The present proposal addresses the hypothesis that cardioprotective agents activate a specific isoform of PKC which modifies cardiac myofilament protein(s) and decreased myocyte ATP consumption by slowing actin-myosin cycling kinetics. Reduced ATP utilization by myosin is cardioprotective due to maintained function of critical ATP-dependent channels and pumps responsible for a low intracellular [Ca/2+]. To test this hypothesis 5 specific questions/aims will be pursued: (1). Are slowed kinetics of the actomyosin ATPase a common effect of cardioprotective agents? (2.) Are agonist-induced decreases in myocyte velocity of unloaded shortening (Vmax) due to activation of PKC, increased cGMP, and/or inhibition of cAMP? (3.) Does activation of different PKC isoforms and/or the level of PKC activity lead to differential effects on myocyte mechanical function? (4.) Do cardioprotective agents affect myofilament function by altering the activity of a phosphatase, or increasing the phosphorylation of specific myofilament function by altering the activity of a phosphatase, or increase the phosphorylation of specific myofilament proteins? (5.) Does actomyosin ATP consumption decreased under cardioprotective conditions in isolated hearts? To answer these questions the effects of known cardioprotective agents (opioid, muscarinic, adenosine, and alpha- adrenergic receptor agonists) on ventricular myocyte Vmax, and myofibrillar protein phosphate incorporation will be determined. Second messenger involvement will be established by sequentially blocking select second messenger pathways prior to cardioprotective agent activation and re-determining Vmax and myofibrillar phosphatase incorporation. To determine whether cardioprotective agents cause activation and subsequent translocation of specific isoforms of PKC, monoclonal antibodies to PKC isoforms will be utilized to probe Western blots of cell fractions and in confocal microscopy of myocytes before and after stimulation with a cardioprotective agent. The relationship between cardioprotective ability and myofibrillar ATP utilization will be established by determining isolated heart post-ischemic function and actomyosin ATPase following receptor agonist stimulation or an ischemic pre-conditioning protocol. We anticipate any intervention which improves post-ischemic function will decreases actomyosin ATPase. These experiments will allow a better understanding of cardioprotective molecular mechanism(s) and facilitate design of effective and selective clinical therapies. The proposed study is novel since it is the first to examine a link between cardioprotective agents, second messenger pathways, altered myofilament function, and whole hear function following ischemia.
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