ABSTRACT Mechano-stimulation in the musculoskeletal system plays a major role in the homeostasis of all tissues and organs. Particularly in tendons, the proper physiologic exercise load has an anabolic effect with increased tenogenic gene expression in humans; however, the molecular mechanisms that sense mechanical stimuli remain unclear. Piezo1 is a mechano-sensitive ion channel involved in mechano-sensing in various organs and tissues; however, it has not yet been examined in tendons or ligaments. In mice, the R2482H mutation of Piezo1 is an active form mimicking a gain-of-function (GOF) PIEZO1 variant found in humans. To test the potential effects of Piezo1 in physical performance, we generated systemic GOF Piezo1 (Piezo1systemic-GOF) mice, muscle-specific GOF Piezo1 (Piezo1muscle-GOF) mice, and tendon-specific GOF Piezo1 (Piezo1tendon-GOF) mice and found that the jumping power of Piezo1tendon-GOF mice as well as Piezo1systemic-GOF mice at 12-weeks- old was ~1.6 times greater than in both wild-type mice and Piezo1muscle-GOF mice. Consistent with this, significantly altered macroscopic and histological phenotypes were observed in the tendons of Piezo1tendon-GOF mice with increased tendon-specific gene expression. We also found that upregulation of aging related ossification gene expression was significantly reduced in aged Piezo1tendon-GOF mice. This suggests that constitutively enhanced PIEZO1 activity promotes physical performance via tendons/ligaments. These preliminary data underlie our hypothesis that PIEZO1 acts as a critical mechano-sensor in tenocytes, maintaining and promoting tendon/ligament functions by orchestrating tendon/ligament-specific gene expression. To test this hypothesis, we will pursue the following Aims: Aim 1: Analyze the function of Piezo1 in human and mouse tendon homeostasis and aging. We will examine the function of Piezo1 in tenocytes for tendon homeostasis and in aging by morphological, histological and gene expression analysis of tendons from Piezo1tendon-GOF mice and tendon specific loss of function (LOF) Piezo1 (Piezo1tendon-LOF) mice at different aging points. We will also test the role of PIEZO1 in human tenocytes by transcriptome analysis with PIEZO1 siRNA knockdown or a small molecule PIEZO1 activator. Aim 2: Evaluate the role of Piezo1 in tenocytes on physiological performance and exercise in mice. We will evaluate the role of Piezo1 in tenocytes to potentiate physical performance using postnatally induced tendon specific gain of function and loss of function Piezo1 mice at different ages. Thereafter, we will examine whether this physical ability could be strengthened by physiological exercise inducing anabolic mechanical stimulation. Aim 3: Analyze the biomechanical properties of tendon that are regulated by Piezo1. We will uncover how the kinetic energy for the enhanced jumping ability of the Piezo1tendon-GOF mouse is produced by biomechanical analysis of tendon stored energy and muscle contraction power...