Differential Proteoform Regulation by Alternative Splicing in Cardiac Aging and Hypertrophy

项目摘要 选择性剪接是心脏衰老的标志，也与心脏衰老有关。 病理性心脏肥大的发展，表明差异表达的 同种型可能构成了一个主要的分子机制，驱动发病和进展， 心血管功能障碍和疾病。然而，大多数替代品的生物功能 剪接事件目前还不确定，许多替代转录本被认为是降解的， 通过无义介导的衰变途径。为了弥合这一知识差距，需要 确定可变剪接如何在蛋白质组水平上调节心脏功能，并评估 不同的蛋白质异构体如何与它们的主要标准形式不同地起作用。 因此，本建议的目标是研究蛋白质异构体的差异调节 在年轻与老年心脏和正常与肥大心脏中，以及询问功能性 同种型对心肌肥大发生和进展的影响。我的赞助商Maggie医生 Lam的团队最近开发了一种集成的实验和计算组学工作流程 它使用RNA-seq来指导蛋白质组学鉴定替代亚型。在这里我将测试 假设选择性剪接通过重新连接 肌节和代谢蛋白相互作用组和定位。为了实现这一目标，在目标1中， 我会将联合收割机RNA-seq和高分辨率质谱技术结合起来， 在老年心脏和病变心脏中差异表达。在目标2中，我将使用荧光 成像技术来比较细胞内定位和相互作用的合作伙伴， 典型的和替代的蛋白质亚型，并辨别是否遗传操作的 替代亚型可以改善对肥大刺激的病理反应。 这项研究的预期结果将是提高对心脏替代疗法的理解。 剪接，这可能有助于深入了解参与心脏衰老的分子途径， 病理性肥大同时，研究培训计划将为我提供 在蛋白质组学和数据分析（与林博士）和心脏生物学方面的宝贵培训机会 和小鼠模型（与共同赞助者Peter Buttrick博士和合作者Timothy McKinsey博士）， 这将补充我现有的成像专业知识，并帮助我实现我的职业目标， 成为心血管生物学领域的独立研究者。

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.