Thick and Thin Filament Dysfunction in Obese Heart Failure with Preserved Ejection Fraction

射血分数保留的肥胖性心力衰竭的粗细丝功能障碍

• 批准号: 10678204
• 负责人: Vivek Pinakin Jani
• 金额: $ 5.27万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678204
• 关键词: Actins; Affect; Basic Science; Biological Assay; Biomechanics; Biopsy; Body mass index; Calcium; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular system; Cells; Clinical; Complex; Data; Depressed mood; Disease; EFRAC; Enhancers; Enzymes; Exhibits; Exposure to; Filament; Freezing; Functional disorder; Future; Generations; Goals; Head; Heart failure; Human; Hypertension; Hypertrophy; Label; Laboratories; Link; Location; Measures; Mediating; Medicine; Mental Depression; Metabolic syndrome; Microfilaments; Molecular Biology Techniques; Molecular Conformation; Morbidity - disease rate; Muscle Cells; Mutagenesis; Myocardium; Myosin ATPase; Myosin Type I; Obesity; Patients; Pattern; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physicians; Physiologic pulse; Positioning Attribute; Production; Protein Kinase C; Protein phosphatase; Protein-Serine-Threonine Kinases; Proteins; Relaxation; Reporting; Research; Role; Sarcomeres; Scientist; Serine/Threonine Phosphorylation; Site; Skin; Structure; Synchrotrons; Syndrome; Techniques; Test Result; Testing; Thick; Thick Filament; Thin Filament; Thinness; Threonine; Training; Transgenic Organisms; Tropomyosin; Troponin; Troponin I; Ventricular; Work; X ray diffraction analysis; career; comorbidity; effective therapy; heart preservation; mimetics; novel; novel strategies; novel therapeutics; obese patients; pandemic disease; phosphoproteomics; preservation; skills; small molecule; systolic hypertension

PROJECT SUMMARY Heart Failure with Preserved Ejection (HFpEF) is one of the largest unmet needs of all cardiovascular disease. Although it now is the most common form of heart failure, to date, it has little to no specific effective therapy. An obesity pandemic has now changed its phenotype, with obesity and metabolic syndrome now significant drivers of the disease. We recently reported that an obese-HFpEF phenotype exhibits striking depression of right ventricular myocyte tension generation at higher (contraction-related) levels of calcium. Critically, the mechanism by which this occurs is unknown. Myocyte tension is regulated by both the thick filament, consisting of myosin, and the thin filament, consisting of actin, tropomyosin, and cardiac troponins. In the thick filament, approximately half of all myosin heads are in a conformation known as the super-relaxed (SRX) state, and the proportion of myosin in this state is an important regulator of tension. The thin filament regulates tension by altering calcium sensitivity, and one regulator is phosphorylation of cardiac troponin I (cTnI). In exciting new preliminary data, I find that thick filament structure and phosphorylation of myofilament proteins are altered in obese-HFpEF. This proposal derives from these data and aims to elucidate how obesity alters the thick and thin filament in human HFpEF. In Aim 1, I will test the hypothesis that structural inactivation of the thick filament in obese-HFpEF results from an excess of SRX myosin. To assess thick filament structure, I use small angle x-ray diffraction, a technique that leverages the ordered structure of cardiac muscle to quantify distances between sarcomere proteins. This technique is performed at the synchrotron at Argonne National Laboratory, one of few locations globally that can perform the assay, and this proposal describes the first application of this technique to endomyocardial biopsies from human HFpEF patients. While informative, X-ray diffraction on its own cannot prove the presence of excess SRX myosin. For this, I will measure the myosin ATP turnover from single cardiomyocytes from HFpEF patients. I will then explore whether obesity is a driver of excess SRX myosin by measuring both assays in HFpEF patients with both obesity and hypertension/hypertrophy. In Aim 2, I explore the mechanism underlying how hyperphosphorylation alters calcium activated tension. My preliminary data finds that the exposure to enzymatically active protein phosphatase 2A (PP2A) partially reverses the deficit observed in calcium activated tension in obese HFpEF, but the mechanism is unknown. I will test if this is from thick filament activation by measuring x-ray diffraction patterns and myosin ATP turnover after PP2A exposure. I also test if this results from thin filament hyperphosphorylation, specifically at cTnI, in HFpEF. We have identified a novel threonine 181 residue of cTnI to be hyperphosphorylated in HFpEF, but its function is unknown. Phospho-null/mimetic transgenic cTnI Thr181 will be swapped into skinned myocytes from HFpEF patients, and myocyte tension measured. These studies will advance our understanding of the thick and thin filament in HFpEF and could pave the way for new therapies with small molecule sarcomere enhancers that target these mechanisms.

项目概要 保留射血性心力衰竭（HFpEF）是所有心血管疾病中最大的未满足需求之一。 尽管它现在是最常见的心力衰竭形式，但迄今为止，几乎没有具体的有效治疗方法。一个 肥胖流行现在已经改变了其表型，肥胖和代谢综合征现在是重要的驱动因素 的疾病。我们最近报道，肥胖 HFpEF 表型表现出明显的右侧抑郁症 在较高（收缩相关）钙水平下心室肌细胞张力产生。关键的是，该机制 发生这种情况的原因尚不清楚。肌细胞张力由由肌球蛋白组成的粗丝调节， 细丝由肌动蛋白、原肌球蛋白和心肌肌钙蛋白组成。在粗丝中，大约 所有肌球蛋白头的一半处于称为超松弛 (SRX) 状态的构象，并且 这种状态下的肌球蛋白是张力的重要调节剂。细丝通过改变钙来调节张力 敏感性，其中一个调节因子是心肌肌钙蛋白 I (cTnI) 的磷酸化。在令人兴奋的新初步数据中，我 发现肥胖 HFpEF 中肌丝蛋白的粗丝结构和磷酸化发生了改变。这 该提案源自这些数据，旨在阐明肥胖如何改变人类的粗丝和细丝 HFpEF。在目标 1 中，我将检验肥胖 HFpEF 中粗丝结构失活的假设 来自过量的 SRX 肌球蛋白。为了评估粗丝结构，我使用小角度 X 射线衍射，这是一种技术 利用心肌的有序结构来量化肌节蛋白之间的距离。这 该技术是在阿贡国家实验室的同步加速器上进行的，该实验室是全球少数几个可以进行同步加速器的地方之一 进行测定，该提案描述了该技术在心内膜心肌活检中的首次应用 来自人类 HFpEF 患者。虽然 X 射线衍射提供了丰富的信息，但它本身并不能证明过量的存在 SRX 肌球蛋白。为此，我将测量 HFpEF 患者单个心肌细胞的肌球蛋白 ATP 周转率。 然后，我将通过测量 HFpEF 患者的两项检测来探讨肥胖是否是 SRX 肌球蛋白过量的驱动因素 患有肥胖症和高血压/肥大。在目标 2 中，我探索了其背后的机制 过度磷酸化改变钙激活张力。我的初步数据发现，暴露于 酶活性蛋白磷酸酶 2A (PP2A) 部分逆转了钙激活中观察到的缺陷 肥胖 HFpEF 中存在紧张，但其机制尚不清楚。我将测试这是否是由粗丝激活引起的 测量 PP2A 暴露后的 X 射线衍射图案和肌球蛋白 ATP 周转率。我还测试了这是否是由于 HFpEF 中的细丝过度磷酸化，特别是 cTnI 处。我们已经鉴定出一种新的苏氨酸 181 cTnI 残基在 HFpEF 中被过度磷酸化，但其功能尚不清楚。磷酸化无效/模拟 转基因 cTnI Thr181 将被交换到 HFpEF 患者的带皮肌细胞中，并且肌细胞张力 测量。这些研究将增进我们对 HFpEF 中粗丝和细丝的理解，并可能为 针对这些机制的小分子肌节增强剂的新疗法的方法。