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.