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: 10319087
• 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/10319087
• 关键词: 26S proteasome; Adenylate Cyclase; Adrenergic Agents; Adrenergic Agonists; Arrhythmia; Binding; Binding Proteins; Binding Sites; Biochemical; Biology; Biotin; Bungarotoxins; Bypass; Calcium Channel; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular Physiology; Congestive Heart Failure; Coupling; Cyclic AMP-Dependent Protein Kinases; Defect; Development Plans; Disease; Disinhibition; Doxycycline; 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; Label; Lead; 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; 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; 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.

这项提案概述了一项为期5年的综合培训计划，以将医学博士贾里德·库什纳培养成一名 独立翻译调查员。库什纳博士是一位普通心脏病专家和内科科学家，他 最终目标是改善对心脏病患者的护理。为了实现这个目标，他感兴趣的是 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不适应变化。为了 成为一名独立的翻译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 发展计划，旨在填补他在培训中的具体教育和经验空白。这些短期的 目标包括：1)接受细胞电生理学方面的高级培训，重点是技术 该探测器离子通道在其自然环境中起作用；2)在大数据集分析方面获得经验， 目标是解释基因和蛋白质表达的大规模变化；3)开发专业知识 蛋白质组学和质谱学的使用；4)完善他现有的技能并开发新的技能 心血管生理学，重点是负荷非依赖性措施的实验评估 收缩和舒张期功能；5)获得探索蛋白质降解途径的专业知识， 强调破坏健康和疾病中的离子通道；以及5)培养对他的长期健康至关重要的技能。 学期成功，特别强调他的拨款撰写技能。哥伦比亚大学，凭借其丰富的 支持性的研究环境，被证明是库什纳博士组织多学科研究的理想环境 具有完成这些目标的专业知识的导师团队。 在他提出的研究项目中，库什纳博士将使用邻近标记的结果来标记 L型钙通道，CaV1.2，深入了解对其正常功能至关重要的通道调节 生物学和心力衰竭(HF)，一种以收缩功能和肾上腺素储备减少为特征的状况， 病人常经历因劳累而加重的呼吸短促。使用转基因小鼠 CaV1.2亚基与工程抗坏血酸过氧化物酶APEX2、He融合的心脏特异性表达 探讨慢性心衰小鼠心脏经络微环境的变化。他确认了 在HF的CaV1.2附近，2000多种蛋白质中有71种发生了显著变化。以确定这些蛋白质是否 扰乱CaV1.2功能，他将分析遗传学中的通道活动、收缩和肾上腺素能反应性 APEX识别的蛋白质的敲除。为了测量对CaV1.2表达的影响，他将使用转基因 表达含有银环蛇毒素结合位点的yfp标记的α1C的小鼠 α1C)。用荧光团结合的银环蛇毒素处理分离的心肌细胞，他可以追踪总和表面 流式细胞仪检测CaV1.2的表达。在BBS-α1C小鼠中产生HF与APEX-1基因敲除杂交 在识别出HF中的蛋白质后，他可以识别那些改变HF CaV1.2表达的邻近蛋白质。