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）Ca释放通道（也称为心脏ryanodine受体，RyR 2）功能异常，使RyR 2过度活跃，即Ca“泄漏”。在HF中，RyR 2由于SR内部的Ca对通道的异常调节而变得泄漏，这是一种正常运作以促进RyR 2在SR Ca释放后转变为不应状态的机制。RyR 2管腔Ca调节涉及与来自管腔侧的RyR 2相关的几种蛋白质的合作，包括三联蛋白1、连接蛋白和钙螯合蛋白。RyR 2活性增加将导致SR中的Ca耗尽，减少可用于收缩的Ca，可能导致HF中心脏收缩力减弱。此外，持续的Ca泄漏可能能够激活Ca依赖性激酶和磷酸酶，这些激酶和磷酸酶可以反馈RyR 2，从而导致更多的泄漏和Ca释放机制的进一步紊乱。提出了一个全面的研究计划，以确定负责这种RyR 2功能障碍的特定分子原因，及其在失败的心肌细胞的异常钙处理和HF的自然发展中的作用。我们的研究将使用体内技术和细胞和分子生理学方法的独特组合，包括膜片钳测量，细胞溶质和SR隔室中的Ca成像和单个重建RyR 2通道的记录。我们将使用与人类HF高度相关的慢性HF大型动物模型。本研究计划中将解决的具体问题包括：1）在钙处理改变中，SERCA 2介导的摄取改变、NCX清除减少和SR钙泄漏增加的相对作用是什么？2）衰竭心肌细胞的钙稳态缺陷能否通过RyR 2抑制剂或通过基因靶向参与RyR 2管腔钙依赖性调节的调节蛋白来正常化？3）异常的RyR 2门控行为是由RyR 2的异常磷酸化/去磷酸化和/或由与参与管腔Ca传感的管腔辅助蛋白的改变的相互作用引起的吗？和5）向终末期HF的转变是否涉及增强的RyR渗漏，以及治疗性干预例如心脏硬化治疗是否通过使异常RyR 2功能正常化而起作用。相关性：当心脏无法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，并且是65岁以上住院患者中最常见的诊断。我们建议研究心肌钙调节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.