Regulators and interactomes of CaV1.2 in health and disease

CaV1.2 在健康和疾病中的调节因子和相互作用组

• 批准号: 10534139
• 负责人: Jared S Kushner
• 金额: $ 16.55万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-15 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10534139
• 关键词: 26S proteasome; APEX1 gene; Adenylate Cyclase; Adrenergic Agents; Adrenergic Agonists; Arrhythmia; Binding; Binding Proteins; Binding Sites; Biochemical; Biology; Biotin; Bungarotoxins; Bypass; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular Physiology; Congestive Heart Failure; Coupling; Cyclic AMP-Dependent Protein Kinases; Defect; Development Plans; Disease; Disinhibition; Doxycycline; Education; Electrophysiology (science); Engineering; Environment; Enzymes; Exertion; Flow Cytometry; Forskolin; Frequencies; Functional disorder; Galectin 1; Gene Expression; Genetic; Genetic Models; Goals; Grant; Health; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Ion Channel; Knock-out; Knockout Mice; L-type calcium channel alpha(1C); Label; Lectin; Ligation; Longevity; MG132; Maps; Masks; Mass Spectrum Analysis; Measures; Mediating; Membrane; Mentorship; Molecular; Mus; Muscle Cells; Neighborhoods; Organ; Pathway interactions; Patient Care; Patients; Perfusion; Peroxidases; Pharmaceutical Preparations; Phosphodiesterase Inhibitors; Phosphorylation; Physicians; Play; Probability; Proteasome Inhibitor; Proteins; Proteome; Proteomics; RNA Splicing; Regulation; Research; Research Project Grants; Role; Scientist; Shortness of Breath; Signal Pathway; Signal Transduction; Site; Surface; Sympathetic Nervous System; Systolic heart failure; Techniques; Training; Training Programs; Transgenic Mice; Transgenic Organisms; Ubiquitination; Universities; Up-Regulation; Variant; Work; Writing; alpha-bungarotoxin receptor; ascorbate; beta-adrenergic receptor; cardioprotection; career development; density; desensitization; design; experience; experimental study; extracellular; fluorophore; improved; inhibitor; innovation; insight; interest; large datasets; mouse model; multidisciplinary; novel; prevent; protein activation; protein degradation; protein expression; protein reconstitution; response; skills; success; trafficking; translational scientist

This proposal outlines a comprehensive 5-year training program to develop Jared Kushner, MD, into an independent translational investigator. Dr. Kushner is a general cardiologist and physician-scientist whose ultimate goal is to improve care for patients with heart disease. To reach this goal, he is interested in understanding how abnormal ion channel function in the heart can lead to cardiovascular disease and conversely, how cardiovascular disease can cause maladaptive changes in ion channel regulation. In order to become an independent translational scientist and leader in this field, Dr. Kushner has developed a career development plan designed to fill specific educational and experiential gaps in his training. These short-term goals include: 1) Receiving advanced training in cellular electrophysiology, with an emphasis on techniques that probe ion channel function in their native milieu; 2) Acquiring experience in the analysis of large datasets, with the goal of interpreting large-scale changes in gene and protein expression; 3) Developing expertise in proteomics and the use of mass spectrometry; 4) Refining his current skills and developing new skills in cardiovascular physiology, with emphasis on the experimental assessment of load-independent measures of systolic and diastolic function; 5) Acquiring expertise in probing pathways of protein degradation, with emphasis on the destruction of ion channels in health and disease; and 5) Developing skills critical to his long- term success, with particular emphasis on his grant-writing skills. Columbia University, with its rich and supportive research environment, proved to be an ideal setting for Dr. Kushner to organize a multi-disciplinary mentorship team with the expertise to accomplish these goals. In his proposed research project, Dr. Kushner will use results from proximity-labeling the interactome of the L-type Ca2+ channel, CaV1.2, to gain insights into regulators of the channel essential to its function in normal biology and in heart failure (HF), a condition characterized by reduced systolic function and adrenergic reserve, often experienced by patients as increased shortness of breath with exertion. Using transgenic mice with cardiac-specific expression of CaV1.2 subunits fused to the engineered ascorbate peroxidase, APEX2, he probed the changing channel microenvironment in hearts of mice that developed chronic HF. He identified significant changes in 71 of over 2000 proteins in the vicinity of CaV1.2 in HF. To determine if those proteins perturb CaV1.2 function, he will analyze channel activity, contractility, and adrenergic responsiveness in genetic knockouts of APEX-identified proteins. To measure effects on CaV1.2 expression, he will use a transgenic mouse expressing a YFP-tagged α1C containing a bungarotoxin-binding site on an extracellular loop (BBS- α1C). Treating isolated myocytes with fluorophore-conjugated bungarotoxin, he can track total and surface expression of CaV1.2 with flow-cytometry. Generating HF in BBS-α1C mice crossed with knockouts of APEX- identified proteins in HF, he can identify those neighboring proteins that alter CaV1.2 expression of HF.

该提案概述了一项为期 5 年的综合培训计划，旨在将医学博士贾里德·库什纳 (Jared Kushner) 培养成一名 库什纳博士是一位普通心脏病学家和医师科学家。 最终目标是改善对心脏病患者的护理。为了实现这一目标，他很感兴趣。 了解心脏离子通道功能异常如何导致心血管疾病和 相反，心血管疾病如何导致离子通道调节的适应不良变化。 成为一名独立的转化科学家和该领域的领导者，库什纳博士发展了自己的职业生涯 旨在填补这些短期培训中特定教育和经验空白的发展计划。 目标包括： 1) 接受细胞电生理学的高级培训，重点是技术 探测离子通道在其原生环境中的功能；2) 获得分析大型数据集的经验， 目标是解释基因和蛋白质表达的大规模变化；3) 发展专业知识； 蛋白质组学和质谱分析的使用； 4) 完善现有技能并开发新技能 心血管生理学，重点是与负荷无关的测量的实验评估 收缩和舒张功能；5) 获得探索蛋白质降解途径的专业知识， 强调健康和疾病中离子通道的破坏；以及 5) 发展对他的长期健康至关重要的技能。 学期的成功，尤其强调了他在哥伦比亚大学的丰富的资助写作技巧。 支持性研究环境，被证明是库什纳博士组织多学科研究的理想环境 具有实现这些目标的专业知识的指导团队。 在他提出的研究项目中，库什纳博士将使用邻近标记相互作用组的结果 L 型 Ca2+ 通道 CaV1.2，深入了解对其正常功能至关重要的通道调节因子 生物学和心力衰竭（HF），一种以收缩功能和肾上腺素储备降低为特征的疾病， 使用转基因小鼠时，患者经常会出现呼吸急促的情况。 CaV1.2 亚基与工程抗坏血酸过氧化物酶 APEX2 融合的心脏特异性表达 他研究了慢性心力衰竭小鼠心脏通道微环境的变化。 HF 中 CaV1.2 附近的 2000 多种蛋白质中有 71 种发生显着变化。 干扰 CaV1.2 功能，他将分析遗传中的通道活动、收缩性和肾上腺素能反应性 为了测量对 CaV1.2 表达的影响，他将使用转基因技术。 表达 YFP 标记的 α1C 的小鼠，该 α1C 在细胞外环上含有银环蛇毒素结合位点 (BBS- α1C)。用荧光团结合的银环蛇毒素处理分离的肌细胞，他可以追踪总的和表面的。 使用流式细胞术在与 APEX- 敲除杂交的 BBS-α1C 小鼠中产生 HF。 识别 HF 中的蛋白质后，他可以识别那些改变 HF 中 CaV1.2 表达的邻近蛋白质。