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.

说明(申请人提供)：CaV1.2，肌膜L型钙通道，在心肌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迟钝，与心力衰竭和肥厚有关。CaV1.2的激活增加，进而触发钙反应信号通路，包括那些影响基因表达的信号通路，从而导致心力衰竭和肥厚的发病机制。不足为奇的是，CaV1.2受到它锚定的细胞类型特定的大分子复合体成分的严格调控。然而，由于不能概括和剖析异源表达系统中CaV1.2在心肌细胞中功能的关键方面，阻碍了对CaV1.2在心肌细胞中调控的详细分子理解。我们的目标是更好地了解这些大分子复合体的组成成分对CaV1.2的调制如何影响心肌收缩能力、肥厚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进展的主要障碍，并允许我们使用生化和电生理技术在心肌细胞的背景下探索CaV1.2调控的分子方面，但具有异源表达系统的能力。利用转基因(TG)方法，在成年心肌细胞和发育的所有阶段，能够选择性和可靠地表达标志表位标记的、二氢吡啶抗性的CaV1.2通道亚单位，这些亚单位在关键调控部位存在突变或共价连接到调控成分，我们建议在心肌细胞中确定：(A)1C末端的蛋白水解性切割的作用，负责CaV1.2电流的肾上腺素能调节的分子机制，以及是否需要蛋白水解性裂解来调节心脏内的钙离子内流；(B)钙/钙调蛋白依赖性蛋白激酶(CaMKII)在CaV1.2电流调节中与钙通道亚单位的结合和磷酸化的作用；以及(C)钙调素(CaM)是否以钙依赖的方式调节心肌细胞的通道生物合成。利用新的方法分离TG通道中的钙电流，并将这些电流与同一心肌细胞中的内源性通道进行比较，我们将确定心肌细胞中CaV1.2的肾上腺素能和CaMKII调节的分子机制，并确定在生理条件下和心力衰竭开始后，CaM对钙的敏感性如何影响CaV1.2在心肌细胞中的运输。这三个目标对于理解心脏病理和心肌收缩调节的分子机制具有重要意义，为心肌细胞内钙内流的调控提供了关键的新认识。