Pathways of Apoptosis in Hypoxic Cardiac Myocytes

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DESCRIPTION (provided by applicant): C-Jun N-terminal kinase (JNK) is activated by reoxygenation or reperfusion in all models of myocardial ischemia in vitro and in vivo. JNK can exert strong modulation over cell survival and may determine the extent of myocardial cell loss during reperfusion. Controversy has existed over the role of activated JNK in different models of reperfusion with some groups describing pro-apoptosis and others protection. We present new data demonstrating that JNK functions are metabolically regulated that may resolve this controversy; JNK is protective when intracellular [ATP] is high and pro-apoptotic when [ATP] is low. As such JNK acts as a metabolic sensor in determining cell fate. We hypothesize that the targets that promote survival are different from those that promote death. In aim 1 of this proposal we will use the JNK functional switch as a tool to identify the phospho-proteins that mediate these different effects on hypoxic cardiac myocytes. Hypoxia and acidosis are integral features of ischemic heart disease. This combination is one of the strongest stimuli to activate programmed death of cardiac myocytes in culture. The pathway involves the Bcl-2 family protein BNIP3 that is strongly induced by hypoxia. Hypoxic cardiac myocytes containing induced BNIP3 do not undergo programmed death until the environment becomes acidic. We hypothesize that BNIP3 is induced and activated by hypoxia and acidosis respectively. Experiments are proposed to determine the mechanism and whether there are secondary effects of acidosis on mitochondrial signaling. Because we have established the role of BNIP3 in cultured cardiac myocytes, a major goal is to determine whether the same pathway promotes cell death during ischemia and infarction in vivo. The signals for JNK activation by reoxygenation have not been determined but are thought to involve reactive oxygen species (ROS). Our preliminary data suggest that coupled mitochondrial electron transport and membrane potential, but not ROS are essential components of this signaling pathway. We hypothesize that the pathway involves calcium loading activated by the mitochondrial membrane potential, coupled with myofilament calcium transients. Calcium is proposed to activate the kinase Pyk2 followed by Rac-1 and TAK- 1 and then JNK. Experiments proposed to test this hypothesis include measurements of membrane potential and intracellular calcium during hypoxia and reoxygenation and relating these to Pyk2 and JNK.