EXCEED THE SPACE PROVIDED. During the current funding period, we have discovered a number of m olecular mechanisms that underlie exercise- induced late PC. During the next funding period, we will build upon our previous work and address the following five specific Aims: 1. To establish the role of the JAK-STAT signaling pathway in exercise-induced late P C. Using our well- established murine model, we will dptermine whether exercise PC is abrogated in mice with constitutive targeted deletion of the STAT1 gene (STATI'), constitutive cardiac-specific targeted deletiop of the STAT3 gene (STAT3'), and conditional cardiac-specific targeted deletion of the STAT3 gene (cond-STAT3'). 2. To determine the mechanism whereby STAT3 is activated by exercise. We will use two genetically- engineered mice: a transgeni_._puse expressing a dominant negative STAT3 in which Tyr-705 has been replaced by phenylalanine (DN-STAT3 ''wr) and a mutant mouse in W_ Ser-727 of STAT3 has been replaced by alanine (STAT3 SALSA). We will determine whether the DN-STAT3 ..... and STAT3 SALSA mutations abrogate protection and STAT3 activation after exercise, whether the JAK and o_hePKCs-Raf-1-MEK-1/2-p44/42 MAPK cascades are activated by exercise, and whether inhibition of PKCs (PKC6' mice) blocks Ser-727 phosphorylation of STAT3. 3. To determine the relative roles of apoptosis and necrosis in the protection afforded by exercise-induced late PC. We will utilize state-of-the-art confocal microscopy techniques to identify apoptosis and necrosis with hairpin-l/caspase-3 and hairpin-2/caspase-3 dual-labeling assays, respectively. 4. To decipher the molecular mechanism by which exercise suppresses cardiac apoptosis. We will assess the effect of exercise on the expression of the major pro-apoptotic (Bak, Bax, Bid, and Bad) and anti-apoptotic (Bcl-2, Bcl-xL, Mcl-1, c-FLIPL, c-FLIPs, ARC, XIAP, clAP-l, and clAP-2) proteins in the heart. We will also assess the influence of exercise on the extrinsic and intrinsic pathways of apoptosiso 5. To determine the role of ecSOD in exercise-induced late PC and the mechanism of ecSOD upregulation. We will determine whether exercise upregulates ecSOD expression in cardiac myocytes. We will then determine whether the cardioprotective effects of exercise PC are abrogated by genetic deletion of ecSOD (ecSOD' mice) and, conversely whether they can be mimicked by cardiac-specific overexpression of ecSOD (ecSOD t,ransgenic mice). We will also investigate whether exercise activates cardiac Nrf2. Then, we will use Nrf2_ mice to conclusively establish the role of this transactivating protein in the upregulation of ecSOD by exercise. The studies outlined above will extend and complete our current analysis of the mechanism of exercise-induced late PC. Together, they will furnish novel insights into the molecular mechanisms whereby exercise promotes the shift of the heart to a defensive (preconditioned) phenotype that confers tolerance to ischemia/reperfusion injury. Besides ischemja/reperfusion injury, these data will also have broad conceptual implications for our understanding of the mechanism whereby the heart adapts to stress in general. PERFORMANCE SiTE ========================================Section End===========================================
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