Targeted membrane integrity in cardiac ischemia and reperfusion

心肌缺血/再灌注（I/R）损伤是一个主要的人类健康问题，每年导致数百万人死亡。心肌I/R涉及严重的心肌细胞功能障碍和心肌细胞死亡，目前尚无治愈方法。由于I/R改变了许多细胞过程，包括活性氧（ROS）升高、膜不稳定、细胞内Ca 2+处理不当、线粒体解偶联等，因此分析I/R中特定机制途径的作用具有挑战性。尽管存在这些挑战，但肌细胞产生的ROS被广泛认为是导致心脏I/R中细胞损伤的关键起始事件。我们提供了令人兴奋的新证据，证明心肌细胞产生的ROS与心脏I/R损伤显著解偶联。因此，这项建议集中在一个新的范例，在测试的假设，肌膜稳定性，增加活性氧的生产，是中央的I/R损伤。为了探究I/R机制，我们将实施肌膜稳定剂，其在初步工作中显著限制肌细胞渗漏、Ca 2+处理不当、线粒体膜去极化并保持肌细胞活力，尽管I/R介导的ROS增加。这些结果通过强调肌膜稳定在I/R损伤途径中的中心地位，挑战了I/R损伤机制的教条。在此提出了通过细胞表面相互作用合成共聚物稳定肌膜，作为机械解剖I/R中心肌膜完整性的直接作用的工具。基于共聚物的膜稳定剂是两亲性长链大分子共聚物，其在应力期间与细胞膜相互作用并保护细胞膜。该建议的首要假设是，独立于I/R介导的ROS产生，合成的肌膜稳定剂的功能是显著限制肌细胞Ca 2+失调和线粒体去极化，以增加体内心脏泵的性能。换句话说，我们将检验ROS单独不足以引起I/R损伤的假设。如果这一假设正确，将通过提供直接证据来确立膜完整性在I/R发病机制中的中心地位，从而改变该领域。这项工作的影响来自于使用合成肌膜稳定剂作为一种可行的新治疗选择，可以很容易地转化为I/R的临床设置。具体目标是：目标1。为了检验以下假设：在I/R期间，独立于肌细胞ROS产生，共聚物肌膜稳定剂将显著限制膜渗漏、细胞内Ca 2+错误处理和线粒体膜去极化，以促进体外啮齿动物成体肌细胞和人iPSC衍生的心肌细胞中的细胞活力。目标2.在临床相关的猪体内心肌I/R损伤模型中，检验共聚物肌膜稳定化将促进心肌细胞活力并保护心肌功能的假设。

DESCRIPTION (provided by applicant): Myocardial ischemia/reperfusion (I/R) injury is a major human health problem causing millions of deaths per year. Myocardial I/R involves severe cardiac myocyte dysfunction and myocyte death for which there is no cure. It has been challenging to dissect the role of specific mechanistic pathways in I/R owing to the multitude of cellular processes altered in I/R, including elevated reactive oxygen species (ROS), membrane instability, intracellular Ca2+ mishandling, mitochondrial uncoupling and others. Despite these challenges, myocyte generated ROS is widely considered a key initiating event leading to cellular damage in cardiac I/R. We provide exciting new evidence of a significant uncoupling of myocyte-generated ROS and damage in cardiac I/R. Thus, this proposal focuses on a new paradigm in testing the hypothesis that sarcolemma stability, independent of increased ROS production, is central to I/R injury. To interrogate I/R mechanisms we will implement sarcolemma stabilizers that in preliminary work significantly limit myocyte leak, Ca2+ mishandling, mitochondrial membrane depolarization and preserve myocyte viability despite I/R-mediated increased ROS. These results challenge the dogma of the mechanism of I/R damage by highlighting sarcolemma stabilization as central in the I/R injury pathway. Sarcolemma stabilization by cell surface interacting synthetic copolymers is proposed here as a tool for mechanistic dissection of the direct role of cardiac muscle membrane integrity in I/R. Copolymer-based membrane stabilizers are amphiphilic long-chain macromolecular copolymers that interact with and protect cellular membranes during stress. The overarching hypothesis of this proposal is that, independent of I/R-mediated ROS production, synthetic sarcolemma stabilizers function to significantly limit myocyte Ca2+ dysregulation and mitochondrial depolarization to increase heart pump performance in vivo. Stated differently, we will test the hypothesis that ROS alone is insufficient to cause I/R injury. This hypothesis, if correct, will change the field by providing direct evidence in establishing membrane integrity as central in I/R pathogenesis. The impact of this work derives from using synthetic sarcolemma stabilizers as a viable new therapeutic option that could be readily translated to clinical settings of I/R. The Specific Aims are: Aim 1. To test the hypothesis that during I/R, independent of myocyte ROS production, copolymer sarcolemma stabilizers will significantly limit membrane leak, intracellular Ca2+ mishandling and mitochondrial membrane depolarization to promote cell viability in rodent adult myocytes and human iPSC-derived cardiac myocytes in vitro. Aim 2. To test the hypothesis that copolymer sarcolemma stabilization will promote myocyte viability and preserve myocardial function in a clinically relevant porcine model of myocardial I/R injury in vivo.