A novel approach integrating proteomics and metabolomics to understand diabetic cardiomyopathy

项目摘要/摘要 糖尿病心肌病(DCM)是美国发病率和死亡率的主要原因，并且正在上升 在全球范围内肥胖和2型糖尿病(T2 DM)流行的同时，也出现了全球性的流行病。 然而，扩张型心肌病的潜在分子机制仍然知之甚少。据信， 高胰岛素血症可能促进T2 DM和肥胖者DCM心脏的不良后果。最近， 我们的合作者Kevin翔博士的实验室发现了一种新的机制，高胰岛素血症可能会损害 通过抑制肾上腺素能信号来抑制心肌收缩能力，并发现胰岛素和 高脂饮食(HFD)诱导的T2 DM C57J/B6小鼠模型心脏的肾上腺素能通路。 重要的是，他们已经证明，心肌肌钙蛋白I(CTnI)的PKA磷酸化是一种关键的肌小节 调节蛋白)对肾上腺素能刺激的反应在HFD动物中受损。尽管后- 肌瘤蛋白的翻译修饰(PTM)是已知的一种重要机制。 在心脏收缩和松弛的调节中，它在扩张型心肌病心脏中的具体作用在很大程度上仍不清楚。 考虑到更好的血糖控制可以改善舒张期功能障碍和心肌细胞肥大 在糖尿病小鼠中，我假设代谢蛋白将经历各种PTM，如乙酰化和 磷酸化，导致低效代谢状态和能量减少状态，这最终将 最终导致多个肌节PTM改变和收缩能力降低。在此，我建议使用一种新颖的超 基于高分辨质谱学(MS)的自上而下蛋白质组学平台的全面表征 扩张型心肌病心脏的肌节蛋白PTM。此外，我建议调查 扩张型心肌病的肌节状PTMS及其代谢状态。这项建议的具体目标包括：1)确定PTM T2 DM相关高胰岛素血症心肌组织中关键调控蛋白的变化 使用自上而下的蛋白质组学策略，并将肌节的变化与心脏收缩能力的变化联系起来 通过对HFD和正常饮食(对照)小鼠的超声心动图测量发现。2)确定中的更改 代谢能量存储与脂类能量存储并行，注意特定的酰链信息，如 酰基长度、不饱和度和脂头基，利用全面的代谢组学和 扩张型心肌病样本中的脂类组学提取方法。3)用两种方法鉴定代谢蛋白的PTM变化 基于维液相色谱质谱的自上而下蛋白质组学平台 测量收缩功能障碍的变化。这项拟议研究的成功将提供新的见解 研究扩张型心肌病的分子机制，并可能确定新的治疗靶点。

Project Summary/Abstract Diabetic cardiomyopathy (DCM) is a major cause of morbidity and mortality in the United States and is a rising global epidemic in parallel with the worldwide prevalence of obesity and type-2 diabetes mellitus (T2DM). However, the underlying molecular mechanisms in DCM remain poorly understood. It is believed that hyperinsulinemia might promote adverse consequences in the DCM hearts with T2DM and obesity. Recently, our collaborator, Dr. Kevin Xiang’s lab has discovered a novel mechanism that hyperinsulinemia might impair myocardial contractility by inhibiting adrenergic signaling and identified a direct interaction between insulin and adrenergic pathways of the heart in a T2DM C57J/B6 mouse model generated by feeding a high fat diet (HFD). Importantly, they have shown that the PKA phosphorylation of cardiac troponin I (cTnI, a critical sarcomeric regulatory protein) in response to adrenergic stimulation was impaired in HFD animals. Although post- translational modifications (PTM) of the sarcomeric proteins are known to be an important mechanism in the regulation of cardiac contraction and relaxation, its specific role in DCM hearts remains largely uncharacterized. Given that diastolic dysfunction and cardiomyocyte hypertrophy were ameliorated by a greater glycemic control in diabetic mice, I hypothesize that metabolic proteins will undergo various PTMs, like acetylation and phosphorylation, leading to a less efficient metabolic state and a reduced energy state, which will ultimately culminate in multiple altered sarcomeric PTMs and reduced contractility. Herein, I propose to utilize a novel ultra- high resolution mass spectrometry (MS)-based top-down proteomics platform to comprehensively characterize the sarcomeric proteins PTMs in DCM hearts. Moreover, I propose to investigate the interplay between sarcomeric PTMs and the metabolic state in DCM. The specific aims of this proposal include: 1) Identify PTM changes of key regulatory sarcomeric proteins in response to T2DM-associated hyperinsulinemia in heart tissue using a top-down proteomics strategy and link the sarcomeric changes with alterations in cardiac contractility found by echocardiography measurements of HFD and normal chow (control) mice. 2) Identify changes in metabolic energy stores in parallel with lipid energy stores with attention to specific acyl chain information like acyl length, degrees of unsaturation, and lipid head group by utilizing a comprehensive metabolomics and lipidomics extraction protocol in DCM samples. 3) Identify PTM changes of metabolic proteins using a two- dimensional liquid chromatography mass spectrometry-based top-down proteomics platform and link the measured changes to the contractile dysfunction. The success of the proposed research will offer new insights into the molecular mechanism underlying DCM and may identify new therapeutic targets.