IP3R-Dependent Signaling in Excitation-Contraction Coupling during Heart Failure

描述(申请人提供)：心肌兴奋-收缩偶联(ECC)是由钙诱导的钙释放介导的，通过电压门控钙通道的钙内流打开兰尼定受体并触发细胞内钙的大量释放。ECC细胞事件的改变使心脏易于发生心律失常。IP3受体(IP3R)活性对心房肌细胞ECC具有正性变力和致心律失常作用。在心力衰竭期间，IP3R的表达增加；这是否会导致ECC的变化或心律失常尚不清楚。该方案的目的是研究IP3R依赖的信号在心力衰竭期间ECC(特异性目标1)和心律失常发生(特异性目标2)中的作用。研究将利用从正常兔和具有良好特征的兔心力衰竭模型中分离出的心室肌细胞。IP3R依赖的信号转导可通过应用PS释放激动剂Endothelin-1或直接应用IP3来诱导。抑制IP3R依赖的信号将通过药物抑制和IP3亲和陷阱的表达来实现，IP3亲和陷阱结合和缓冲细胞内的IP3。使用钙敏感染料的荧光显微镜将监测细胞内钙(荧光显微镜，INDO-1)和亚细胞钙释放事件(激光扫描共聚焦荧光显微镜，FUIO-4)。此外，膜片钳技术将被用来记录膜电位和离子通道电流。具体目的1研究IP3R依赖的信号对心室肌细胞ECC产生正性变力作用的假说，特别是在心力衰竭时。为了达到这个目的，我们将研究IP3R依赖的信号对基础细胞内钙、动作电位诱导的钙瞬变和基本钙释放事件(钙火花和钙喷雾)的影响。特异性目标2验证了IP3R依赖的信号转导有助于心力衰竭心肌细胞心律失常的假说。在这里，我们将研究IP3R依赖的信号对致心律失常钙信号(自发钙释放、钙波和钙交替)频率和膜电位(早期和延迟后除极、自发动作电位)的影响。心律失常是心力衰竭时猝死的主要原因。预计这项提案的结果将使人们对心力衰竭期间心律失常的机制有必要的了解，这可能有助于心脏病患者的治疗。

DESCRIPTION (provided by applicant): Cardiac Excitation-contraction coupling (ECC) is mediated by calcium-induced calcium release, where calcium influx through voltage gated calcium channels opens ryanodine receptors and triggers massive release of calcium from intracellular stores. Alterations in the cellular events of ECC predispose the heart to arrhythmia. IP3 receptor (IP3R) activity exerts positive inotropic and arrhythmogenic effects on ECC in atrial myocytes. During heart failure, the expression of IP3R is increased; whether this results in changes in ECC or arrhythmogenesis remains unclear. The goal of this proposal is to investigate the role of IP3R-dependent signaling on ECC (Specific Aim 1) and arrhythmogenesis (Specific Aim 2) during heart failure. Studies will utilize ventricular myocytes isolated from normal rabbits as well as from the well-characterized rabbit heart failure model. IP3R-dependent signaling will be induced using application of the I PS-liberating agonist Endothelin-1 or via direct application of IP3. Inhibition of IP3R-dependent signaling will be achieved using both pharmacological inhibition and the expression of an IP3 affinity trap which binds and buffers intracellular IP3. Fluorescence microscopy with calcium sensitive dyes will monitor intracellular calcium (epifluorescence microscopy, indo-1) and subcellular calcium release events (laser scanning confocal fluorescence microscopy, fluo-4). Further, patch clamp techniques will be used to record membrane potential and ion channel currents. Specific Aim 1 investigates the hypothesis that IP3R-dependent signaling exerts positive inotropic effects on ECC in ventricular myocytes, particularly during heart failure. In this Aim the effects of IP3R-dependent signaling on basal intracellular calcium, action potential-induced calcium transients, and elementary calcium release events (calcium sparks and puffs) will be examined. Specific Aim 2 tests the hypothesis that IP3R-dependent signaling contributes to arrhythmogenesis in heart failure ventricular myocytes. Here, the effects of IP3R-dependent signaling on the frequency of arrhythmogenic calcium signals (spontaneous calcium release, calcium waves, and calcium alternans) and changes in membrane potential (Early and Delayed Afterdepolarizations, spontaneous action potentials) will be investigated. Arrhythmia represents the major cause of sudden death during heart failure. It is expected that results from this proposal will give needed insight into the mechanisms of arrhythmia during heart failure, which may aid treatment of individuals with cardiac disease.