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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要保留射血（HFPEF）的心力衰竭是所有心血管疾病中最大的未满足需求之一。尽管现在是心力衰竭的最常见形式，但迄今为止，它几乎没有特定的有效疗法。一个肥胖大流行现在改变了其表型，肥胖和代谢综合征现在重要的驱动力疾病。我们最近报道说，肥胖的-HFPEF表型表现出右凹陷钙的较高（收缩相关）水平的室心肌细胞张力产生。至关重要的是机制发生这种情况是未知的。肌细胞张力受肌球蛋白组成的厚细丝调节，薄丝，包括肌动蛋白，肌动蛋白和心脏肌钙蛋白。在厚的细丝中，大约所有肌球蛋白头的一半处于一个被称为超级省的（SRX）状态的构象，而比例在这种状态下的肌球蛋白是张力的重要调节因子。细丝通过改变钙来调节张力敏感性和一个调节剂是心脏肌钙蛋白I（CTNI）的磷酸化。在令人兴奋的新初步数据中，我发现在肥胖-HFPEF中，肌丝蛋白的厚细丝结构和磷酸化发生了改变。这提案来自这些数据，旨在阐明肥胖症如何改变人类的厚和细丝 HFPEF。在AIM 1中，我将检验以下假设：肥胖-HFPEF结果中厚细丝的结构灭活从过量的SRX肌球蛋白中。为了评估厚丝结构，我使用小角度X射线衍射，这是一种技术这利用心肌的有序结构来量化肌动蛋白之间的距离。这技术是在Argonne National Laboratory的同步加速器上进行的，Argonne National Laboratory是全球少数几个位置之一执行测定法，该提案描述了该技术在心肌活检中的首次应用来自人类HFPEF患者。虽然内容丰富，但X射线衍射本身并不能证明过多的存在 SRX肌球蛋白。为此，我将测量来自HFPEF患者的单个心肌细胞的肌球蛋白ATP更新。然后，我将通过测量HFPEF患者的两个测定法肥胖和高血压/肥大。在AIM 2中，我探讨了如何高磷酸化改变了钙激活的张力。我的初步数据发现酶活性蛋白质磷酸酶2a（PP2A）部分逆转了钙激活中观察到的缺陷肥胖HFPEF的张力，但该机制尚不清楚。我将测试这是否是从厚细丝激活的 PP2A暴露后测量X射线衍射模式和肌球蛋白ATP周转。我还测试了这是否结果瘦细丝高磷酸化，特别是在CTNI中，在HFPEF中。我们已经确定了一种新颖的苏氨酸181 CTNI的残基在HFPEF中被过度磷酸化，但其功能尚不清楚。磷酸无效/模拟物转基因CTNI THR181将换成HFPEF患者的皮肤肌细胞和心肌细胞张力测量。这些研究将提高我们对HFPEF中厚和细丝的理解，并可以铺路针对这些机制的小分子肌节增强剂的新疗法的方式。