Connexin-based Signaling in the Heart: Cellular and Exosomal

项目摘要/摘要 缝隙连接(GJ)蛋白连接蛋白43(Cx43)长期以来一直被认为是动作传导的必备蛋白 心室肌的电位(AP)。然而，在过去20年里积累的证据表明 非哺乳动物、Cx43基因敲除小鼠和人类Cx43突变表明Cx43 GJS不提供 对AP如何在心脏传播的充分解释-实际上，Cx43 GJS对于 心脏传导。根据它的短短半小时(~1.5小时)，我们最近估计，平均而言，人类女性 心脏每年产生超过80克的Cx43--这个速度是肌动蛋白的30倍以上，肌动蛋白是最丰富的 心肌蛋白质处于稳定状态。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在脑室的传播机制是什么 这种现象？第二，如果心脏传导不是绝对需要的，心脏有什么目的？ 以如此高的速度生产Cx43？在最近报道的研究中，我们确定：a)反式- 电压门控钠通道相互作用复合体(TRANS-VGSCs)位于GJ边缘(周围神经节) 有助于心脏传导；以及B)Cx43羧基末端(CT)在调节 心脏对缺血损伤的反应。基于这些发现，该项目的目标是：1)确定 反式VGSCs在心脏AP传播中的作用及其作用程度 独立于基于Cx43 GJ的偶联；2)开发一种以反式VGSC为靶点的药理学方法 新的抗心律失常策略；3)确定心脏高水平产生Cx43的程度 这可能是因为最近的研究发现，Cx43在外切体中输出；4)确定循环 胞外体Cx43，包括Cx43 CT亚型，是调节缺血的自然信号机制的一部分 损伤严重程度；以及5)开发基于外切体的治疗心肌梗死的新方法 携带Cx43 CT模拟多肽，包括在大型动物模型-猪身上进行翻译测试。该项目 将涉及生物学家、药物化学家、生物医学工程师和 临床医生--包括人类和兽医。PI有真正的翻译诚意，有一种药物针对 Cx43 CT是在他的实验室发明的，目前正在进行多个第三阶段临床试验。将使用最先进的方法 包括超分辨率成像，在硅胶药物设计，转基因病毒，ipscs和测试 在大型动物模型上的基于外显子的疗法。我们假设心肌细胞Cx43的初步定位 可能不在AP传播中。相反，我们的假设是，对Cx43的高能承诺 由心脏和其他组织(如皮肤、血管和大脑)产生的物质主要与 Cx43在基于外体的信号机制中的作用，该机制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项目试图挑战关于GJ蛋白Cx43作用的主流观点，并翻译 为临床治疗心律失常和心肌梗死提供了新的视角。

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.