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

描述（由申请人提供）：心脏兴奋-收缩偶联（ECC）由钙诱导的钙释放介导，其中通过电压门控钙通道的钙内流打开ryanodine受体并触发细胞内钙库的大量钙释放。ECC的细胞事件的改变使心脏易患心律失常。IP 3受体（IP 3R）活性对心房肌细胞ECC具有正性肌力和促心律失常作用。在心力衰竭期间，IP 3R的表达增加;这是否导致ECC或血管生成的变化仍不清楚。本提案的目标是研究心力衰竭期间IP 3 R依赖性信号传导对ECC（特定目标1）和心律失常发生（特定目标2）的作用。研究将使用从正常家兔以及充分表征的家兔心力衰竭模型中分离的心室肌细胞。IP 3R依赖性信号传导将使用IPS释放激动剂内皮素-1的应用或通过直接应用IP 3来诱导。IP 3R依赖性信号传导的抑制将使用药理学抑制和结合并缓冲细胞内IP 3的IP 3亲和陷阱的表达来实现。使用钙敏感染料的荧光显微镜将监测细胞内钙（落射荧光显微镜，indo-1）和亚细胞钙释放事件（激光扫描共聚焦荧光显微镜，fluo-4）。此外，膜片钳技术将用于记录膜电位和离子通道电流。具体目标1研究IP 3R依赖性信号传导对心室肌细胞ECC发挥正性肌力作用的假设，特别是在心力衰竭期间。在这个目标中，将检查IP 3R依赖性信号传导对基础细胞内钙、动作电位诱导的钙瞬变和基本钙释放事件（钙火花和喷流）的影响。具体目标2测试IP 3R依赖性信号传导有助于心力衰竭心室肌细胞中的心肌发生的假设。在此，将研究IP 3R依赖性信号传导对促钙信号频率（自发钙释放、钙波和钙交替）和膜电位变化（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期间猝死的主要原因。预计该提案的结果将对心力衰竭期间心律失常的机制提供所需的见解，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.