Abnormal Intracellular Calcium Release in Heart Failure

描述(由申请人提供)：本提案的总体目标是了解细胞内钙处理改变在心力衰竭病理生理学中的作用。该提案的重点是最近发现的肌浆网(SR)钙释放通道(也称为心脏Ryanodine受体，RyR2)功能异常，使RyR2过度活跃，即钙“泄漏”。在HF中，由于SR内部的Ca对通道的异常调制，RyR2变得泄漏，这是一种通常操作以促进RyR2在SR Ca释放后转变为难解状态的机制。RyR2腔内钙调节涉及腔侧与RyR2相关的几种蛋白质的协同作用，包括Triadin 1、Junctin和Calequestrin。RyR2活性的增加将导致SR中钙的耗竭，减少可用于收缩的钙，潜在地导致心力衰竭时心肌收缩力量的减弱。此外，持续的钙泄漏可能能够激活钙依赖的激酶和磷酸酶，这些酶可以反馈到RyR2，从而导致更多的钙泄漏和钙释放机制的进一步紊乱。一个全面的研究计划被提出，以确定导致这种RyR2功能障碍的特定分子原因，以及它在衰竭心肌细胞的异常钙处理和HF的自然发展中所起的作用。我们的研究将使用细胞和分子生理学的体内技术和方法的独特组合，包括膜片钳测量、胞浆和SR室的钙成像以及从单个重组的RyR2通道记录。我们将使用与人类心力衰竭高度相关的慢性心力衰竭的大型动物模型。这项研究将解决的具体问题包括：1)改变的SERCA2介导的摄取、减少的NCX清除和增加的SR钙泄漏在改变的钙处理中起什么相对作用；2)衰竭心肌细胞的钙稳态缺陷能否通过RyR2抑制剂或通过基因靶向参与RyR2管腔钙依赖调节的调节蛋白来正常化；3)异常的RyR2门控行为是由RyR2的异常磷酸化/去磷酸化和/或与参与管腔钙感知的管腔辅助蛋白的改变相互作用引起的；4)异常的钙处理是心衰的原因还是结果；5)过渡到终末期心衰是否涉及RyR漏增强，心脏再同步化治疗等治疗干预是否通过使异常RyR2功能正常化起作用。相关性：当心脏不能泵出足够的血液来满足身体的需要时，就会发生心力衰竭。心力衰竭在美国人口中持续增加，是65岁以上住院患者最常见的诊断。我们建议研究心肌钙调节的异常如何导致心力衰竭。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the role of altered intracellular Ca handling in the pathophysiology of heart failure. The proposal focuses on a recently discovered abnormality in the function of the sarcoplasmic reticulum (SR) Ca release channel (also known as the cardiac ryanodine receptor, RyR2) that makes the RyR2 overly active, i.e. "leaky" for Ca. In HF, the RyR2 becomes leaky due to abnormal modulation of the channel by Ca from inside the SR, a mechanism that normally operates to facilitate the transition of the RyR2s into a refractory state following SR Ca release. RyR2 luminal Ca regulation involves cooperation of several proteins associated with the RyR2 from the luminal side, including triadin 1, junctin and calsequestrin. Increased RyR2 activity would lead to Ca depletion in the SR, reducing Ca available for contraction, potentially contributing to the weakened cardiac contractile force in HF. Additionally, a sustained Ca leak might be capable of activating Ca dependent kinases and phosphatases that can feed back on RyR2s to cause more leak and a further derangement of the Ca release machinery. A comprehensive research plan is proposed to define the specific molecular causes responsible for this RyR2 dysfunction and its role in abnormal Ca handling of failing myocytes and in the natural development of HF. Our studies will use a unique combination of in vivo techniques and methods of cellular and molecular physiology, including patch clamp measurements, Ca imaging in the cytosolic and SR compartments and recording from single reconstituted RyR2 channels. We will use a large animal model of chronic HF which is highly relevant to human HF. The specific questions that will be addressed in this research proposal include: 1) What are the relative roles of altered SERCA2-mediated uptake, reduced NCX removal and enhanced SR Ca leak in altered Ca handling; 2) Can the defective Ca homeostasis of failing myocytes be normalized by RyR2 inhibitors or by genetically targeting regulatory proteins involved in RyR2 luminal Ca-dependent modulation; 3) Is abnormal RyR2 gating behavior is caused by abnormal phosphorylation/dephosphorylation of the RyR2 and/or by altered interactions with luminal auxiliary proteins involved in luminal Ca sensing; 4) Is abnormal Ca handling a cause or a consequence of HF; and 5) Does transition to end stage HF involve an enhanced RyR leak, and do therapeutic interventions such as cardiac resynchronization therapy act by normalizing abnormal RyR2 function. RELEVANCE: Heart failure occurs when the heart is unable to pump enough blood to meet the needs of the body. Heart failure continues to increase in the U.S. population and is the most common diagnosis in hospitalized patients over the age of 65. We propose to study how abnormalities in the regulation of calcium in the heart muscle contribute to heart failure.