Investigation of calcium modulation in cardiomyocytes by novel methods

通过新方法研究心肌细胞钙调节

• 批准号: 8435742
• 负责人: Steven O Marx
• 金额: $ 54.93万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8435742
• 关键词: Action Potentials; Adrenergic Agents; Adult; Affect; Anabolism; Arrhythmia; Binding; Biochemical; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcium; Calcium/calmodulin-dependent protein kinase; Calmodulin; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Characteristics; Chemosensitization; Complex; Coupling; Cyclic AMP-Dependent Protein Kinases; Development; Dihydropyridines; Disease; Doxycycline; Epitopes; Feedback; Frequencies; Functional disorder; Gene Expression; Goals; Health; Heart; Heart Hypertrophy; Heart failure; Homeostasis; Hormonal; Hypertrophy; Individual; Investigation; Link; Macromolecular Complexes; Methodology; Methods; Molecular; Molecular Probes; Mus; Muscle Cells; Mutation; Pathogenesis; Pathologic; Phase; Phosphorylation; Phosphorylation Site; Physiological; Play; Regulation; Reporter; Resistance; Role; Signal Pathway; Site; Staging; Stress; System; Techniques; Testing; Transgenic Mice; Transgenic Organisms; adrenergic; base; calmodulin-dependent protein kinase II; cell type; dihydropyridine; molecular pathology; mutant; novel; prevent; public health relevance; sensor; therapeutic target; tool; trafficking; voltage

DESCRIPTION (provided by applicant): CaV1.2, the sarcolemmal L-type Ca2+ channel, plays a key role in cardiac excitation-contraction coupling. Abnormalities in CaV1.2 function, including increased long-opening-mode gating and blunted adrenergic responsiveness, are associated with heart failure and hypertrophy. The increased activation of CaV1.2, in turn, triggers Ca2+-responsive signaling pathways, including those affecting gene expressions, which contribute to the pathogenesis of heart failure and hypertrophy. Not surprisingly, CaV1.2 is tightly regulated by components of cell type-specific macromolecular complexes that it anchors. A detailed molecular understanding of CaV1.2 regulation in myocytes has been hampered, however, by the inability to recapitulate and then dissect in heterologous expression systems key aspects of CaV1.2 function in myocytes. Our goals are to gain a better understanding of how CaV1.2 modulation by components of these macromolecular complex impacts cardiac contractility, the development of hypertrophy and heart failure, and the associated electrophysiological complications. We have developed novel tools to surmount major obstacles that have limited progress in the field, and allow us to probe molecular aspects of CaV1.2 regulation, using biochemical and electrophysiological techniques, within the context of cardiomyocytes, but with the power of a heterologous expression system. Using a transgenic (TG) approach that enables selective and reliable expression of FLAG-epitope tagged, dihydropyridine-resistant CaV1.2 channel subunits, harboring mutations at key regulatory sites or covalently linked to regulatory components, in adult cardiomyocytes and at all stages of development, we propose to determine in cardiomyocytes: (a) the role for proteolytic cleavage of the ¿1C C-terminus, the molecular mechanisms responsible for adrenergic regulation of CaV1.2 current and whether proteolytic cleavage is required for adrenergic regulation of Ca2+ influx in the heart; (b) the rol of Ca2+/calmodulin-dependent protein kinase (CaMKII) association with, and phosphorylation of, Ca2+ channel subunits in the regulation of CaV1.2 current; and (c) whether calmodulin (CaM) associated with the C-terminus of ¿1C regulates channel biosynthesis in cardiomyocytes in a Ca2+-dependent manner. Using novel methodologies to isolate Ca2+ currents from the TG channels and compare these currents to endogenous channels in the same cardiomyocyte, we will determine the molecular mechanisms of adrenergic and CaMKII regulation of CaV1.2 in cardiomyocytes and define how the Ca2+-sensitivity of CaM affects CaV1.2 trafficking in cardiomyocytes, both under physiological conditions and after initiation of heart failure. The three Aims, which should provide key new understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding cardiac pathologies and the molecular mechanisms responsible for the modulation of cardiac contractility.

描述（由申请人提供）：CaV1.2，肌膜 L 型 Ca2+ 通道，在心脏兴奋-收缩耦合中发挥关键作用 CaV1.2 功能异常，包括长开放模式门控增加和肾上腺素能反应性减弱，与心力衰竭和肥大相关。CaV1.2 激活的增加反过来会触发 Ca2+ 响应信号通路，包括影响基因的信号通路。毫不奇怪，CaV1.2 受到其锚定的细胞类型特异性大分子复合物成分的严格调控，对肌细胞中 CaV1.2 调控的详细分子了解受到阻碍。然而，由于无法在异源表达系统中重述和剖析 CaV1.2 在肌细胞中功能的关键方面，我们的目标是更好地了解 CaV1.2 如何调节。这些大分子复合物的成分影响心脏收缩力、肥厚和心力衰竭的发展以及相关的电生理并发症。我们开发了新的工具来克服该领域进展有限的主要障碍，并使我们能够探索 CaV1 的分子方面。 2 调节，使用生化和电生理学技术，在心肌细胞的背景下，但具有异源表达系统的力量，使用转基因（TG）方法，能够选择性和可靠地表达 FLAG 表位。在成年心肌细胞和所有发育阶段，标记的二氢吡啶抗性 CaV1.2 通道亚基在关键调节位点含有突变或与调节成分共价连接，我们建议在心肌细胞中确定：（a）蛋白水解裂解的作用这 1C C 末端，负责 CaV1.2 电流的肾上腺素调节的分子机制，以及心脏中 Ca2+ 流入的肾上腺素调节是否需要蛋白水解裂解；(b) Ca2+/钙调蛋白依赖性蛋白激酶 (CaMKII) 关联的作用； Ca2+通道亚基的磷酸化在CaV1.2电流的调节中的作用；以及(c)是否与 ¿ C 末端相关的钙调蛋白 (CaM) 1C 以 Ca2+ 依赖性方式调节心肌细胞中的通道生物合成。使用新方法从 TG 通道中分离 Ca2+ 电流，并将这些电流与同一心肌细胞中的内源性通道进行比较，我们将确定 CaV1 的肾上腺素能和 CaMKII 调节的分子机制。 2 在心肌细胞中，并定义 CaM 的 Ca2+ 敏感性如何影响心肌细胞中的 CaV1.2 运输，无论是在生理条件下还是在启动后这三个目标应该提供有关心肌细胞中 Ca2+ 内流调节的关键新理解，与理解心脏病理学和负责调节心肌收缩力的分子机制高度相关。