Connexin-based Signaling in the Heart: Cellular and Exosomal

项目摘要/总结 差距连接（GJ）蛋白连接蛋白43（Cx43）长期以来被认为是传导作用的专性蛋白 心室肌电位（AP）。然而，在过去的二十年里， 非哺乳动物、Cx43敲除小鼠和人Cx43突变表明，Cx43 GJ不提供 这充分解释了AP如何在心脏中传播-事实上，Cx43 GJ可能是由于 心脏传导基于其短半（约1.5小时），我们最近估计，平均而言， 心脏每年产生超过80克的Cx43--比最丰富的肌动蛋白的速率高出30倍以上。 稳定状态下的心肌蛋白。我的实验室专注于这些数据提出的两个问题。第一、 如果仅基于Cx43 GJ的偶联不能解释AP在心室中传播的机制， 这种现象？第二，如果心脏传导不是绝对需要的，那么心脏传导的目的是什么？ 产生Cx43的速度如此之快在最近报道的研究中，我们已经确定：A）反式- 位于GJ边缘的电压门控钠通道（trans-VGSC）相互作用复合物（perinexus） 有助于心脏传导;和B）Cx43羧基末端（CT）在调节心脏传导中具有关键作用。 心脏对缺血性损伤的反应基于这些发现，本项目的目标是：1）确定 trans-VGSC对心脏AP传播的贡献以及这种机制的作用程度 独立于基于Cx43 GJ的偶联; 2）开发靶向trans-VGSC的药理学方法， 新的抗心律失常策略; 3）确定心脏产生高水平Cx43的程度， 可以通过最近发现Cx43在外来体中输出来解释; 4）确定是否循环 外泌体Cx43，包括Cx43 CT同种型，是调节缺血性心脏病的天然信号传导机制的一部分。 损伤严重程度;以及5）开发基于外来体的治疗心肌梗死的新治疗方法 携带Cx43 CT模拟肽，包括在大型动物模型-猪中的翻译测试。项目 将涉及生物学家，药物化学家，生物医学工程师和 临床医生-人类和兽医。PI具有真正的翻译诚意，药物靶向 Cx43 CT在他的实验室发明，目前在多个III期临床试验。将采用最先进的方法 包括超分辨率成像、计算机药物设计、转基因病毒、iPSC和 在大型动物模型上进行基于外泌体的治疗。我们认为，心肌Cx43的主要分配， 可能不在AP传播中。相反，我们的假设是，对Cx43的高能量承诺 由心脏和其它组织（例如，皮肤、脉管系统和脑），主要涉及 Cx43在基于外泌体的信号传导机制中调节人类和其他动物对缺血性损伤的反应 哺乳动物这个R35项目旨在挑战关于GJ蛋白Cx43作用的流行观点，并翻译 为治疗心律失常和心肌梗死的临床获益提供了新的视角。

PROJECT ABSTRACT/SUMMARY The gap junction (GJ) protein Connexin 43 (Cx43) has long been held to be obligate for conduction of action potential (AP) in the ventricular myocardium. However, accumulating evidence during the last two decades from non-mammals, Cx43 knockout mice and human Cx43 mutations suggests that Cx43 GJs do not provide a sufficient explanation for how AP propagates in the heart – and indeed, that Cx43 GJs may be dispensable for cardiac conduction. Based on its short half (~1.5 hours), we recently estimated that on average the human female heart produces more than 80 grams of Cx43 per year – a rate more than 30-times that of actin, the most abundant myocardial protein in steady state. My lab is focused on two questions that are raised by these data. First, what is the mechanism of AP propagation in the ventricle, if Cx43 GJ-based coupling alone cannot account for this phenomenon? Second, if not absolutely required for cardiac conduction, for what purpose does the heart produce Cx43 at such high rates? In recently reported studies, we have determined that: A) Trans- interacting complexes of voltage-gated sodium channels (trans-VGSCs) located at the GJ edge (the perinexus) contribute to cardiac conduction; and B) That the Cx43 carboxyl terminus (CT) has key roles in modulating the heart's response to ischemic injury. Building on these findings, the goals of this project are to: 1) Determine the contribution of trans-VGSCs to cardiac AP propagation and the extent to which this mechanism operates independent of Cx43 GJ-based coupling; 2) Develop a pharmacologic approach to targeting trans-VGSCs as a novel anti-arrhythmic strategy; 3) Determine the extent to which the high levels of Cx43 production by the heart may be accounted for by recent findings that Cx43 is exported in exosomes; 4) Determine whether circulating exosomal Cx43, including Cx43 CT isoforms, are part of a natural signaling mechanism that modulates ischemic injury severity; and 5) Develop a novel therapeutic approach to treat myocardial infarction based on exosomes carrying Cx43 CT mimetic peptides, including translational testing in a large animal model – the pig. The project will involve cross-disciplinary collaboration between biologists, medicinal chemists, biomedical engineers and clinicians – both human and veterinary. The PI has genuine translational bona-fides, with a drug targeting the Cx43 CT invented in his lab currently in multiple Phase III clinical trials. State-of-the-art approaches will be used including super resolution imaging, in silico drug design, genetically modified viruses, iPSCs and testing of exosome-based therapeutics on large animal models. We posit that the primary assignment of myocardial Cx43 may not be in AP propagation. Rather, it is our hypothesis that the high energetic commitment to Cx43 production made by the heart, and other tissues (e.g., skin, vasculature and brain), relates mainly to functions of Cx43 in an exosome-based signaling mechanism that modulates response to ischemic injury in humans and other mammals. This R35 project seeks to challenge prevailing views on the role of the GJ protein Cx43 and translate the new perspectives gained for clinical benefit in treatment of arrhythmia and myocardial infarction.