Cardiac Myosin Binding Protein-C in Development and Reversal of Heart Failure

心肌肌球蛋白结合蛋白-C 在心力衰竭发生和逆转中的作用

• 批准号: 10544998
• 负责人: Carl Wei-Chan Tong
• 金额: $ 46.18万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-15 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10544998
• 关键词: Acceleration; Adenoviruses; Adrenergic Agents; Aging; American; Biochemistry; Biological Assay; Blood Vessels; C10; Calcium; Cardiac; Cardiac Myosins; Cardiac Surgery procedures; Companions; Death Rate; Dependovirus; Deterioration; Development; Diagnosis; Disease; EFRAC; Echocardiography; Exclusion; Exhibits; Focal Adhesion Kinase 1; Functional disorder; Heart; Heart Rate; Heart failure; High Fat Diet; Hour; Hypotension; Immunoprecipitation; In Vitro; Knock-in Mouse; Mass Spectrum Analysis; Measurement; Mechanical Stress; Mediating; Mind; Modeling; Mus; Muscle Proteins; Mutagenesis; Mutate; Mutation; Myocardial dysfunction; Myocardium; Myofibrils; NG-Nitroarginine Methyl Ester; Nitric Oxide; Obesity; Operative Surgical Procedures; Pharmaceutical Preparations; Phenotype; Phenylalanine; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Preparation; Prevalence; Prevention; Protein Dephosphorylation; Pulmonary Edema; Recombinants; Relaxation; Research Project Grants; Running; Sarcomeres; Serine; Signal Transduction; Site; Stress; Structure; Techniques; Thick Filament; Translating; Treatment Failure; Tyrosine; Tyrosine Phosphorylation; Use Effectiveness; age related; aorta constriction; density; effective therapy; efficacy evaluation; experimental study; gene therapy; heart function; heart preservation; improved; in vivo; inhibitor; insight; mimetics; mortality; mouse model; myosin-binding protein C; novel; overexpression; papillary muscle; preservation; pressure; prevent; response; stem

Heart failure (HF) afflicted 6.5 million Americans in 2017, carries 5-year mortality of ~50%, and will likely increase to > 8 million by 2030. The lack of significant improvement in mortality for the last 46 years, the reality of needing to discontinue effective medications due to hypotension in heart failure with reduced ejection fraction (HFrEF), and the paucity of effective treatment for heart failure with preserved ejection fraction (HFpEF) combine to suggest that new treatments are needed. Cardiac myosin binding protein-C (cMyBPC) resides on the thick filament of the heart muscle. Phosphorylation of cMyBPC at its M-domain increases cross-bridge cycling rate. Because increasing cross-bridge cycling rate can improve both contractility and lusitropy, we hypothesize that phosphorylation of cMyBPC provides a novel central mechanism that can prevent and treat HF due to different causes. Using mouse models that mimic de-phosphorylated and phosphorylated cMyBPC at 3 serine (S) sites in the M-domain, we recently discover that cMyBPC phosphorylation mitigates both aged-related development of HFpEF and trans-aortic constriction (TAC) surgery induced HFrEF, as seen by improved survival and better preservation of diastolic function. Thus, cMyBPC phosphorylation holds potential to treat both HFrEF and HFpEF. However, we find evidence that 2 new S phosphorylation sites outside our study of 3 S sites add significant functional effects. Consequently, we have made 2 new knock-in mouse models to include the new sites for elucidating the effects of maximally phosphorylated cMyBPC(5SD) and dephosphorylated cMyBPC(5SA) M-domain. We also find evidence that pressure stress can activate focal adhesion kinase (FAK) to phosphorylate tyrosine (Y) residue(s) in cMyBPC. This discovery leads to a new supporting hypothesis that pressure stress triggers FAK to phosphorylate cMyBPC to increase contractility as a compensatory response. With these discoveries in mind, we intend to determine the efficacy of maximal M-domain phosphorylation and elucidate the novel FAK-cMyBPC mechanism. Aim #1: Determine the efficacy and companion mechanisms of phosphorylated cMyBPC to preserve cardiac function under HF inducing conditions of aging, pressure stress, and obesity. We will challenge mice with 2-yr aging, TAC, or high fat diet. We will use a combination of functional and biochemistry techniques to determine the underlying mechanisms. Ability of cMyBPC(5SD) mouse to resist deterioration and effectiveness of using cMyBPC(5SD) gene therapy to reverse failing WT hearts will quantify the efficacy of cMyBPC phosphorylation for prevention and treatment respectively. Aim #2: Elucidate signaling mechanism and functional results of pressure stress induced tyrosine phosphorylation of cMyBPC. We will identify Y site(s), confirm FAK-cMyBPC interaction, and elucidate effects of FAK phosphorylating cMyBPC on models ranging from intact papillary muscle to new knock-in mouse. Impact: Efficacy results and mechanistic insights stemming from this study provide evidence for translating cMyBPC phosphorylation to new HF treatment.

心力衰竭（HF）在2017年遭受了650万美国人的折磨，5年死亡率约为50％，可能会增加 到2030年到800万> 800万。在过去的46年中，死亡率缺乏显着改善，需要 由于心力衰竭减少的射血分数（HFREF）而导致的有效药物（HFREF）导致有效药物， 以及对保留的射血分数（HFPEF）的有效治疗的缺乏性治疗 建议需要新的治疗方法。心脏肌球蛋白结合蛋白-C（CMYBPC）位于厚的 心肌的细丝。 CMYBPC在其M域中的磷酸化增加了跨桥循环速率。 由于提高跨桥循环速率可以提高收缩性和lusitropy，因此我们假设 CMYBPC的磷酸化提供了一种新型的中心机制，可以预防和治疗HF，原因不同 原因。使用模仿3丝氨酸位点上磷酸化和磷酸化的CMYBPC的小鼠模型 在M域中，我们最近发现CMYBPC磷酸化会降低与老年相关的发展 HFPEF和反式运动收缩（TAC）手术诱导的HFREF，可通过提高生存和更好 保存舒张功能。因此，CMYBPC磷酸化具有治疗HFREF和 HFPEF。但是，我们发现证据表明，在3秒内的研究之外，有2个新的S磷酸化位点增加了 显着的功能效应。因此，我们制作了2个新的敲门鼠标模型，以包括新的 阐明最大磷酸化的CMYBPC（5SD）和去磷酸化的CMYBPC（5SA）影响的位点 M域。我们还发现了压力应力可以激活局灶性激酶（FAK）以磷酸化的证据 CMYBPC中的酪氨酸（Y）残基。这一发现导致了一个新的支持假设，即压力压力 触发FAK磷酸化CMYBPC以增加收缩力作为补偿性反应。与这些 考虑到发现，我们打算确定最大M域磷酸化的功效并阐明 新型FAK-CMYBPC机制。 AIM＃1：确定磷酸化的功效和伴随机制 CMYBPC在HF诱导衰老，压力和肥胖状态下保留心脏功能。我们 将以2年衰老，TAC或高脂肪饮食挑战小鼠。我们将结合功能和生物化学 确定潜在机制的技术。 CMYBPC（5SD）小鼠抵抗恶化和 使用CMYBPC（5SD）基因疗法逆转WT心脏的有效性将量化CMYBPC的功效 分别用于预防和治疗的磷酸化。目标＃2：阐明信号传导机制和 压力应力诱导CMYBPC酪氨酸磷酸化的功能结果。我们将确定Y网站， 确认FAK-CMYBPC相互作用，并阐明FAK磷酸化CMYBPC对范围型号的效果 从完整的乳头肌到新的敲入鼠标。影响：功效结果和机理见解 这项研究提供了将CMYBPC磷酸化转化为新的HF治疗的证据。

项目成果

Isolation of Adult Mouse Cardiomyocytes Using Langendorff Perfusion Apparatus.

使用 Langendorff 灌注装置分离成年小鼠心肌细胞。

• DOI: 10.1007/978-1-0716-1480-8_16
• 发表时间: 2021
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Liu,Yang;Dostal,DavidE;Tong,CarlW
• 通讯作者: Tong,CarlW

Preparation and Identification of Cardiac Myofibrils from Whole Heart Samples.

• DOI: 10.1007/978-1-0716-1480-8_2
• 发表时间: 2021-01-01
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Creed, Heidi A;Tong, Carl W
• 通讯作者: Tong, Carl W