Reperfusion of the myocardium following an ischemic episode is associated with profound contractile and metabolic dysfunction, referred to as myocardial stunning. Reperfusion also increases the activity of the Na+/H+ exchanger (NHE), which restores intracellular pH (pHi) towards normal following ischemia-induced acidosis. However, activation of NHE also produces undesirable secondary effects leading to the exacerbation of tissue injury, a phenomenon termed the "pH paradox". Increased generation of oxygen free radicals (OFR) plays an important role in reperfusion-induced myocardial stunning and NHE activation. An in vitro model for studying the effects of OFRs on cultured neonatal rat ventricular myocytes (NRVM) has been defined, in which low concentrations of H2O2 (similar to those generated during reperfusion) cause contractile dysfunction, Ca2+ overload, and NHE activation. There is considerable interest in identifying signaling events that link H2O2 to myocardial dysfunction. H2O2 and hypoxia activate members of the mitogen activated protein kinase (MAPK) family, including p38, c-jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinases (ERK1/2). Low doses of H2O2 decrease myocyte contractility and stimulate NHE activity in an ERK1/2-dependent manner. Preliminary data indicate that exposure of cardiac myocytes to H2O2 induces myofilament disassembly, Ca2+ overload, and the activation of the nonreceptor tyrosine kinase src. The hypothesis of this proposal is that the MAPK family modulates NHE activity, Ca2+ overload and contractile dysfunction induced by H2O2. In Aim 1, experiments with synthetic inhibitors and antisense oligonucleotides will determine whether MAP kinase inhibition blocks H2O2-induced phosphorylation of NHE, since phosphorylation of the exchanger protein is associated with its activation. NHE activation will be measured by fluorimetric imaging of intracellular pH and by examining the phosphorylation state of the NHE protein in vitro and in vivo. In Aim 2, the link between H2O2-induced contractile dysfunction, Ca2+ overload and MAPK activation will be investigated using pharmacological inhibitors and antisense oligonucleotides against p38, JNK, and ERK MAPKs. Contractile dysfunction will be defined as a decrease in myocyte contractility (using video edge detection), and by immunocytochemistry to measure myofibrillar assembly. In Aim 3, a studies using immunecomplex kinase assays, immunoprecipitation and Western blot analysis will identify regulatory components upstream of MAPKs that are activated by H2O2. This aim will focus on src, protein kinase C, and the Ras superfamily of monomeric GTP-binding proteins. The proposed investigations are fundamentally important to the development of therapeutic strategies targeted to signaling pathways involved in oxidant-induced injury and may have important clinical implications in the treatment myocardial ischemia.