Temporal Changes in the Muscle Extracellular Matrix Due to Volumetric Muscle Loss Injury Promote Functional Fibrosis

Abstract

Skeletal muscle exhibits a remarkable ability to adapt and recover from a variety of stressors placed upon it. For example, numerous models of muscle injury, such as freeze, crush, and myotoxin, display immediate robust physical damage to the tissue when observed histologically. Yet in all three models the damaged tissue is able to completely regenerate back to pre-injury levels of fiber numbers and function. A complex process of cellular signaling mediates the successful regeneration seen in these models, which is ultimately achieved through satellite cell proliferation and fusion into new and existing myofibers. However, in more severe cases of traumatic injury, such as volumetric muscle loss (VML), there is a degree of damage that cannot be overcome endogenously. Instead of muscle regeneration, VML results in extensive fibrotic deposition and prolonged inflammatory signaling that dramatically reduces muscle function. The persistent fibrotic (TGF-β1, CCN2/CTGF) and inflammatory (IL-6, TNF-α) signaling further limits the ability of current treatment options to make significant improvements in patient outcomes. Moreover, the temporal changes in these processes have not been well documented following VML injury. Therefore, a study design using terminal time points of 3, 7, 14, 21 and 48 days following VML injury was created to evaluate the changes in extracellular proteins leading to fibrosis. Briefly, an initial significant increase in the proportion of collagen III was observed, which was subsequently reduced and overtaken by collagen I after 48 days post-VML. Similarly, histological results indicated an increase in loosely packed collagen through 21 days, before switching to an overwhelmingly dense collagen matrix by 48 days post-VML. Taken together, the window for successful intervention is likely before this shift to densely packed collagen I. Future work will evaluate immediate and delayed CCN2/CTGF inhibition combined with minced muscle graft implantation post-VML injury for effectiveness as interventions. Furthermore, the relationship between impaired neuromuscular junctions and fibrotic signaling will be assessed.