A novel approach integrating proteomics and metabolomics to understand diabetic cardiomyopathy

整合蛋白质组学和代谢组学来了解糖尿病心肌病的新方法

• 批准号: 10360416
• 负责人: Benjamin Wancewicz
• 金额: $ 3.53万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10360416
• 关键词: Acetylation; Adrenergic Agents; Animals; Attention; Back; Branched-Chain Amino Acids; Cardiac; Cardiac Myocytes; Ceramides; Complex; Contractile Proteins; Cyclic AMP-Dependent Protein Kinases; Data; Detection; Development; Diabetic mouse; Echocardiography; Energy-Generating Resources; Enzymes; Epidemic; Functional disorder; Future; Head; Heart; Hexoses; High Fat Diet; Hyperinsulinism; Hypertrophy; Impairment; Insulin; Knowledge; Length; Link; Lipids; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Metabolic Control; Methods; Molecular; Morbidity - disease rate; Mus; Muscle Cells; Myocardial; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Pathway interactions; Patients; Phosphorylation; Phosphorylcholine; Polyunsaturated Fatty Acids; Post-Translational Protein Processing; Prevalence; Proteins; Proteomics; Protocols documentation; Regulation; Relaxation; Research; Risk; Risk Factors; Role; Sampling; Sarcomeres; Serial Extraction; Signal Transduction; Testing; Therapeutic; Tissue Sample; Tissues; Triglycerides; Troponin; Troponin I; United States; acylcarnitine; adverse outcome; base; diabetic; diabetic cardiomyopathy; fatty acid-binding proteins; feeding; genetic regulatory protein; glycemic control; insight; lipidomics; liquid chromatography mass spectrometry; metabolomics; mortality; mouse model; new therapeutic target; novel; novel strategies; protein metabolite; response; success; surfactant; two-dimensional; ultra high resolution

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.

项目总结/摘要 糖尿病心肌病（DCM）是美国发病率和死亡率的主要原因， 全球流行与肥胖和2型糖尿病（T2 DM）的全球患病率平行。 然而，DCM的潜在分子机制仍然知之甚少。据信 高胰岛素血症可能会促进DCM心脏与T2 DM和肥胖的不良后果。最近， 我们的合作者Kevin Xiang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传导来增强心肌收缩力，并确定了胰岛素与 通过喂食高脂饮食（HFD）产生的T2 DM C57 J/B6小鼠模型中心脏的肾上腺素能途径。 重要的是，他们已经表明，心肌肌钙蛋白I（cTnI，一种重要的肌节蛋白）的PKA磷酸化， 调节蛋白）对肾上腺素能刺激的响应在HFD动物中受损。虽然后- 已知肌节蛋白质的翻译修饰（PTM）是肌节蛋白质中的重要机制。 由于其对心脏收缩和舒张的调节作用，其在DCM心脏中的具体作用在很大程度上仍未被表征。 考虑到舒张功能障碍和心肌细胞肥大可以通过更好的血糖控制来改善， 在糖尿病小鼠中，我假设代谢蛋白质将经历各种PTM，如乙酰化， 磷酸化，导致效率较低的代谢状态和降低的能量状态，这将最终 最终导致多个改变的肌节PTM和收缩性降低。在此，我建议使用一种新的超- 基于高分辨率质谱（MS）自上而下的蛋白质组学平台， DCM心脏中的肌节蛋白PTM。此外，我建议调查以下因素之间的相互作用 肌节PTM和DCM中的代谢状态。本提案的具体目标包括：1）确定PTM 2型糖尿病相关高胰岛素血症时心脏组织中关键调节性肌节蛋白的变化 使用自上而下的蛋白质组学策略，并将肌节变化与心肌收缩力的改变联系起来， 通过HFD和正常食物（对照）小鼠的超声心动图测量发现。2)确定以下方面的变化 代谢能量储存与脂质能量储存并行，注意特定的酰基链信息，如 酰基长度、不饱和度和脂质头基， DCM样品中的脂质组学提取方案。3)使用双- 三维液相色谱质谱为基础的自上而下的蛋白质组学平台，并连接 测量收缩功能障碍的变化。拟议研究的成功将提供新的见解 深入研究DCM的分子机制，并可能发现新的治疗靶点。