We have made the novel observation that an elevation in intracellular zinc ion concentration ([Zn2+]int) in the isolated cardiomyocyte leads to improved relaxation dynamics. Interestingly, cardiomyocyte [Zn2+]int rises naturally in response to oxidative stress, which diminishes cardiac function and occurs frequently in conditions (such as diabetes, hypertension coronary artery disease or myocardial infarction) that lead to related cardiomyopathies or heart failure. The rise in [Zn2+]int after oxidative stress may represent a functionally important mediator of cardiac function and/or a signaling mechanism to compensate for the detrimental effects of oxidative stress. However, the physiological role of [Zn2+]int in modifying cardiomyocyte function in response to oxidative stress has not been identified. Our proposed experiments are designed to uncover Zn2+-sensitive mechanisms that affect cardiomyocyte contraction-relaxation dynamics and to test the relevance of Zn2+ in the recovery from oxidative stress including its signaling gene expression. [Zn2+]int most likely improves cardiomyocyte function via its effects on Ca2+ regulation, myofilament force-production, and/or proximal second messengers. We will use conventional fluorescence microscopy to measure SR Ca2+ load and the rate of Na+-dependent Ca2+ efflux in isolated cardiomyocytes exposed to various extracellular Zn2+ concentrations (Aim 1). These experiments will demonstrate whether Zn2+ lowers SR Ca2+ load, enhances Na+-dependent Ca2+ efflux and thereby enhances diastolic function. We will use the X-ray fluorescence microprobe to measure total calcium content in isolated cardiomyocytes exposed to various extracellular Zn2+ concentrations and test whether zinc accumulation lowers total calcium content in a dose-dependent manner (Aim 2).
Aim 2 complements Aim 1 by providing an independent measure of intracellular calcium load, which adversely affects diastolic function. Using chemically-skinned myocardium, we will measure the myofilament tension vs. Ca2+ relationship, acto-myosin crossbridge kinetics, velocity of shortening and mechanical power production at various Zn2+ concentrations (Aim 3). Using the myosin motility assay, we will measure the velocities of regulated and unregulated actin filaments.
Aims 3 and 4 are designed to complement each other as independent measures of crossbridge kinetics and thin filament Ca2+ sensitivity. We will furthermore undertake the same measures proposed in Aims 1-4 under conditions of oxidative stress and test the importance of Zn2+ in preventing the subsequent diminished function of the respective molecular mechanisms. Finally, we will use micro-array techniques to detect the [Zn2+]int-dependency of the cardiomyocyte gene expression profile elicited by oxidative stress. The current proposal represents a comprehensive and multi-disciplinary approach to examining the physiological role of cardiac zinc.
We will uncover the physiological relevance of zinc as a mediator of heart function and gene expression in response to oxidative stress, which has a detrimental effect on heart function and occurs with advanced age, diabetes, hypertension coronary artery disease, myocardial infarction and heart failure. This study could therefore provide a basis for therapies for several diseases or conditions that affect a large component of the population.
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