Extracellular integration of a cohesive myotendinous junction

力通过一个称为肌腱连接的特殊界面从肌肉传递到肌腱 （MTJ）。MTJ由跨膜、细胞间和细胞外蛋白质组成，其将肌肉连接到 细胞和细胞外基质（ECM）。ECM是一种大分子的3D网络， 肌肉和肌腱的连续体，促进力的无缝传递。指状交错 MTJ的界面在从肌肉到肌腱的力传递中是关键的，并且被假设为减少应力; 然而，MTJ损伤可由各种事件引起，包括体力劳动、运动和创伤， 通常由于过大的偏心力而发生。工程技术临床转化的挑战 由于难以整合两种不同的组织以形成 无缝MTJ。本提案的目的是确定ECM和机械载荷在以下方面的作用： 建立和维持肌肉和肌腱之间的牢固联系。这些知识将是必不可少的 用于开发治疗方法以恢复受损MTJ的功能。 拟议的研究将测试这一假设，即机械坚固的MTJ的形成依赖于 对肌肉和结缔组织细胞分泌的ECM在界面处的整合有影响， 循环加载。我们将从两个不同的目标来检验我们的假设。在目标1中，我们将识别细胞， 建立MTJ特异性ECM的相互作用。我们将使用细胞特异性代谢标记， 蛋白质组学，以确定肌肉和结缔组织细胞独立贡献的组成部分， MTJ（目标1a）。由于目前在技术上不可行的是，仅靶向表达MTJ特异性蛋白， 在体内的肌肉-肌腱界面，我们将使用体外共培养来研究和干扰相互作用。 生肌细胞和结缔组织细胞之间的信号传导（Aim 1b）。 在目标2中，我们将确定机械力在创建和维持功能性MTJ中的作用。一是 将通过使用肌肉发育不全小鼠直接研究胚胎运动如何影响发育 肌肉收缩被取消的模型（目标2a）。然后，我们将直接比较如何卸载（后肢 悬吊）和增加负荷（跑步机训练）影响ECM在界面处的重塑， 代谢标记和蛋白质组学。接下来，我们将基于纤维蛋白凝胶生成3D肌肉肌腱结构， 细胞产生的ECM（目标2b），因为在体内完全去除所有机械力是不可行的。的 构造将使我们能够测试卸载、静态和循环加载对MTJ形成的影响。 成功完成拟议的研究将确定细胞外参数， 保持肌肉和肌腱之间的界面，这对于开发治疗方法以恢复 功能损坏的MTJ。

Force is transmitted from muscle to tendon across a specialized interface called the myotendinous junction (MTJ). The MTJ consists of transmembrane, intercellular and extracellular proteins that connect muscle to the cells and extracellular matrix (ECM) of tendon. The ECM is a 3D network of macromolecules that forms a continuum integrating muscle and tendon, facilitating the seamless transmission of force. The interdigitating interface of the MTJ is critical in force transmission from muscle to tendon and is hypothesized to reduce stress; nevertheless, MTJ injuries can arise from diverse events, including physical work, sports and trauma, which typically occur due to excessive eccentric force. Challenges in the clinical translation of engineering technologies targeted toward the muscle-tendon interface arise from the difficulty in integrating two disparate tissues to form a seamless MTJ. The objective of this proposal is to identify the role of the ECM and mechanical loading in establishing and maintaining the strong linkage between muscle and tendon. This knowledge will be essential for developing therapies to restore functionality to damaged MTJs. The proposed studies will test the hypothesis that the formation of a mechanically robust MTJ is dependent on the integration of ECM secreted by both muscle and connective tissue cells at the interface and is maintained by cyclic loading. We will test our hypothesis in two separate aims. In Aim 1, we will identify the cells and reciprocal interactions that establish an MTJ-specific ECM. We will use cell-specific metabolic labeling and proteomics to identify the components that muscle and connective tissue cells independently contribute to the MTJ (Aim 1a). Since it is currently technically infeasible to target the expression of MTJ-specific proteins only at the muscle-tendon interface in vivo, we will use in vitro co-cultures to investigate and perturb the reciprocal signaling between myogenic and connective tissue cells (Aim 1b). In Aim 2, we will determine role of mechanical forces in creating and maintaining a functional MTJ. First, we will directly investigate how embryonic motility affects development by using the muscular dysgenesis mouse model in which muscle contraction is abrogated (Aim 2a). Then we will directly compare how unloading (hindlimb suspension) and increased loading (treadmill training) affect the remodeling of ECM at the interface using metabolic labeling and proteomics. Next, we will generate 3D muscle-tendon constructs based on fibrin gels and cell-generated ECM (Aim 2b), since is not feasible to completely remove all mechanical forces in vivo. The constructs will enable us to test the effect of unloading, and static and cyclic loading on MTJ formation. Successful completion of the proposed studies will identify the extracellular parameters that establish and maintain the interface between muscle and tendon, which is essential for developing therapies to restore functionality to damaged MTJs.