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.

该提案概述了一项全面的5年培训计划，以开发医学博士Jared Kushner， 独立翻译研究员。库什纳博士是一名普通心脏病专家和身体科学家 最终目标是改善心脏病患者的护理。为了实现这一目标，他对 了解心脏异常离子通道的功能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。为了 成为该领域的独立翻译科学家和领导者，库什纳博士发展了职业 开发计划旨在填补他的培训中特定的教育和经验丰富的空白。这些短期 目标包括：1）接受细胞电生理学的高级培训，重点是技术 该探测离子渠道在其本地环境中的功能； 2）获得大型数据集分析的经验， 目的是解释基因和蛋白质表达的大规模变化； 3）发展专业知识 蛋白质组学和质谱法的使用； 4）完善他当前的技能并发展新技能 心血管生理学，重点是对负载无关测量的实验评估 收缩和舒张功能； 5）获得探测蛋白质降解途径的专业知识，并具有 强调健康和疾病中离子通道的破坏； 5）发展技能对他的长期至关重要 术语成功，特别着重于他的赠款技巧。哥伦比亚大学，有钱 支持性研究环境，被证明是库什纳博士组织多学科的理想环境 拥有专业知识来实现​​这些目标的属团团队。 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 l型Ca2+通道Cav1.2，以洞悉通道的调节因子在正常中必不可少的函数 生物学和心力衰竭（HF），这种疾病以降低的收缩功能和肾上腺素储备为特征， 患者经常经历，因为劳累的呼吸急促。将转基因小鼠与 CAV1.2亚基的心脏特异性表达与工程抗坏血酸酯过氧化物酶Apex2，He 探测了发展出慢性HF的小鼠心脏中不断变化的通道微环境。他确定了 在HF中，Cav1.2附近的2000多个蛋白质中的71种发生了显着变化。确定这些蛋白是否 扰动CAV1.2功能，他将分析通用的通道活动，收缩力和肾上腺素的响应能力 顶端识别蛋白的敲除。为了衡量对CAV1.2表达的影响，他将使用转基因 表达YFP标记的α1C的小鼠在细胞外环上包含bungarotoxin结合位点（BBS- α1c）。用荧光团偶联的bungarotoxin治疗孤立的肌细胞，他可以跟踪总和表面 Cav1.2的表达使用流循环仪。在BBS-α1C小鼠中产生HF，并与Apex-敲除 在HF中鉴定出的蛋白质，他可以鉴定出改变HF的CAV1.2表达的那些相邻的蛋白质。