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: 9065601
• 负责人: Steven O Marx
• 金额: $ 55.7万
• 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 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9065601
• 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; Electrophysiology (science); Epitopes; Feedback; Frequencies; 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; Myocardial dysfunction; Pathogenesis; Pathologic; Phase; Phosphorylation; Phosphorylation Site; Physiological; Play; Regulation; Reporter; Resistance; Role; Signal Pathway; Site; Staging; Stress; System; Techniques; Testing; Transgenic Mice; Transgenic Organisms; base; calmodulin-dependent protein kinase II; cell type; molecular pathology; mutant; novel; novel therapeutics; prevent; sensor; targeted treatment; 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是肌膜L型Ca 2+通道，在心脏兴奋-收缩偶联中起关键作用。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，与心力衰竭和心肌肥厚相关。CaV1.2的激活增加，反过来，触发Ca 2+响应信号通路，包括那些影响基因表达，这有助于心力衰竭和肥大的发病机制。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的组分的严格调控。然而，由于无法概括并在异源表达系统中剖析肌细胞中CaV1.2功能的关键方面，对肌细胞中CaV1.2调节的详细分子理解受到阻碍。我们的目标是更好地了解这些大分子复合物的组分如何调节CaV1.2影响心肌收缩力，肥大和心力衰竭的发展以及相关的电生理并发症。我们已经开发了新的工具来克服在该领域取得有限进展的主要障碍，并使我们能够在心肌细胞的背景下，使用生物化学和电生理技术，但具有异源表达系统的力量，来探测CaV1.2调节的分子方面。使用转基因（TG）方法，使选择性和可靠的表达FLAG-表位标记，二氢吡啶抗性CaV1.2通道亚基，窝藏在关键调控位点突变或共价连接到调控成分，在成年心肌细胞和发育的所有阶段，我们建议在心肌细胞中确定：（a）蛋白水解切割的作用？1C C-末端，负责肾上腺素能调节CaV1.2电流的分子机制，以及心脏中肾上腺素能调节Ca 2+内流是否需要蛋白水解裂解;（B）Ca 2 +/钙调蛋白依赖性蛋白激酶（CaMK II）与Ca 2+通道亚基的结合以及Ca 2+通道亚基的磷酸化在CaV 1.2电流调节中的作用;和（c）与1C的C-末端相关的钙调蛋白（CaM）是否以Ca 2+依赖性方式调节心肌细胞中的通道生物合成。使用新的方法来分离钙电流从TG通道和比较这些电流在同一心肌细胞的内源性通道，我们将确定肾上腺素和CaMKII调节心肌细胞中的CaV1.2的分子机制，并定义钙调素的钙敏感性如何影响CaV1.2在心肌细胞中的运输，无论是在生理条件下，并开始心力衰竭。这三个目标，这应该提供关键的新的理解有关的Ca 2+流入心肌细胞的调节，是高度相关的了解心脏病理和负责心脏收缩力的调制的分子机制。