Extracellular integration of a cohesive myotendinous junction

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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.