Differential Proteoform Regulation by Alternative Splicing in Cardiac Aging and Hypertrophy

项目总结 选择性剪接是心脏老化的一个标志，也与 病理性心肌肥厚的发展，提示存在差异表达 异构体可能构成了一种主要的分子机制，推动了骨肉瘤的发生和发展。 心血管功能障碍和疾病。然而，大多数替代品的生物学功能 剪接事件目前还不确定，许多替代转录被认为是退化的 通过胡言乱语介导的衰变途径。为了弥合这一知识鸿沟，有必要 在蛋白质组学水平上确定选择性剪接如何调节心脏功能，并评估 替代蛋白质异构体如何与它们的主要标准型不同地发挥作用。 因此，这项建议的目标是研究蛋白质异构体的不同调控。 在年轻心脏与老年心脏和正常心脏与肥厚心脏之间，以及询问功能 异构体对心肌肥厚发生和发展的影响。我的赞助人玛吉医生 林的团队最近开发了一种集成的实验和计算组学工作流 它使用RNA-SEQ来指导可选异构体的蛋白质组学鉴定。在这里，我将测试 假设选择性剪接通过重新连接心脏细胞来驱动心脏衰老和病理 肌节蛋白和代谢蛋白的相互作用组和定位。为了实现这一目标，在目标1中， 我将结合rna-seq和高分辨率质谱学来确定蛋白质亚型 在老年心脏和疾病心脏中有差异表达。在目标2中，我将使用荧光 比较细胞内定位和相互作用伙伴的成像技术 标准的和替代的蛋白质异构体，并辨别基因操作是否 替代的异构体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对心脏替代方案的更好理解 剪接，这可能有助于深入了解参与心脏老化和 病理性肥大。同时，研究培训计划将为我提供 宝贵的蛋白质组学和数据分析培训机会(与林博士合作)和心脏生物学 和老鼠模型(与彼得·巴特里克博士和合作者蒂莫西·麦肯锡博士共同赞助)， 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员。

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.