Molecular mechanisms of mechanosensation in the cardiac pacemaker

项目摘要/摘要 在这个项目中，我们将测试整个假设，即压电通道介导心脏的机械感觉。 他们是心脏起搏器，是机械拉伸引起的心率加速的重要参与者。我们 将利用我们在心脏起搏器研究方面的专业知识，为我们的 实验室：机械传感和机电耦合。心脏是机械上最活跃的生物之一 身体里的器官。心脏除了是一种非常有效的泵外，还能感知它的机械环境 并调整其性能以匹配生理需求。在一种被称为“班布里奇”的机制中 反射“，心脏起搏器对静脉回流增加所引起的牵拉反应为 加快它的脚步，有效地排空心脏。为了感觉到伸展过程中的这些持续变化， 起搏器配备了伸展激活的通道，然而，它们的分子身份仍然难以捉摸。 到目前为止，在检测到其表达的每一种细胞类型中，压电通道都参与了机械转导。 尽管在起搏器中表达并被认为是调解起搏器的候选者 机械转导，压电通道在这个组织中的作用还没有被探索过。这项提议将 直接测试压电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特定起搏器的小鼠品系来测试压电波的效果 在细胞、组织和动物水平上抑制和过度表达起搏器的活动。我们会 结合免疫检测、原位杂交、电生理学、高分辨率成像、钙成像、 和遥测以：(目标1)表征丰度、异构体相对表达、细胞表达特异性、 Piezo1和Piezo2通道在起搏器组织和隔离起搏器中的亚细胞定位 细胞。(目的2)评价压电通道在起搏器牵张激活电流中的作用。 起搏细胞的自发放电频率、自律性及其阈值下钙离子的激活增加 活动。(目标3)。确定压电通道在牵张激活的电和钙反应中的作用 在完整的起搏器组织中及其在体内正常心脏功能中的作用。完成上面列出的目标 将为起搏细胞机械转导的分子机制提供新的见解，并将有助于 寻找检测和治疗相关心律失常的新靶点。我们的结果还将提供多样化的 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压电波通道。

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.