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

项目摘要 心肌梗死的死亡率正在下降;然而，存活者发生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， 缺血性损伤会产生一种不同步的收缩，这种收缩不能用 常规治疗（心脏复苏治疗，CRT）。然而，主要研究者 以前发现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。这项提案将利用分子机制 在CRT中发现，在对它没有反应的患者群体中，ICM患者。初步数据显示， 人和小鼠ICM样品显示肌丝钙脱敏， 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库，定位于 在人ICM中显著减少的肌丝，其与钙的减少相关 灵敏度重要的是，额外的初步数据表明GSK-3β在肌丝的定位是 通过GSK-3β在酪氨酸216处的磷酸化介导。这可以允许有针对性地调节 GSK-3β作为改善肌丝功能的治疗策略。因此，根据这些 新的初步数据，这一建议解决了中心假设，缺血失活， 通过改变酪氨酸216（Y216）磷酸化，降低GSK-3β的肌丝库 其肌丝靶点的磷酸化和抑制肌丝功能。有三 具体目标。目的1将阐明ICM降低GSK-3β抑制剂中肌丝功能的假设。 依赖的方式。将对改变GSK-3β活性的遗传小鼠模型进行手术诱导， 心肌梗死形成ICM后检测心肌肌丝功能和GSK-3β活性。人类 将类似地研究来自ICM患者的组织。目的2将解决的假设，磷酸化， GSK-3β上的Y216调节其与肌丝的结合，ICM降低GSK-3 β的量和活性。 肌丝3β。Y216位点不可磷酸化或模拟的GSK-3β突变形式 组成性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 到肌丝还将评估肌丝功能，以确定这些突变形式的肌丝蛋白是否具有免疫原性。 GSK-3β可使GSK-3β基因敲除小鼠的功能恢复。最后一个目标将解决的假设， GSK-3β可使ICM患者和ICM小鼠组织使用状态的肌丝磷酸化蛋白质组正常化 现有技术的质谱方法。该项目的长期目标是确定 GSK-3β影响ICM心脏中肌丝功能，目的是发现治疗 预防心肌梗死后转变为缺血性心肌病的方法。

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.