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: 8849960
• 负责人: Steven O Marx
• 金额: $ 54.86万
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8849960
• 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; 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; adrenergic; base; calmodulin-dependent protein kinase II; cell type; dihydropyridine; molecular pathology; mutant; novel; 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（由适用提供）：CAV1.2，Sarcolemmal L型Ca2+通道，在心脏兴奋 - 收缩耦合中起关键作用。 CAV1.2功能的异常，包括增加的长期开放模式门控和钝性的肾上腺素反应性，与心力衰竭和肥大有关。 CAV1.2的激活增加，反过来触发Ca2+​​反应信号传导途径，包括影响基因表达的那些，这有助于心力衰竭和肥大的发病机理。毫不奇怪，CAV1.2受锚定的细胞类型特异性大分子复合物的成分严格调节。然而，对肌细胞中CAV1.2调节的详细分子理解受到阻碍，但是，无法概括并在异源表达系统中剖析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通过这些大分子复合物的组成部分调节如何影响心脏收缩力，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该领域进展有限的主要障碍，并让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探测Cav1.2调节的分子方面，并使用生化和电生理技术，但使用转基因（TG）方法，可实现经过的转基因（TG）方法，以实现选择性和可靠的标记标签的表达。亚基，在关键的监管部位有突变或与调节成分，成人心肌细胞中的共同关联以及在各个发育阶段的共同关系，我们提议在心肌细胞中确定：（a）pec- terminus and covuntion praventimist and covuntion intressigant and pavenrigation的蛋白水解裂解的作用。是对心脏中Ca2+影响的肾上腺素调节所必需的； （b）Ca2+/钙调蛋白依赖性蛋白激酶（CAMKII）的ROL与Cav1.2电流调节中Ca2+通道亚基的磷酸化和磷酸化； （c）与1C的C末端相关的钙调蛋白（CAM）是否以Ca2+依赖性方式调节心肌细胞中的通道生物合成。使用新的方法将Ca2+电流与TG通道分离并将这些电流与同一心肌细胞中的内源通道进行比较，我们将确定心肌细胞中CAV1.2的肾上腺素和CAMKII调节的分子机制心力衰竭。这三个目标应提供有关心肌细胞中Ca2+影响的关键新理解，与理解心脏病理和负责调节心脏收缩力的分子机制高度相关。