Differential Proteoform Regulation by Alternative Splicing in Cardiac Aging and Hypertrophy

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PROJECT SUMMARY Alternative splicing is a hallmark of cardiac aging and is also causally implicated in the development of pathological cardiac hypertrophy, suggesting the differential expression of isoforms may constitute a major molecular mechanism that drives the onset and progression of cardiovascular dysfunction and diseases. However, the biological functions of most alternative splicing events are currently uncertain, with many alternative transcripts thought to be degraded through nonsense mediated decay pathways. To bridge this knowledge gap, there is a need to identify how alternative splicing regulates cardiac functions on a proteome scale and evaluate how alternative protein isoforms function differently from their main canonical forms. Accordingly, the goal of this proposal is to examine the differential regulation of protein isoforms in young vs. aged hearts and normal vs. hypertrophic hearts as well as to interrogate the functional impact of isoforms on the onset and progression of cardiac hypertrophy. My Sponsor Dr. Maggie Lam’s group recently developed an integrated experimental and computational omics workflow which uses RNA-seq to guide the proteomics identification of alternative isoforms. Here I will test the hypothesis that alternative splicing drives cardiac aging and pathology by rewiring the interactome and localization of sarcomeric and metabolic proteins. To achieve this goal, in Aim 1, I will combine RNA-seq and high-resolution mass spectrometry to determine protein isoforms that are differentially expressed in aged heart and diseased heart. In Aim 2, I will use fluorescence imaging technologies to compare the intracellular localization and interacting partners of canonical and alternative protein isoforms, and discern whether genetic manipulation of alternative isoforms can ameliorate pathological responses to hypertrophic stimuli. The anticipated outcome of this study will be an improved understanding of cardiac alternative splicing, which may contribute insights into molecular pathways involved in cardiac aging and pathological hypertrophy. At the same time, the research training plan will provide me with valuable training opportunities in proteomics and data analysis (with Dr. Lam) and cardiac biology and mouse models (with Co-Sponsor Dr. Peter Buttrick and Collaborator Dr. Timothy McKinsey), which will complement my existing expertise in imaging and help me achieve my career goal of becoming an independent investigator in the field of cardiovascular biology.