir 6.2 subunits) treated with H2S and subjected to MI-R. Specific Aim 2: To investigate the role of antioxidants in H2S-mediated cardioprotection. Studies will evaluate the acute and chronic effects of H2S on Nrf-2 activation and oxidative stress during MI-R. Studies will also investigate the effects of H2S on the induction of antioxidant signaling pathways in the myocardium prior to MI-R. Specific Aim 3: To investigate the role of the RISK pathway in H2S-mediated cardioprotection. Studies will investigate the effects of H2S on RISK pathway (PI3K, Akt, PKC5, and Erk 1/2) activation, downstream anti-apoptotic signaling, MPTP opening, and myocardial cell death following MI-R. The proposed studies will significantly extend our current understanding of the molecular and cellular pathophysiology of MI-R injury and provide the foundation for the development of H2S therapy for the treatment of acute myocardial infarction.