Mechano signals regulating tendon and ligament homeostasis

摘要 肌肉骨骼系统中的机械刺激在所有组织和组织的动态平衡中起主要作用 器官。特别是在肌腱中，适当的生理性运动负荷会随着增加而产生合成代谢作用。 张力基因在人类中的表达；然而，感觉机械刺激的分子机制 目前仍不清楚。PIEZO1是一种机械敏感的离子通道，参与各种器官的机械传感和 组织；然而，它还没有在肌腱或韧带中进行检测。在小鼠中，R2482H突变 PIEZO1是一种活性形式，模仿在人类中发现的一种功能增益(GOF)PIEZO1变体。要测试 Piezo1在体能表现上的潜在影响，我们产生了全身性GOF Piezo1(Piezo1系统-GOF) 小鼠、肌肉特异性GOF Piezo1(Piezo1肌肉-GOF)小鼠和肌腱特异性GOF Piezo1(Piezo1tendon-GOF) 发现Piezo1tendon-GOF小鼠和Piezo1全身GOF小鼠在12周时的跳跃能力- OLD是野生型小鼠和Piezo1肌肉GOF小鼠的1.6倍。与此一致的是， 在Piezo1肌腱-GOF的肌腱中观察到明显的宏观和组织学表型变化 肌腱特异性基因表达增加的小鼠。我们还发现，衰老的上调与 老年Piezo1 Tenon-GOF小鼠成骨基因表达显著降低。这表明 结构性增强的PIEZO1活动通过肌腱/韧带促进身体表现。这些 初步数据支持了我们的假设，即PIEZO1在肌腱细胞中起着关键的机械传感器的作用， 通过协调肌腱/韧带特异性基因维持和促进肌腱/韧带功能 表情。为了验证这一假说，我们将追求以下目标：目标1：分析 PIEZO1在人和小鼠肌腱动态平衡和衰老中的作用。我们将研究Piezo1在 肌腱细胞在肌腱稳态和衰老中的形态、组织学和基因表达分析 Piezo1肌腱-GOF小鼠和肌腱特异性功能丧失(LOF)Piezo1(Piezo1肌腱-LOF)小鼠 不同的老化点。我们还将通过转录组分析来测试PIEZO1在人类腱细胞中的作用 PIEZO1 siRNA敲除或小分子PIEZO1激活剂。目的2：评估Piezo1在 腱细胞对小鼠生理表现和运动的影响。我们将评估Piezo1在 肌腱细胞利用出生后诱导的肌腱特异性功能获得和增强体能 不同年龄段Piezo1小鼠功能丧失。之后，我们将研究这种体能是否可能 通过生理性运动诱导合成代谢机械刺激而增强。目标3：分析 肌腱受Piezo1调控的生物力学特性。我们将揭开动能是如何 为了提高Piezo1肌腱的跳跃能力，通过肌腱的生物力学分析产生了GOF小鼠 储存能量和肌肉收缩能力。完成这些目标将支持Piezo1的概念 可作为维持或增强肌肉骨骼系统结构和功能的治疗靶点。

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. Completion of these aims will support the concept that Piezo1 could be a therapeutic target to maintain or enhance structure and function of the musculoskeletal system.