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.

项目概要 心肌梗塞死亡率正在下降；然而，幸存者患缺血性心脏病的风险很高 心肌病（ICM）。了解这种转变所涉及的机制可能有助于开发方法 防止它。缺血性损伤会产生一种无法用药物治疗的不同步收缩 常规疗法（心脏再同步疗法，CRT）。然而，首席研究员 先前发现了 CRT 的一个关键分子机制：它重新激活糖原合成酶激酶 3β (GSK-3β) 从而恢复肌丝功能。该提案将利用分子机制 在 CRT 中发现无法对其做出反应的患者群体，即 ICM 患者。初步数据显示 人类和小鼠 ICM 样本表现出肌丝钙脱敏作用，以及外源性治疗 GSK-3β 恢复钙敏感性，表明功能缺陷与 GSK-3β 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 池，该池位于 肌丝在人类 ICM 中显着减少，这与钙的减少相关 敏感性。重要的是，额外的初步数据表明 GSK-3β 在肌丝中的定位是 由 GSK-3β 在酪氨酸 216 处的磷酸化介导。这可以允许靶向调节 GSK-3β 肌丝池作为改善肌丝功能的治疗策略。因此，基于这些 新的初步数据，该提案提出了一个中心假设，即缺血使 GSK-3β 肌丝池通过改变酪氨酸 216 (Y216) 磷酸化，减少 其肌丝靶标磷酸化并抑制肌丝功能。有三个 具体目标。目标 1 将解决 ICM 降低 GSK-3β 中肌丝功能的假设 依赖方式。改变 GSK-3β 活性的基因小鼠模型将接受手术诱导 心肌梗塞产生ICM，然后检测肌丝功能和GSK-3β活性。人类 来自 ICM 患者的组织也将进行类似的研究。目标 2 将解决以下假设：磷酸化位于 GSK-3β 上的 Y216 调节其与肌丝的结合，ICM 降低 GSK-3β 的数量和活性 3β位于肌丝上。 GSK-3β 的突变形式，其中 Y216 位点不可磷酸化或类似 组成型磷酸化将在心肌细胞中表达，以确定 GSK-3β 的结合位置和方式 到肌丝。还将评估肌丝功能以确定这些突变形式是否 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可以使用状态使ICM患者和ICM小鼠组织中的肌丝磷酸化蛋白质组正常化 最先进的质谱方法。该项目的长期目标是确定机制 GSK-3β 通过影响 ICM 心脏中的肌丝功能，目标是发现一种治疗方法 预防心肌梗死后转变为缺血性心肌病的方法。