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 ...