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

项目总结 心肌梗死的死亡率正在下降；然而，幸存者发生缺血的风险很高。 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来 防止它发生。缺血性损伤会产生一种不能治疗的不同步收缩 常规治疗(心脏再同步治疗，CRT)。然而，首席调查员 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K3β (葛兰素史克-3β)，从而恢复肌丝功能。这项提议将利用分子机制 在CRT中发现对其无反应的患者群体，ICM患者。初步数据显示， 人和小鼠ICM样本显示肌丝钙脱敏，并外源性处理 葛兰素史克-3β可恢复钙敏感性，提示功能缺陷与葛兰素史克-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葛兰素史克-3β池，定位于 人ICM中显著减少的肌丝，这与钙的减少有关 敏感度。重要的是，更多的初步数据表明，gsk-3β在肌丝的定位是 GSK-3β酪氨酸第216位磷酸化。这可能允许有针对性地调制 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，这项建议解决了中心假说，即缺血去激活 通过改变酪氨酸216(Y216)磷酸化减少葛兰素史克-3β的肌丝池 其肌丝靶标的磷酸化和抑制肌丝功能。一共有三个 明确的目标。目标1将解决在葛兰素史克-3β中ICM降低肌丝功能的假设 依赖的态度。改变葛兰素史克-3β活性的遗传小鼠模型将受到手术诱导 心肌梗死后产生心肌间充质，然后测定肌丝功能和GSK-3β活性。人类 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：磷酸化在 GSK-3β上的Y216调节其与肌丝的结合，ICM减少GSK-3的数量和活性 3肌丝β。Y216位不能磷酸化或模拟的葛兰素史克-3β突变形式 结构性磷酸化将在心肌细胞中表达，以确定葛兰素史克-3β在哪里以及如何结合 到肌丝。还将对肌丝功能进行评估，以确定这些突变形式的 葛兰素史克-3β可恢复β基因敲除小鼠的功能。最后一个目标将解决这样的假设 葛兰素史克-3β可使脑梗塞患者和脑梗塞小鼠组织肌丝磷酸化蛋白质组正常化 最先进的质谱学方法。该项目的长期目标是确定 通过GSK-3β影响ICM心脏的肌丝功能，目的是发现一种治疗方法 预防心肌梗死后向缺血性心肌病过渡的方法。

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.