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.
