Using collagenase to target collagen architecture in fibrotic skeletal muscle to reduce stiffness and promote tissue regeneration. - PROJECT SUMMARY In healthy skeletal muscle the extracellular matrix (ECM) provides structural support, facilitates lateral force transmission, and contributes to cell signaling. However, certain muscle diseases can alter the ECM to the detriment of muscle function. Duchenne Muscular Dystrophy (DMD) is one such disease which is caused by a mutation on the X-chromosome to the gene encoding dystrophin. The lack of functional dystrophin in DMD leads to a severe loss of muscle fiber structural integrity, resulting in repeated cycles of muscle damage with incomplete regeneration. This leads to the accumulation of ECM materials, termed fibrosis, which hinders muscle stem cell (MuSC) function, decreases muscle strength, and increases muscle stiffness that all lower the quality of life for patients with DMD. Fibrosis is also generally seen as irreversible, so there is a great need for treatments that slow or prevent the progression of fibrosis in patients with DMD and other musculoskeletal diseases. While previous research has shown that the architecture of collagen, the primary component and load-bearer of the ECM, drives deficits in MuSC function and muscle mechanical properties, few studies have tried to alter collagen architecture to restore muscle health. Thus, the proposed research aims to further elucidate the mechanisms by which fibrotic collagen architecture drives muscle stiffness and impairs MuSC regenerative capacity, and assess the ability of Collagenase Clostridium histolyticum (collagenase) to ablate collagen architecture, reduce fibrosis, and improve muscle health. Our central hypothesis is that mechanical and regenerative deficits in muscle function due to fibrotic collagen architecture are reversible through collagenase digestion of collagen fibers. Using the D2.mdx mouse, an established model of DMD, we will perform experiments that clarify the stretch dependence of collagen alignment on muscle stiffness, demonstrate the ability of collagenase to alter MuSC function on the fibrotic ECM, and evaluate the efficacy of collagenase in reducing fibrosis and restoring muscle health. Aim 1 will utilize the label-free imaging method of second harmonic generation (SHG) microscopy with concurrent mechanical testing to connect dynamic changes in collagen architecture to changes in passive mechanics of D2.mdx mouse muscles. Aim 2 will assay the ability of MuSC to proliferate and differentiate on healthy and fibrotic decellularized matrices with or without a collagenase treatment. Finally, Aim 3 will involve the use of intramuscular injections of collagenase to reduce fibrosis and muscle stiffness while improving muscle strength in anterior and posterior hindlimb muscles of D2.mdx mice. The results of these experiments will provide new insights into the importance of collagen architecture to muscle mechanical and regenerative function, and the usefulness of collagenase in treating muscle fibrosis.
