The advent of procedures that produce reperfusion of ischemic heart muscle has made post-ischemic myocardial dysfunction of """"""""stunning"""""""" an increasingly relevant clinical phenomenon. While significant progress has been made describing the contractile and electrophysiologic changes that occur in stunned myocardium, our understanding of the mechanisms underlying these changes is far from complete. In this myocardium, our understanding of the mechanisms underlying these changes is far from complete. In this proposal, we present a unique model of cardiac stunning in chronically instrumented pigs. Preliminary data shows this model has significant advantages for monitoring stunning in vivo. Furthermore, myocytes isolated from stunned myocardium retain a """"""""stunned"""""""" phenotype which allows us to undertake an in-depth investigation of mechanisms underlying post-ischemic dysfunction. Based on the preliminary data, our initial hypothesis is that transient ischemia and reperfusion compromises contractility of cardiac muscle by decreasing the Ca2+ sensitivity of the contractile apparatus along with a concomitant decrease in Ca2+ decreasing the Ca2+ sensitivity of the contractile apparatus along with a concomitant decrease in Ca2+ sequestration by the sarcoplasmic reticulum and that these changes in Ca2+ handling induce chaotic electrophysiologic behavior which promote early after depolarizations (EADs). To test this hypothesis, we will examine: 1) contractile function in skinned cardiac muscle preparations and ventricular myocytes isolated from stunned myocardium. Experiments will determine if Ca2+ sensitivity, force development and cross-bridge kinetics Are altered in stunned muscle and, if so, the content and/or phosphorylation of contractile proteins that might cause these effects; 2) Ca2+ flux pathways and E-coupling in single myocytes loaded with fluorescent Ca2+ indicators. Myocytes from control and stunned muscle will be voltage -clamped while intracellular Ca2+ is measured with microfluorimetric and confocal microscopic techniques to determine precisely how specific Ca2+ regulatory mechanisms are affected by stunning. Any changes observed at the cellular level will then be corroborated by determining whether the content and/or phosphorylation state of specific Ca2+ regulatory proteins is altered in stunned myocardium; 3) changes in the action potential, underlying membrane currents and intracellular Ca2+ handling that promote arrhythmogenic behavior. These experiments will be analyzed with conventional kinetic and non-linear dynamic algorithms. The resulting data will then be used to test and develop computer simulations of the pig action potential to determine if altered Ca2+ dynamics in stunned muscle cells could be responsible for the generation of EADs. Given the similarity of pig and human myocardium, the results of this project should yield critical insights into the mechanisms underlying post-ischemic dysfunction in human heart.
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