Molecular mechanisms of mechanosensation in the cardiac pacemaker

项目总结/摘要 在这个项目中，我们将测试PIEZO通道介导心脏机械感觉的整体假设， 心脏起搏器，他们是必不可少的球员对心率加速引起的机械拉伸。我们 将利用我们在心脏起搏器研究方面的专业知识，为我们进入两个新的研究领域， 实验室：机械感觉和机电耦合。心脏是人体内最活跃的 身体里的器官。除了作为一个非常有效的泵，心脏感觉它的机械环境 并调整其性能以匹配生理需求。在一个被称为“班布里奇”的机制中 心脏起搏器对静脉回流增加引起的牵拉反应， 加速其步伐，以有效地清空心脏。为了感知这些持续的拉伸变化， 起搏器配备有牵张激活通道，然而，它们的分子身份仍然难以捉摸。 PIEZO通道介导迄今为止已检测到其表达的每种细胞类型中的机械转导。 尽管在起搏器中表达，并被认为是介导起搏器的候选者， 尽管PIEZO通道在机械转导中的作用尚未被探索。这项建议会 直接测试PIEZO通道在心脏起搏器牵张激活反应中的作用。 我们的创新方法包括开发起搏器特异性小鼠品系来测试PIEZO的效果 在细胞、组织和动物水平上的起搏器活性的敲低和过表达。我们将 联合收割机免疫检测、原位杂交、电生理学、高分辨率成像、钙成像， （目的1）表征所述基因的丰度、同种型相对表达、细胞表达特异性， Piezo 1和Piezo 2通道在起搏器组织和离体起搏器中的定位和亚细胞定位 细胞(Aim 2）评估PIEZO通道在起搏器牵张激活电流、牵张激活电流和牵张激活电流中的作用。 激活增加内在放电率，起搏细胞的自律性，并在其阈下钙 活动(Aim 3）。确定PIEZO通道在牵张激活的电和钙反应中的作用 以及它们在体内正常心脏功能中的作用。完成上述目标 将为起搏细胞中机械传导的分子机制提供新的见解， 以识别用于检测和治疗相关心律失常的新靶点。我们的研究结果还将提供一个多样化的 用于识别起搏器伸展转换为心率背后的多种重要机制的工具包 加速，为我们的实验室研究受调控的下游信号通路开辟了新的途径 通过机械激活这个组织中的PIEZO通道。

Project Summary/Abstract In this project, we will test the overall hypothesis that PIEZO channels mediate mechanosensation in the cardiac pacemaker and that they are essential players on the heart rate acceleration evoked by mechanical stretch. We will leverage our expertise in the study of the cardiac pacemaker to enter into two new research fields for our laboratory: mechanosensation and mechano-electrical coupling. The heart is one of the most mechanically active organs in the body. Besides being a remarkably effective pump, the heart senses its mechanical environment and adjusts its performance to match the physiological demands. In a mechanism known as the “Bainbridge Reflex”, the cardiac pacemaker responds to the stretch induced by the increase in venous return with an acceleration of its pace to empty the heart effectively. To sense these constant changes in stretch, the pacemaker is equipped with stretch-activated channels, however, their molecular identity remains elusive. PIEZO channels mediate mechanotransduction in every cell type where its expression has been detected so far. Despite being expressed in the pacemaker and being considered the candidate to mediate pacemaker mechanotransduction, the role of PIEZO channels in this tissue has not been explored yet. This proposal will directly test the role of PIEZO channels in the stretch-activated response of the cardiac pacemaker. Our innovative approach includes the development of pacemaker-specific mouse lines to test the effect of PIEZO knockdown and overexpression in the pacemaker activity at the cellular, tissue, and animal level. We will combine immunodetection, in-situ hybridization, electrophysiology, high-resolution imaging, calcium imaging, and telemetry to: (Aim 1) Characterize the abundance, isoform relative expression, cell-expression specificity, and subcellular localization of Piezo1 and Piezo2 channels in the pacemaker tissue and isolated pacemaker cells. (Aim 2) Evaluate the role of PIEZO channels in the pacemaker stretch-activated current, the stretch- activated increase in intrinsic firing rate, the automaticity of pacemaker cells, and in their subthreshold calcium activity. (Aim 3). Determine the role of PIEZO channels in the stretch-activated electrical and calcium responses in the intact pacemaker tissue and their role in normal heart function in vivo. Completing the aims listed above will provide new insight into the molecular mechanisms of mechanotransduction in pacemaker cells and will help to identify novel targets for detecting and treating associated arrhythmias. Our results will also provide a diverse toolkit to identify multiple important mechanisms behind the translation of pacemaker stretch into heart rate acceleration, opening new avenues for our lab to study the downstream signaling pathways that are regulated by the mechanical activation of PIEZO channels in this tissue.