E-C Coupling and Ca2+ Regulation in atrial myocytes

描述（由申请人提供）：心律失常（包括房颤）是卒中和心源性猝死的高风险因素。心脏电交替是诱发心脏折返和致命性心律失常的关键致心律失常因素。在细胞水平，交替发生电（动作电位时程，APD），机械和钙交替。在心房细胞中，兴奋-收缩偶联期间的Ca释放在空间上是不均匀的。此外，心房细胞中的Ca交替显示亚细胞梯度和亚细胞区域交替异相，导致自发促钙释放和Ca波的高倾向。膜电压（Vm）和[Ca]i调节是双向耦合的，使得Vm（APD恢复特性）和细胞内Ca循环动力学的不稳定性导致机电和Ca交替。偶联方式决定了交替的形式和染色体畸变的严重程度。本提案的总体目标是在单细胞水平上通过实验测试Vm和[Ca]i之间的双向耦合如何导致APD和Ca交替并产生有利于房性心律失常的条件。具体目标1：确定Vm中的不稳定性如何影响[Ca]i并导致交替（Vm？[Ca]i耦合）。假设：心房肌细胞APD恢复受损导致起搏诱导的APD和Ca交替。具体目标2：确定钙循环中的不稳定性如何影响Vm并导致交替（[Ca]i？Vm耦合）。假设：心搏间钙循环的两个关键参数的相互依赖性-（1）SR钙释放和SR负荷（部分释放）之间的非线性关系和（2）钙螯合效率（细胞溶质钙缓冲、SERCA的钙螯合、线粒体钙缓冲、钙排出）-导致APD和钙交替。具体目标#3：确定钙释放的恢复特性（全局钙瞬变和基本钙火花）如何与交替相关。假设：在有利于[Ca]i驱动的交替的条件下，基本Ca释放事件的恢复减慢，这有利于Ca交替。为了实现这些目标，将使用多种实验技术：通过激光扫描共聚焦显微镜在单个心房肌细胞中进行高分辨率成像以测量全细胞和亚细胞[Ca]i、[Ca]mito和[Ca]SR，全细胞电压和电流钳技术以研究膜电流和Vm（APD），笼状Ca的亚细胞光解，腺病毒基因转移以及Ca转运和缓冲的药理学操作。实验将在成年猫心房肌细胞和分离自Ca处理蛋白（钙螯合蛋白、受磷蛋白）表达改变的转基因小鼠的心房肌细胞上进行。该研究将为理解房性心律失常的APD和交替钙的细胞机制提供新的基础信息。公共卫生相关性：心脏电交替（T波电交替，脉冲电交替）已被认为是心律失常和心脏性猝死的危险因素，并已直接与心房颤动，最常见的心律失常形式。在细胞水平，电（动作电位时程）和细胞内钙交替与促炎性钙释放相关。本研究旨在确定在心房肌细胞，在细胞和亚细胞水平上，有利于交替和房性心律失常的发生的电活动和钙调节的紊乱的机制。

DESCRIPTION (provided by applicant): Cardiac arrhythmias, including atrial fibrillation, are a high risk factor for stroke and sudden cardiac death. Cardiac alternans is a key arrhythmogenic factor predisposing the heart to re-entry and lethal arrhythmias. At the cellular level, alternans occurs as electrical (action potential duration, APD), mechanical and Ca alternans. In atrial cells Ca release during excitation-contraction coupling is spatially inhomogeneous. Furthermore, Ca alternans in atrial cells reveals subcellular gradients and subcellular regions alternating out-of-phase, leading to a high propensity of spontaneous pro-arrhythmic Ca release and Ca waves. Membrane voltage (Vm) and [Ca]i regulation are bidirectionally coupled, such that instabilities in Vm (APD restitution properties) and intracellular Ca cycling dynamics result in electromechanical and Ca alternans. The mode of coupling determines the form of alternans and severity of arrhythmogenesis. The overall goal of this proposal is to test experimentally at the single cell level how bidirectional coupling between Vm and [Ca]i leads to APD and Ca alternans and generates conditions that favor atrial arrhythmia. The following specific aims are proposed: Specific aim 1: determine how instabilities in Vm affect [Ca]i and lead to alternans (Vm?[Ca]i coupling). Hypothesis: impaired APD restitution in atrial myocytes leads to pacing-induced APD and Ca alternans. Specific aim 2: determine how instabilities in Ca cycling affect Vm and lead to alternans ([Ca]i ?Vm coupling). Hypothesis: the interdependence of 2 key parameters of beat-to-beat Ca cycling - (1) the non-linear relationship between SR Ca release and SR load (fractional release) and (2) the efficiency of Ca sequestration (cytosolic Ca buffering, Ca sequestration by SERCA, mitochondrial Ca buffering, Ca extrusion) - lead to APD and Ca alternans. Specific aim #3: determine how the restitution properties of Ca release (global Ca transient and elementary Ca sparks) are related to alternans. Hypothesis: under conditions that favor [Ca]i -driven alternans the restitution of elementary Ca release events is slowed which favors Ca alternans. To achieve these aims a multitude of experimental techniques will be used: high resolution imaging by laser scanning confocal microscopy in single atrial myocytes to measure whole cell and subcellular [Ca]i, [Ca]mito and [Ca]SR, whole-cell voltage and current clamp techniques to study membrane currents and Vm (APD), subcellular photolysis of caged Ca, adenoviral gene-transfer, and pharmacological manipulation of Ca transport and buffering. Experiments will be conducted on adult cat atrial myocytes and atrial myocytes isolated from transgenic mice with altered expression of Ca handling proteins (calsequestrin, phospholamban). The proposed research will provide fundamental new information on the cellular mechanism of APD and Ca alternans relevant to the understanding of atrial arrhythmias. PUBLIC HEALTH RELEVANCE: Cardiac alternans (T-wave alternans, pulsus alternans) has been recognized as a risk factor for cardiac arrhythmia and sudden cardiac death, and has been linked directly to atrial fibrillation, the most common form of cardiac arrhythmia. At the cellular level electrical (action potential duration) and intracellular calcium alternans correlate with arrhythmogenic calcium release. This study seeks to determine in atrial myocytes, at the cellular and subcellular level, the mechanisms of disturbances of electrical activity and calcium regulation that favor the occurrence of alternans and atrial arrhythmia.