E-C Coupling and Ca2+ Regulation atrial myocytes

描述（由申请人提供）：在兴奋-收缩（e-c）偶联期间，心房肌细胞中肌浆网（SR）的Ca 2+释放与心室肌细胞显着不同。心房肌细胞缺乏横小管，并有两种不同类型的SR，交界性（j-SR）和非交界性SR（nj-SR）。钙离子在电-钙耦合过程中的释放是空间不均匀的。Ca 2+诱导的j-SR和nj-SR的Ca 2+释放（CICR）由明显不同的机制调节。在心房肌细胞中，IP 3依赖性Ca 2+信号传导调节电偶联期间的Ca 2+信号传导，心肌收缩力受自主神经系统调节。β-肾上腺素能受体（betaa-AR）信号传导通过涉及G蛋白、蛋白激酶、一氧化氮（NO）和Ca 2+的细胞内信号传导途径介导心脏功能的交感神经调节。Ca 2+交替反映了在正常起搏频率下，导致电机械交替的Ca 2+瞬时幅度的变化。心房Ca 2+交替与房性心律失常的产生直接相关，房性心律失常是心血管发病率和死亡率的主要原因。拟议研究的总体目标是阐明与正常心房e-c偶联及其扰动相关的机制和信号通路，这些扰动导致Ca 2+交替，从而导致心房组织中的致心律失常行为。建议的具体目标如下： 具体目标#1：确定肌醇-磷酸（IP 3）信号转导在e-c偶联过程中控制Ca 2+信号转导的亚细胞机制。 具体目标#2确定a-肾上腺素能信号调节e-c偶联过程中j-SR和nj-SR的Ca 2+释放的机制。 具体目标#3阐明IP 3-、a-AR-和NO-依赖性信号转导障碍导致Ca 2+交替的机制。 为了实现这些目标，将使用多种实验技术：在单个心房肌细胞中通过激光扫描共聚焦显微镜进行的高分辨率[Ca 2 +]i成像，研究膜电流的全细胞电压钳技术，通过重构成平面脂质双层的心脏SR Ca 2+释放通道进行的单通道记录，笼状Ca 2+和IP 3的亚细胞光解，以及α-肾上腺素能调节的药理学操作，IP 3信号传导和Ca 2+进入、释放和摄取。 这项研究将为在正常和改变的房性心律失常相关条件下调节心房e-c偶联和Ca 2+释放提供基本的新信息。

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.