A systems biology approach to understanding the role of myofilaments in cardiac regeneration

 描述(申请人提供)：心力衰竭(HF)是美国男性和女性的主要死亡原因，最终是由心脏损伤后心脏功能心肌细胞的丧失引起的，目前唯一解决心肌细胞丧失的治疗策略是心脏移植。不幸的是，供者心脏的需求远远大于供应，因此，心脏再生领域的目标是修复损伤后功能不全的心肌，这对减轻心衰相关的发病率和死亡率具有很大的希望。事实上，最近的几项临床前研究已经报道，在心脏损伤的大型动物模型以及因心肌梗死(MI)而患有心功能障碍的患者中，干细胞(SC)治疗后的心功能得到改善；然而，改善心功能的机制仍不清楚。鉴于将干细胞移植到受损心脏中可显著改善严重缺血心脏的收缩性能，以及心肌细丝在信号转导和接收中扮演的重要角色，我们假设干细胞与梗死周边区(BZ)高危心肌细胞之间的细胞信号转导，可能会刺激参与肌丝蛋白翻译后修饰改变的信号通路，而这些变化可以提高心肌的收缩能力，减少应激诱导的心肌细胞死亡。在此，我们采用一种系统的方法，结合体外和体内的功能研究，利用基于自上而下的质谱学(MS)的蛋白质组学来描述肌丝在SC相关的心脏再生中的作用。这项建议的具体目标包括：1)通过一种新的自上而下的蛋白质组学策略，确定与SC治疗相关的肌丝PTM变化，并通过对对照猪左室和心肌梗塞周围BZ分离的去皮心肌标本进行机械测量，将观察到的变化与心肌细胞收缩能力的变化相关联 和干细胞治疗的心肌梗塞猪。2)采用自上而下的蛋白质组学方法，确定心肌梗塞和干细胞处理的心肌梗塞猪心肌梗死周围区肌丝中PKA和PKC的磷酸化部位，并利用去皮心肌标本确定这些修饰对心肌收缩性能的影响。3)通过将重组肌丝蛋白与皮肤纤维束中的内源性肌丝蛋白交换，评估心脏再生相关PTM如何影响心肌收缩能力。这项拟议研究的成功将为干细胞相关心脏再生的分子机制提供新的见解，并可能促进改进的基于细胞的治疗方法的开发，这可能有助于对抗心肌梗死后发生的心衰。

 DESCRIPTION (provided by applicant): Heart failure (HF), the leading cause of death for both men and women in the US, is ultimately precipitated by a loss of functional cardiomyocytes from the heart following cardiac injury, and currently the only therapeutic strategy that addresses cardiomyocyte loss is heart transplantation. Unfortunately, the demand for donor hearts far outweighs the supply and, thus, the field of cardiac regeneration, which aims to repair dysfunctional myocardium following injury, holds great promise for the alleviation HF-associated morbidity and mortality. Indeed, several recent preclinical studies have now reported improved cardiac function following stem cell (SC) treatment in large animal models of cardiac injury as well as patients suffering from cardiac dysfunction as a consequence of myocardial infarction (MI); however, the mechanism underlying the improved cardiac function still remains unclear. Given that the contractile performance of severely ischemic hearts was significantly improved by engrafting SCs into the injured hearts, and the essential role that cardiac myofilaments play in signal transduction and reception, we hypothesize that cellular signaling between SCs and at-risk cardiomyocytes within the peri-infarct border zone (BZ), results in the stimulation of signaling pathways involved in the alteration of myofilament protein post-translational modifications, and that these changes can improve contractility and reduce stress-induced cardiac cell death. Herein, we are undertaking a systems approach to delineate the role of myofilaments in SC-associated cardiac regeneration utilizing top-down mass spectrometry (MS)-based proteomics in combination with in vitro and in vivo functional studies. The specific aims of this proposal include: 1) Identify myofilament PTM changes associated with SC treatment via a novel top-down proteomics strategy and correlate the observed changes with alterations in myocyte contractility by performing mechanical measurements of skinned myocardial preparations isolated from control pig left ventricle and from the peri-infarct BZ of MI and stem cell-treated MI pigs. 2) Identify the sites of PKA and PKC phosphorylation in myofilaments from the peri-infarct BZ of MI and stem cell-treated MI pig myocardium using top-down proteomics and determine the effects of these modifications on cardiac contractility using skinned myocardial preparations. 3) Assess how cardiac regeneration-associated PTMs influence cardiac contractility by exchanging recombinant myofilament proteins, engineered to express site-specific PTMs, with endogenous myofilament proteins in skinned fiber bundles and performing mechanical measurements. The success of the proposed research will offer new insights into the molecular mechanisms underlying SC-associated cardiac regeneration and may facilitate the development of improved cell-based therapies, which could aid in the battle against HF development post-MI.