Ethanol Effects of SR Ca2+ Release in Cardiac Myocytes

描述(由申请人提供)： 酒精滥用最严重的次要影响之一是发展特定的酒精引起的心肌病，这通常会导致充血性心力衰竭。以往的研究结果表明，乙醇降低心功能的机制之一可能与直接抑制心脏兴奋-收缩(E-C)偶联有关。有趣的是，这种直接效应似乎并不涉及心脏收缩触发器的改变(通过L型钙电流)，这增加了乙醇的主要负性肌力作用可能存在于对该触发器的改变的反应中。本项目的目的是研究在心脏周期中，乙醇如何影响肌浆网(SR)内库钙的释放。这将通过测量钙火花来实现，钙火花被认为是负责收缩的基本钙释放单位。本项目的具体目的是：1)确定乙醇的心脏抑制作用是否是直接抑制肌浆网钙释放的结果；2)研究肌浆网钙释放的这些变化是否与慢性酒精性心肌病模型的心功能抑制有关。采用激光扫描共聚焦显微镜结合全细胞电压钳技术，用钙离子敏感的荧光指示剂Fluo-4测定大鼠心室肌细胞内的钙火花。一些实验将在皂素通透性的心肌细胞中进行 不同的细胞内钙离子浓度，以评估在没有功能性肌膜钙电流的情况下，单纯由钙离子激活的钙火花对钙的敏感性。这些方法将有助于研究EtoH如何影响SR钙释放，这既是由于其正常触发(L型钙通道)的结果，也是对E-C偶联的最终共同途径--钙直接激活的反应。除了研究乙醇对肌浆网钙释放的生理和生物物理过程的直接影响外，我们还将把肌浆网钙信号的这些变化与酒精性心肌病的发生联系起来，以确定整个器官水平的心输出量减少是否是由于肌浆网水平的E-C偶联被抑制所致。这一研究结果将有助于我们理解乙醇干扰肌浆网钙释放的机制，以及这一机制是否涉及E-C偶联的触发或对该触发的反应。更重要的是，这些信息可以用来理解乙醇对SR功能的降低如何有助于整体心脏功能的慢性抑制，从而导致与长期酗酒相关的心肌病和心力衰竭的发展。

DESCRIPTION (provided by applicant): One of the most serious secondary effects of alcohol abuse is the development of specific alcohol-induced cardiomyopathies which often lead to congestive heart failure. Previous findings suggest that one of the mechanisms underlying reduced cardiac function by ethanol (EtOH) may involve the direct suppression of cardiac excitation-contraction (E-C) coupling. Interestingly, this direct effect does not appear to involve alterations in the trigger for cardiac contraction (via L-type Ca 2+ current) raising the possibility that the primary negative inotropic effects of EtOH might reside in an altered response to that trigger. The goal of this project is to investigate how EtOH affects Ca2+ release from internal stores in the sarcoplasmic reticulum (SR) during the cardiac cycle. This will be accomplished through measurement of Ca 2+ sparks, which are thought to represent the fundamental Ca 2+ release units responsible for contraction. The Specific Aims of this project are: 1) to determine if the cardiodepressant effects of EtOH occur as the result of a direct suppression of SR Ca 2+ release; and 2) to investigate whether or not these changes in SR Ca 2+ release contribute to the depression in cardiac function that occurs in a chronic model of alcohol-induced cardiomyopathy. Ca 2+ sparks will be measured in rat ventricular myocytes with the Ca2+-sensitive fluorescent indicator fluo-4 using laser scanning confocal microscopy in combination with whole cell voltage clamp techniques. Some experiments will be performed in saponin-permeabilized myocytes exposed to different cytosolic Ca2+ concentrations in order to assess Ca 2+ sensitivity of Ca 2+ sparks activated purely by Ca 2+ in the absence of functional sarcolemmal Ca 2+ current. These methodological approaches will allow the investigation of how EtOH affects SR Ca 2+ release that occurs both as a result of its normal trigger (L-type Ca 2+ channels) and in response to the final common pathway for E-C coupling, direct activation by Ca 2+. In addition to the study of direct effects of EtOH on the physiological and biophysical processes responsible for SR Ca 2+ release, we will also correlate these changes in SR Ca 2+ signaling with the development of alcohol-induced cardiomyopathy in order to determine if the reduction of cardiac output at the whole organ level occurs as the result of a suppression of E-C coupling at the level of the SR. The results of this study will contribute to our understanding of the mechanisms by which EtOH interferes with SR Ca 2+ release and whether or not this mechanism involves the trigger for E-C coupling or the response to that trigger. More importantly, such information can then be applied to understanding how a reduction in SR function by EtOH might contribute to chronic suppression of overall cardiac function, leading to the development of cardiomyopathies and heart failure associated with long-term alcohol abuse.