Furthermore, activation of AMPK protects the heart from myocardial infarction and heart failure. The present study examines whether or not AMPK affects the peroxisome proliferator-activated receptor-␣ (PPAR␣)/mitochondria pathway in response to acute oxidative stress in cultured cardiomyocytes. Cultured H9c2 rat embryonic cardioblasts were exposed to H2O2-induced acute oxidative stress in the presence or absence of metformin, compound C (AMPK inhibitor), GW6471 (PPAR␣ inhibitor), or A-769662 (AMPK activator). Results showed that AMPK activation by metformin reverted oxidative stress-induced inactivation of AMPK and prevented oxidative stress-induced cell death. In addition, metformin attenuated reactive oxygen species generation and depolarization of the inner mitochondrial membrane. The antioxidative effects of metformin were associated with the prevention of mitochondrial DNA damage in cardiomyocytes. Coimmunoprecipitation studies revealed that metformin abolished oxidative stress-induced physical interactions between PPAR␣ and cyclophilin D (CypD), and the abolishment of these interactions was associated with inhibition of permeability transition pore formation. The beneficial effects of metformin were not due to acetylation or phosphorylation of PPAR␣ in response to oxidative stress. In conclusion, this study demonstrates that the protective effects of metformininduced AMPK activation against oxidative stress converge on mitochondria and are mediated, at least in part, through the dissociation of PPAR␣-CypD interactions, independent of phosphorylation and acetylation of PPAR␣ and CypD. H9c2 cardiomyocytes; oxidative stress; mitochondria; metformin; AMPK; PPAR␣ THE ROLE OF MITOCHONDRIA in energy production is well established. Mitochondria are also involved in a range of other processes, such as reactive oxygen species (ROS) production, ion signaling, redox control, lipid metabolism, cell growth, and cell death through apoptosis, necrosis, and autophagy (16). Structural and functional abnormalities are implicated in the pathogenesis of cardiac diseases, including myocardial ischemia (infarction) and heart failure (37). Activation of numerous survival protein kinases, including the AMP-activated protein kinase (AMPK) (43), protein kinase C (28), phosphatidylinositol-4,5-bisphosphate 3-kinase (11), glycogen synthase kinase 3 (24), and cGMP-dependent protein kinase (27), has been demonstrated to protect cardiomyocytes against oxidative stress through a direct or indirect interaction with mitochondria. Specifically, AMPK activation is associated with a reduction in cardiac hypertrophy (49) and infarct size, preservation of cardiac energy sources, and reduction in both necrosis and apoptosis (26). AMPK has been widely accepted as the main cellular energy sensor that both initiates ATPgenerating processes and blocks ATP-consuming processes. Furthermore, AMPK plays a central role in the regulation of mitochondrial metabolism and controls the redox state of the cell, though the underlying mechanisms of its action on ...