Targeting Pannexin 1 as a Novel Mechanism for Arrhythmia and Fibrosis in Duchenne Cardiomyopathy

项目摘要 心血管疾病是杜氏肌营养不良症（DMD）患者死亡的主要原因。 心律失常和心脏纤维化导致扩张型心肌病是心脏病的主要发病机制， mortality.泛膜联蛋白（Pxs）是大电导离子和小分子通道，已经被广泛应用于生物医学领域。 与其他纤维增生性疾病有关，并被认为在其他心脏模型中是致瘤性 疾病DMD中的主要缺陷肌营养不良蛋白的缺失导致细胞内钙（Ca 2+）升高， 也是Pxs的主要效应子。该项目的目标是研究Px 1 在DMD心肌病模型中调节心脏纤维化和血管生成的发展。 我们的初步数据表明，Px 1在心脏纤维化的发展中具有新的作用， 在DMD的D2-mdx模型中观察到的心律失常。D2-mdx模型（mdxPx 1-/-）中Px 1的遗传消除 挽救心脏表型，包括通过组织病理学评估的心脏正常化，fibrosis.as 异丙肾上腺素诱导的心室异位显著减少。根据这些数据，我们假设， 病理性升高的细胞内Ca 2+，这种疾病的标志，导致Px 1激活，并导致 激活凋亡、氧化和炎症通路的信号级联，最终导致 成纤维细胞活化和心脏纤维化的发展。我们还假设Px 1通道代表 通过产生延迟后去极化（DAD）的室性心律失常的独立机制。 我们使用本提案中概述的3个具体目标来测试这些假设。在目标1中，我们将使用 除了药理学Px抑制外，还具有整体Px 1缺失的转基因小鼠，以确定Px 1 激活导致触发的心律失常。在目标2中，我们将确定Px 1有助于 使用药理学和遗传学策略研究DMD心肌病中的心脏纤维化。当Px表示为 在心肌细胞和心脏成纤维细胞中，Aim 3将测试成纤维细胞迁移是否依赖于Px 1 使用共培养技术在心肌细胞和/或成纤维细胞中活化人诱导多能干细胞 细胞心肌细胞（hiPSC-CM）和心脏成纤维细胞。这些研究的完成将有助于改善 我们对DMD心血管疾病机制的理解，将为进一步研究DMD提供基础。 研究一种新的治疗靶点，有可能延迟或预防DMD的心脏死亡 患者此外，这项建议将使一个有前途的年轻医生科学家获得重要的技能， 心脏电生理学、细胞信号传导和炎症/纤维化生物学的基础和转化研究 在一个高度成就和敬业的导师委员会的专家指导下。这些新技能将 为从初级研究人员到独立资助的学术人员的成功过渡奠定基础 医学科学家

PROJECT SUMMARY Cardiovascular disease is the primary cause of death for patients with Duchenne muscular dystrophy (DMD). Arrhythmia and cardiac fibrosis leading to dilated cardiomyopathy are the primary mechanisms of cardiac mortality. Pannexins (Pxs), which are large conductance ion and small molecule channels, have been implicated in other fibroproliferative diseases and are thought to be arrhythmogenic in other model of cardiac disease. Loss of dystrophin, the primary defect in DMD, leads to elevated intracellular calcium (Ca2+) which is also a primary effector of Pxs. The goal of this project is to investigate the mechanisms by which Px1 modulates the development of cardiac fibrosis and arrhythmogenesis in models of DMD cardiomyopathy. Our preliminary data demonstrate a novel role for Px1 in the development of cardiac fibrosis and inducible arrhythmia seen in the D2-mdx model of DMD. Genetic ablation of Px1 in the D2-mdx model (mdxPx1-/-) rescues the cardiac phenotype, including normalization of cardiac fibrosis.as assessed by histopathology and significant reduction in isoproterenol-induced ventricular ectopy. Based on these data, we hypothesize that pathologically elevated intracellular Ca2+, a hallmark of this disease, leads to Px1 activation and results in signaling cascades that activate apoptotic, oxidative, and inflammatory pathways that ultimately lead to fibroblast activation and the development of cardiac fibrosis. We also hypothesize that Px1 channels represent an independent mechanism for ventricular arrhythmia via generation of delayed after-depolarizations (DADs). We with test these hypotheses using the 3 specific aims outlined in this proposal. In Aim 1, we will use transgenic mice with global Px1 deletion in addition to pharmacological Px inhibition to determine if Px1 activation results in triggered arrhythmia. In Aim 2, we will identify the mechanism by which Px1 contributes to cardiac fibrosis in DMD cardiomyopathy using pharmacological and genetic strategies. As Pxs are expressed in both cardiomyocytes and cardiac fibroblasts, Aim 3 will test if fibroblast migration is dependent on Px1 activation in cardiomyocytes and/or fibroblasts using co-culture techniques for human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts. The completion of these studies will help to improve our understanding of the mechanisms of cardiovascular disease in DMD and will provide the basis for further investigation of a novel therapeutic target that has the potential to delay or prevent cardiac mortality in DMD patients. Additionally, this proposal will allow a promising young physician scientist to gain important skill in basic and translational studies in cardiac electrophysiology, cell signaling, and inflammation/fibrosis biology under the expert guidance of a highly accomplished and dedicated mentorship committee. These new skills will provide the foundation for a successful transition from junior investigator to an independently-funded academic physician scientist.