Cardiomyocyte Zinc and its Regulation of Contraction-Relaxation Function

描述（由申请人提供）：我们已经进行了新的观察，即在分离的心肌细胞中细胞内锌离子浓度（[Zn 2 +]int）的升高导致改善的舒张动力学。有趣的是，心肌细胞[Zn 2 +]int在氧化应激反应中自然上升，这会降低心脏功能，并经常发生在导致相关心肌病或心力衰竭的疾病（如糖尿病，高血压冠状动脉疾病或心肌梗死）中。氧化应激后[Zn 2 +]int的升高可能代表心脏功能的功能重要介质和/或补偿氧化应激的有害影响的信号传导机制。然而，[Zn 2 +]int在修饰心肌细胞功能以响应氧化应激中的生理作用尚未被确定。我们提出的实验旨在揭示影响心肌细胞收缩-舒张动力学的Zn 2+敏感机制，并测试Zn 2+在氧化应激恢复中的相关性，包括其信号基因表达。[Zn2+]int最有可能通过其对Ca 2+调节、肌丝力产生和/或近端第二信使的影响来改善心肌细胞功能。我们将使用传统的荧光显微镜来测量SR Ca 2+负荷和Na+依赖性Ca 2+流出率在分离的心肌细胞暴露于各种细胞外Zn 2+浓度（目的1）。这些实验将证明Zn 2+是否降低SR Ca 2+负荷，增强Na+依赖性Ca 2+流出，从而增强舒张功能。我们将使用X射线荧光微探针测量暴露于各种细胞外Zn 2+浓度的离体心肌细胞中的总钙含量，并测试锌积累是否以剂量依赖性方式降低总钙含量（目的2）。目的2通过提供细胞内钙负荷的独立测量来补充目的1，细胞内钙负荷对舒张功能有不利影响。使用化学皮肤的心肌，我们将测量肌丝张力与Ca 2+的关系，肌动蛋白-肌球蛋白过桥动力学，缩短速度和机械功率产生在不同的Zn 2+浓度（目的3）。使用肌球蛋白运动分析，我们将测量调节和未调节的肌动蛋白丝的速度。目的3和4被设计为作为跨桥动力学和细丝Ca 2+敏感性的独立测量而相互补充。我们还将在氧化应激条件下采取目标1-4中提出的相同措施，并测试Zn 2+在防止相应分子机制随后功能减弱方面的重要性。最后，我们将使用微阵列技术来检测由氧化应激引起的心肌细胞基因表达谱的[Zn 2 +] int依赖性。目前的建议是一个全面的和多学科的方法来检查心脏锌的生理作用。公共卫生相关性：我们将揭示锌作为心脏功能和基因表达的介体在氧化应激反应中的生理相关性，氧化应激对心脏功能有不利影响，并与高龄，糖尿病，高血压，冠状动脉疾病，心肌梗死和心力衰竭一起发生。因此，这项研究可以为影响大部分人群的几种疾病或病症的治疗提供基础。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.