GSK-3β Localizes to the Myofilament and Modifies its Function in Ischemic Cardiomyopathy

GSK-3β 定位于肌丝并改变其在缺血性心肌病中的功能

• 批准号: 9287330
• 负责人: JONATHAN A KIRK
• 金额: $ 37.11万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9287330
• 关键词: ATP phosphohydrolase; Address; Adenoviruses; Affect; Animal Model; Binding; Biological Assay; Calcium; Cardiac Myocytes; Cell Culture Techniques; Cells; Co-Immunoprecipitations; Data; Defect; Depressed mood; Deterioration; Dilated Cardiomyopathy; Echocardiography; Exhibits; GYS1 gene; Genetic; Goals; Heart; Heart failure; Heterogeneity; Human; Hybrids; Immunofluorescence Immunologic; Infarction; Ischemia; Knockout Mice; Label; Laboratories; Link; Mass Spectrum Analysis; Measures; Mediating; Methods; Microfilaments; Molecular; Motion; Mus; Muscle Cells; Mutate; Myocardial Infarction; Operative Surgical Procedures; Pacemakers; Patients; Phosphorylation; Phosphorylation Site; Phosphotransferases; Principal Investigator; Protein Kinase; Proteins; Proteome; Recombinant Proteins; Role; Sampling; Site; Skin; Structure; Survivors; Testing; Therapeutic; Therapeutic Intervention; Tissues; Tyrosine; Ventricular; Yeasts; base; cardiac resynchronization therapy; conventional therapy; desensitization; experimental study; functional restoration; glycogen synthase kinase 3 beta; heart cell; high risk; human tissue; improved; in vivo; ischemic cardiomyopathy; mortality; mouse model; mutant; patient population; prevent; repaired; therapeutic target

PROJECT SUMMARY Mortality from myocardial infarction is decreasing; however, survivors are at high risk of developing ischemic cardiomyopathy (ICM). Understanding the mechanisms involved in that transition may help develop methods to prevent it. Ischemic damage produces a form of dyssynchronous contraction that cannot be treated with conventional therapies (Cardiac Resynchronization Therapy, CRT). However, the Principal Investigator previously discovered a critical molecular mechanism of CRT: it reactivates glycogen synthase kinase 3β (GSK-3β) and thus restores myofilament function. This proposal will leverage the molecular mechanism discovered in CRT in a patient population that cannot respond to it, ICM patients. Preliminary data reveals that human and mouse ICM samples exhibit myofilament calcium desensitization, and exogenous treatment with GSK-3β restores calcium sensitivity, suggesting the functional defect is linked to deactivation of GSK-3β. Further, new preliminary data has identified an independently regulated pool of GSK-3β localized to the myofilament that decreases significantly in human ICM, which correlates with the decrease in calcium sensitivity. Importantly, additional preliminary data suggest the localization of GSK-3β to the myofilament is mediated by phosphorylation of GSK-3β at tyrosine 216. This could allow targeted modulation of the myofilament pool of GSK-3β as a therapeutic strategy to improve myofilament function. Thus, based on these new preliminary data, this proposal addresses the central hypothesis that ischemia de-activates a myofilament pool of GSK-3β via altering tyrosine 216 (Y216) phosphorylation, decreasing phosphorylation of its myofilament targets and depressing myofilament function. There are three specific aims. Aim 1 will address the hypothesis that ICM decreases myofilament function in a GSK-3β dependent manner. Genetic mouse models that alter GSK-3β activity will be subjected to surgical induction of myocardial infarction to generate ICM and then myofilament function and GSK-3β activity will assayed. Human tissue from ICM patients will be studied similarly. Aim 2 will address the the hypothesis that phosphorylation at Y216 on GSK-3β modulates its binding to the myofilament and ICM decreases the amount and activity of GSK- 3β at the myofilament. Mutant forms of GSK-3β where the Y216 site is unphosphorylatable or mimic constitutive phosphorylation will be expressed in cardiac myocytes to determine where and how GSK-3β binds to the myofilament. Myofilament function will also be assessed to determine whether these mutant forms of GSK-3β can restore function in the GSK-3β knock-out mouse. The last aim will address the hypothesis that GSK-3β can normalize the myofilament phospho-proteome in ICM patients and ICM mouse tissue using state of the art mass spectrometry approaches. The long-term objective of this project is to identify the mechanisms by which GSK-3β affects myofilament function in the ICM heart, with the goal of discovering a therapeutic approach to prevent the transition to ischemic cardiomyopathy after a myocardial infarction.

项目摘要 心肌梗塞的死亡率正在降低；但是，存活率有脑缺血的高风险 心肌病（ICM）。了解该过渡中涉及的机制可能有助于开发方法 防止它。缺血性损害会产生一种无法用的异步收缩形式 常规疗法（心脏重新同步疗法，CRT）。但是，首席研究员 以前发现了CRT的关键分子机制：它反应糖原合酶激酶3β （GSK-3β），因此恢复了肌丝功能。该建议将利用分子机制 ICM患者在CRT中发现了无法反应的患者。初步数据表明 人和小鼠ICM样品表现出肌丝钙脱敏，并与外源治疗 GSK-3β恢复了钙敏感性，表明功能缺陷与GSK-3β的失活有关。 此外，新的初步数据已经确定了一个独立调节的GSK-3β库。 肌丝在人ICM中显着降低，这与钙的减少相关 灵敏度。重要的是，其他初步数据表明，GSK-3β在肌丝中的定位是 通过在酪氨酸216处的GSK-3β磷酸化介导 GSK-3β的肌丝池作为改善肌丝功能的治疗策略。那，基于这些 新的初步数据，该提案解决了局部假设，缺血使A脱离激活 GSK-3β的肌丝池通过改变酪氨酸216（Y216）磷酸化，减少 其肌丝靶标的磷酸化和降低肌丝功能。有三个 具体目标。 AIM 1将解决以下假设：ICM在GSK-3β中下降肌丝功能 依赖方式。改变GSK-3β活性的遗传小鼠模型将经过手术诱导 心肌梗死将产生ICM，然后将分析肌丝功能和GSK-3β活性。人类 来自ICM患者的组织将同样研究。 AIM 2将解决以下假设 GSK-3β上的Y216调节其与肌丝的结合，ICM降低了GSK-的数量和活性 3β处的肌丝。 GSK-3β的突变形式，其中Y216位点是无磷酸的或模仿的 构成磷酸化将在心肌细胞中表达，以确定GSK-3β在何处以及如何结合 还将评估肌丝功能以确定这些突变形式是否形式 GSK-3β可以恢复GSK-3β敲除小鼠的功能。最后一个目标将解决以下假设 GSK-3β可以使用状态将ICM患者和ICM小鼠组织中的肌丝磷酸蛋白酶归一化 艺术质谱法的方法。该项目的长期目标是确定机制 GSK-3β会影响ICM心脏中的肌丝功能，目的是发现治疗 心肌梗塞后防止过渡到缺血性心肌病的方法。