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中的作用。该建议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，RyR2）功能异常，该异常使RyR2过度活跃，即。在HF中，由于Ca在SR内部对通道的异常调制，RyR2变得泄漏，这一机制通常是在SR Ca释放后促进RyR2s转变为难熔状态。RyR2腔内钙调控涉及与RyR2腔内侧相关的几种蛋白的合作，包括triadin 1、junctin和calsequestrin。RyR2活性的增加会导致SR中的Ca消耗，减少可用于收缩的Ca，可能导致HF中心脏收缩力减弱。此外，持续的钙泄漏可能能够激活钙依赖性激酶和磷酸酶，这些激酶和磷酸酶可以反馈到RyR2s上，导致更多的泄漏和钙释放机制的进一步紊乱。我们提出了一项全面的研究计划，以确定导致RyR2功能障碍的具体分子原因及其在衰竭肌细胞异常钙处理和HF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，细胞质和SR室的Ca成像以及单个重构RyR2通道的记录。我们将使用与人类HF高度相关的大型慢性HF动物模型。本研究计划将解决的具体问题包括：1)在改变的Ca处理中，改变的serca2介导的摄取、减少的NCX去除和增强的SR Ca泄漏的相对作用是什么；2)衰竭肌细胞钙稳态缺陷是否可以通过RyR2抑制剂或基因靶向RyR2腔内钙依赖调节的调控蛋白实现正常化；3)异常的RyR2门控行为是由RyR2的异常磷酸化/去磷酸化和/或与参与腔内Ca感应的腔内辅助蛋白的相互作用改变引起的吗？4)异常Ca处理是HF的原因还是结果；5)过渡到终末期HF是否涉及RyR泄漏的增强，以及心脏再同步化治疗等治疗干预是否通过使异常的RyR2功能正常化而起作用。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岁以上住院患者中最常见的诊断。我们建议研究心肌钙调节异常是如何导致心力衰竭的。

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.