Ethanol Effects of SR Ca2+ Release in Cardiac Myocytes

描述（由申请人提供）： 酒精滥用最严重的副作用之一是发展为特定的酒精诱导的心肌病，其通常导致充血性心力衰竭。先前的研究结果表明，乙醇（EtOH）降低心脏功能的机制之一可能涉及心脏兴奋-收缩（E-C）偶联的直接抑制。有趣的是，这种直接效应似乎并不涉及心脏收缩触发（通过L型Ca 2+电流）的改变，这增加了EtOH的主要负性肌力作用可能存在于对该触发的反应改变中的可能性。本项目的目的是研究乙醇如何影响心肌细胞在心动周期中肌浆网（SR）内钙离子的释放。这将通过测量Ca 2+火花来实现，Ca 2+火花被认为是负责收缩的基本Ca 2+释放单位。本项目的具体目的是：1）确定乙醇的心脏舒张作用是否是由于直接抑制SR Ca 2+释放的结果; 2）研究这些SR Ca 2+释放的变化是否有助于酒精诱导的慢性心肌病模型中发生的心功能抑制。采用激光扫描共聚焦显微镜结合全细胞电压钳技术，以钙敏感荧光指示剂Fluo-4测定大鼠心室肌细胞中的钙火花。一些实验将在暴露于以下物质的皂苷透化的肌细胞中进行： 在缺乏功能性肌膜Ca 2+电流的情况下，用不同的胞浆Ca 2+浓度来评估单纯由Ca 2+激活的Ca 2+火花的Ca 2+敏感性。这些方法将允许调查乙醇如何影响SR Ca 2+释放，这是由于其正常触发（L型Ca 2+通道）和响应于E-C偶联的最终共同途径，由Ca 2+直接激活。除了研究乙醇对负责SR Ca 2+释放的生理和生物物理过程的直接影响外，我们还将把这些SR Ca 2+信号的变化与酒精诱导的心肌病的发展联系起来，以确定是否在整个器官水平上心输出量的减少是E-受体抑制的结果。本研究的结果将有助于我们了解乙醇干扰SR Ca 2+释放的机制，以及该机制是否涉及E-C偶联的触发或对该触发的反应。更重要的是，这些信息可以用于了解EtOH如何降低SR功能可能导致整体心脏功能的慢性抑制，导致与长期酒精滥用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.