Mechanotransduction in Intestinal Smooth Muscle Cells

项目总结/摘要 协调的胃肠道运动是正常胃肠道功能的基础。几种细胞类型 联合收割机调节胃肠动力，平滑肌细胞（SMC）作为提供胃肠动力所需的主力。 收缩的体力。SMC功能的破坏导致常见的GI疾病，可能发生在 感染和炎症，并与罕见但破坏性胃肠道动力障碍，如内脏肌病 和假性梗阻肠壁是一个高度复杂的多层结构，在机械应力下， 基线和不断移动。因此，胃肠道中的细胞经历一系列类型和量的炎症。 机械刺激正常的协调运动需要感知和调整力的能力。在多个 周期的这一补助金，我们已经解剖了平滑肌机械转导机制， 这些发现推进了胃肠道生理学和病理生理学，并提供了新的药物靶点。但我们的 目前对SMC机械感测的理解仍然不完全。据确定，SMC，即使作为单一的 在一个叫做肌原性反射的过程中，细胞会调整收缩以响应力。在血管平滑肌细胞中， 肌源性反射依赖于机械离子通道，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 主要机械离子通道参与GI SMC机械敏感性。为了这个提议，我们创造了一个新的 动物模型，并使用尖端技术来产生令人信服的初步数据。我们的初步 研究表明，最近发现的机械离子通道Tmem 63 a在亚群中表达， 的SMC，其针对力感测进行了优化，并分布在整个组织中以检测力。确实 原代小鼠胃肠道平滑肌细胞中的机械敏感离子电流具有独特的生物物理特性 与Tmem 63 a一致，通过强制激活Tmem 63 a导致Ca 2+增加，调节小的和大的 肠收缩和整个肠道通过时间。有趣的是，我们的数据还显示， 便秘患者Tmem 63 a减少。因此，中心假设是机械门控离子通道 将在两个目标中测试Tmem 63 a对肌源性反射的显著贡献。在目标1中，我们确定 Tmem 63 a的功能，其对力的响应，以及其在使用传统和尖端技术的GI SMC中的作用 电生理学和Ca 2+成像方法。在目标2中，我们提出了实验来定义Tmem 63 a + SMC群体，并确定Tmem 63 a SMC在调节GI平滑肌功能中的作用。以来 Tmem 63 a在SMC的亚群中被发现，我们使用单细胞和空间转录组学，新的Ca 2 + 成像、平滑肌收缩性测定和体内全肠道运输。圆满完成拟议的 创新实验既有基础意义又有临床影响，评价和建立一种新的SMC 机械门控离子通道，有助于SMC功能和肌源性反射，从长远来看， 可能为功能性和运动性GI疾病提供新的靶点。

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.