E-C Coupling and Ca2+ Regulation atrial myocytes

描述(申请人提供)：在兴奋-收缩(e-c)耦合过程中，心房肌细胞从肌浆网(SR)释放的钙明显不同于心室肌细胞。心房肌细胞缺乏横管，有两种不同类型的SR，交界性SR(j-SR)和非交界性SR(NJ-SR)。E-c耦合过程中Ca2的释放在空间上是不均匀的。钙诱导的钙释放(CICR)在j-SR和NJ-SR上的调节机制截然不同。在心房肌细胞中，依赖于IP3的钙信号在e-c偶联过程中调节钙信号，心脏的收缩能力受自主神经系统的调节。β-肾上腺素能受体(β-AR)信号通过G蛋白、蛋白激酶、一氧化氮(NO)和钙离子等细胞内信号通路介导交感神经调节心功能。CA2交替反映了在规则起搏频率下钙瞬变幅度的变化，从而导致机电交替。房性钙交替与房性心律失常的发生直接相关，房性心律失常是心血管疾病发病率和死亡率的主要因素。这项研究的总体目标是阐明与正常的心房e-c偶联相关的机制和信号通路以及它们的扰动，从而导致心房组织中的钙交替，从而导致心律失常的行为。提出了以下具体目标： 具体目标1：确定在e-c偶联过程中，肌醇-磷酸(IP3)信号支配钙信号的亚细胞机制。 具体目的#2.确定在e-c偶联过程中，α-肾上腺素能信号调节j-SR和Nj-SR释放钙的机制。 具体目的#3.阐明依赖IP_3、α-AR和NO的信号传导干扰(S)导致钙离子代谢紊乱的机制。 为了实现这些目标，将使用多种实验技术：单个心房肌细胞高分辨率激光扫描共聚焦显微镜成像，研究膜电流的全细胞电压钳技术，通过重构为平面脂质双层的心脏SR钙释放通道的单通道记录，笼中钙和IP3的亚细胞光解，以及对α-肾上腺素能调节、IP3信号和钙进入、释放和摄取的药物调控。 这项研究将为房性心律失常相关的正常和异常状态下的心房e-c偶联和钙释放的调节提供新的基础信息。

DESCRIPTION (provided by applicant): During excitation-contraction (e-c) coupling Ca2+ release from the sarcoplasmic reticulum (SR) in atrial myocytes differs significantly from ventricular myocytes. Atrial myocytes lack transverse tubules and have two different types of SR, junctional (j-SR) and non-junctional SR (nj-SR). Ca2+ release during e-c coupling is spatially inhomogeneous. Ca2+-induced Ca2+-release (CICR) from j-SR and nj-SR is regulated by distinctly different mechanisms. In atrial myocytes IP3-dependent Ca2+ signalling modulates Ca2+ signaling during e-c coupling and cardiac contractility is regulated by the autonomic nervous system. Beta-adrenergic receptor (betaa-AR) signaling mediates symapathetic regulation of cardiac function through intracellular signaling pathways involving G-proteins, protein kinases, nitric oxide (NO) and Ca2+. Ca2+ alternans reflects the alternations of the Ca2+ transient amplitude at regular pacing frequency which results in electromechanical alternans. Atrial Ca2+ alternans are directly related to the generation of atrial arrhythmias which is a major contributor to cardiovascular morbidity and mortality. The overall goal of the proposed study is to elucidate mechanisms and signalling pathways that are relevant to normal atrial e-c coupling and their perturbations which lead to Ca2+ alternans and therefore arrhythmogenic behavior in atrial tissue. The following Specific Aims are proposed: Specific Aim #1: Determine the subcellular mechanisms by which inositol-phosphate (IP3) signaling governs Ca2+ signaling during e-c coupling. Specific Aim #2. Determine the mechanisms by which a-adrenergic signaling regulates Ca2+ release from j-SR and nj-SR during e-c coupling. Specific Aim #3. Elucidate the mechanisms through which disturbance(s) of IP3-, a-AR- and NO-dependent signaling leads to Ca2+ aiternans. To achieve these aims a multitude of experimental techniques will be used: high resolution [Ca2+]i imaging by laser scanning confocal microscopy in single atrial myocytes, whole-cell voltage clamp techniques to study membrane currents, single channel recordings through cardiac SR Ca2+ release channels reconstituted into planar lipid bilayers, subcellular photolysisof caged Ca2+ and IP3, and pharmacological manipulation of a-adrenergic regulation, IP3 signaling and Ca2+ entry, release and uptake. The proposed research will provide fundamental new information on the regulation of atrial e-c coupling and Ca2+ release under normal and altered conditions relevant to atrial arrhythmias.