Mechanotransduction in Intestinal Smooth Muscle Cells

项目摘要/摘要 协调的胃肠道运动是胃肠道正常功能的基础。几种细胞类型 结合调节胃肠动力，以平滑肌细胞(SMC)为主力，提供 对宫缩的体力。SMC功能中断会导致常见的胃肠道疾病，可能发生在 感染和炎症，并与罕见但破坏性的胃肠动力障碍有关，如内脏肌病 和假性梗阻。在机械应力作用下，肠壁是一个高度复杂的多层结构。 基准线和不断移动。因此，胃肠道中的细胞经历了一系列类型和数量的 机械刺激。正常的协调运动需要有感知和适应力量的能力。在多个 在这笔拨款的循环中，我们剖析了平滑肌机械转导的机制，已经做出了 促进胃肠道生理学和病理生理学的发现，并提供了新的药物靶点。然而，我们的 目前对SMC机械传感的理解仍然不完整。已经确定，即使是作为单身的SMC 细胞在一个被称为肌源性反射的过程中，调整它们的收缩来对力做出反应。在血管内皮细胞中， 肌源性反射依赖于机械化的离子通道，但在胃肠道，细胞和分子。 机制仍然知之甚少。因此，我们研究的总体目标是确定 初级机械化离子通道参与GI-SMC的机械敏感性。为了这个提议，我们创作了一部小说 动物模型，并使用尖端技术生成令人信服的初步数据。我们的预赛 研究表明，最近发现的机械化离子通道TMem63a在一个亚群中表达 SMC，它针对力感应进行了优化，并分布在组织中以检测力。确实如此 原代小鼠胃肠道SMC的机械敏感离子电流具有独特的生物物理特性 与Tem63a一致，它的强制激活会导致钙离子的增加，调节大小 肠道收缩和整个肠道通过时间。有趣的是，我们的数据还显示，中转缓慢的患者 便秘患者Tem63a有减少的趋势。因此，中心假设是一个机械化的离子通道 Tem63a对肌源性反射的显著贡献将在两个目标上进行测试。在目标1中，我们确定 Tem63a的功能，它对力的反应，以及它在使用传统和尖端技术的GI SMC中的作用 电生理学和钙离子成像方法。在目标2中，我们提出了定义Tem63a的实验 目的：探讨Tmem63a SMC在胃肠道平滑肌功能调节中的作用。自.以来 Tem63a是在SMC的一个亚群中发现的，我们利用单细胞和空间转录，新的钙 成像、平滑肌收缩能力测定和活体全肠道运输。圆满完成拟议中的 创新实验既有基础意义又有临床影响，评估和建立新型SMC 有助于SMC功能和肌源性反射的机械化离子通道，从长远来看， 可能为功能性和运动性胃肠道疾病提供一个新的靶点。

PROJECT SUMMARY/ABSTRACT Coordinated gastrointestinal (GI) tract motility is fundamental for normal GI tract function. Several cell types combine to regulate GI motility, with the smooth muscle cell (SMC) as the workhorse required to provide the physical power for contractions. Disruptions in SMC function contribute to common GI disorders, may occur after infections and inflammation, and associate with rare but devastating GI motility disorders like visceral myopathies and pseudo-obstruction. The gut wall is a highly complex multilayered structure under mechanical stress at baseline and constantly moving. Therefore, cells in the GI tract experience a range of types and amounts of mechanical stimuli. The normal coordinated motility requires an ability to sense and adjust to forces. In multiple cycles of this grant, we have dissected mechanisms of smooth muscle mechanotransduction, have made discoveries that advanced GI physiology and pathophysiology, and provided novel drug targets. However, our current understanding of SMC mechanosensing remains incomplete. It is established that SMCs, even as single cells, adjust their contractions in response to force in a process called the myogenic reflex. In vascular SMCs, the myogenic reflex depends on mechanogated ion channels, but in the GI tract, cellular and molecular mechanisms remain poorly understood. Therefore, the overall objective of our research is to determine the primary mechanogated ion channels involved in GI SMC mechanosensitivity. For this proposal, we created novel animal models and used cutting-edge techniques to generate compelling preliminary data. Our preliminary studies show that a recently discovered mechanogated ion channel Tmem63a is expressed in a subpopulation of SMCs, which are optimized for force sensing and distributed across the tissue to detect force. Indeed mechanosensitive ionic currents in a population of primary mouse GI SMCs have unique biophysical properties consistent with Tmem63a, the activation of which by force leads to a Ca2+ increase, modulating small and large bowel contractions and whole gut transit time. Interestingly, our data also show that patients with slow transit constipation have a decrease in Tmem63a. Thus, the central hypothesis that a mechanogated ion channel Tmem63a significantly contributes to the myogenic reflex will be tested in two Aims. In Aim 1, we determine Tmem63a function, its response to force, and its role in GI SMCs using conventional and cutting-edge techniques electrophysiology and Ca2+ imaging approaches. In Aim 2, we propose experiments to define the Tmem63a+ SMC population and to determine the role of Tmem63a SMCs in regulating GI smooth muscle function. Since Tmem63a is found in a subpopulation of SMCs, we use single-cell and spatial transcriptomics, novel Ca2+ imaging, smooth muscle contractility assays and in vivo whole gut transit. Successful completion of the proposed innovative experiments has both basic significance and clinical impact, evaluating and establishing a novel SMC mechanogated ion channel which contributes to SMC function and the myogenic reflex and, in the long term, may provide a novel target for functional and motility GI disorders.