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.

项目摘要 射血功能保留性心力衰竭（HFpEF）是所有心血管疾病中最大的未满足需求之一。 尽管它现在是心力衰竭最常见的形式，但迄今为止，它几乎没有具体有效的治疗方法。一个 肥胖症的流行现在已经改变了它的表型，肥胖症和代谢综合征现在是重要的驱动因素 的疾病。我们最近报道了一种肥胖型HFpEF表型表现出显著的右心室收缩抑制， 心室肌细胞在较高的（收缩相关的）钙水平下产生张力。关键是， 这种情况发生的原因是未知的。肌细胞张力由肌球蛋白组成的粗肌丝， 以及由肌动蛋白、原肌球蛋白和心肌肌钙蛋白组成的细丝。在粗丝中，大约 所有肌球蛋白头部的一半处于被称为超松弛（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 一种调节因子是心肌肌钙蛋白I（cTnI）的磷酸化。在令人兴奋的新的初步数据，我 发现肥胖HFpEF中的粗丝结构和肌丝蛋白的磷酸化改变。这 一项提案源于这些数据，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中粗纤维的结构失活导致 SRX肌球蛋白过量所致为了评估粗灯丝结构，我使用小角度X射线衍射， 利用心肌的有序结构来量化肌节蛋白之间的距离。这 这项技术是在阿贡国家实验室的同步加速器上进行的，阿贡国家实验室是全球少数几个可以 进行测定，该建议描述了该技术首次应用于子宫内膜活检 来自人类HFpEF患者。虽然信息丰富，但X射线衍射本身不能证明存在过量的 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患者的两种检测方法来探索肥胖是否是过量SRX肌球蛋白的驱动因素 患有肥胖症和高血压/肥大症。在目标2中，我探讨了 过度磷酸化改变钙激活张力。我的初步数据显示， 酶活性蛋白磷酸酶2A（PP 2A）部分逆转了在钙激活的细胞中观察到的缺陷。 肥胖HFpEF的张力，但机制尚不清楚。我将测试这是否是由粗灯丝激活引起的， 测量PP 2A暴露后的X射线衍射图和肌球蛋白ATP周转率。我还测试了这是否是由于 HFpEF中细纤维过度磷酸化，特别是cTnI。我们已经鉴定了一种新的苏氨酸181 在HFpEF中，cTnI残基被过度磷酸化，但其功能尚不清楚。磷酸化无效/模拟 将转基因cTnI Thr 181交换到来自HFpEF患者的皮肤肌细胞中， 测定了这些研究将促进我们对HFpEF中粗丝和细丝的理解， 新疗法的方式与小分子肌节增强剂靶向这些机制。