E-C Coupling and Ca2+ Regulation in atrial myocytes

描述(申请人提供)：心律失常，包括房颤，是中风和心脏性猝死的高危因素。心脏交替是心脏易再入和致死性心律失常的关键致心律失常因素。在细胞水平，交替以电(动作电位时程)、机械和钙交替的形式出现。在心房细胞中，兴奋-收缩耦合过程中钙的释放在空间上是不均匀的。此外，心房细胞内的钙交替表现出亚细胞梯度和亚细胞区域的异相交替，导致自发性心律失常前钙释放和钙波的高倾向。膜电压(Vm)和[Ca]i调节是双向耦合的，因此Vm(时程恢复特性)和细胞内钙循环动力学的不稳定性导致机电和钙交替。偶联方式决定了交替性心律失常的形式和心律失常的严重程度。这项建议的总体目标是在单细胞水平上实验测试Vm和[Ca]i之间的双向耦合如何导致动作电位和钙交替，并产生有利于房性心律失常的条件。提出了以下具体目标：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：心房肌细胞时程重建受损导致起搏诱发时程和钙交替。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偶联)。假说：钙循环的两个关键参数相互依赖-(1)肌浆网钙释放与肌浆网负荷(部分释放)之间的非线性关系；(2)钙储存的效率(细胞内钙缓冲、SERCA对钙的储存、线粒体钙缓冲、钙排出)-导致动作电位和钙交替。具体目标#3：确定钙释放的恢复特性(整体钙瞬变和基本钙火花)与交替的关系。假设：在有利于[Ca]i驱动交替的条件下，基本钙释放事件的恢复缓慢，这有利于钙交替。为了实现这些目标，将使用多种实验技术：单个心房肌细胞激光扫描共聚焦显微镜高分辨率成像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研究膜电流和Vm(时程)，笼中钙的亚细胞光解，腺病毒基因转移，以及钙运输和缓冲的药物调控。实验将在成年猫心房肌细胞和从转基因小鼠分离的心房肌细胞上进行，这些转基因小鼠的钙处理蛋白(钙调素、磷蛋白)表达发生了变化。这项研究将为了解房性心律失常的细胞机制提供新的基础信息。公共卫生相关性：心脏交替(T波交替、脉搏交替)已被认为是心律失常和心脏性猝死的危险因素，并与最常见的心律失常形式--房颤直接相关。在细胞水平上，电(动作电位时程)和细胞内钙交替与致心律失常的钙释放相关。本研究试图在细胞和亚细胞水平确定心房肌细胞电活动和钙调节紊乱的机制，以利于交替和房性心律失常的发生。

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.