Małgorzata Zimowska

The injury of adult skeletal muscle initiates series of well-coordinated events that lead to the efficient repair of the damaged tissue. Any disturbances during muscle myolysis or reconstruction may result in the unsuccessful regeneration, characterised by strong inflammatory response and formation of connective tissue, that is, fibrosis. The switch between proper regeneration of skeletal muscle and development of fibrosis is controlled by various factors. Amongst them are those belonging to the transforming growth factor β family. One of the TGF-β family members is TGF-β1, a multifunctional cytokine involved in the regulation of muscle repair via satellite cells activation, connective tissue formation, as well as regulation of the immune response intensity. Here, we present the role of TGF-β1 in myogenic differentiation and muscle repair. The understanding of the mechanisms controlling these processes can contribute to the better understanding of skeletal muscle atrophy and diseases which consequence is fibrosis disrupting muscle function.

BACKGROUND INFORMATION: The regeneration of skeletal muscles involves satellite cells, which are muscle-specific precursor cells. In muscles, injured either mechanically or as a consequence of a disease, such as muscular dystrophy, local release of the growth factors and cytokines leads to satellite cells activation, proliferation and differentiation of the resulting myoblasts, followed by the formation of new myofibres. Various cell types, such as stem and progenitor cells, originating from other tissues different than the muscle, are also able to follow a myogenic program. Participation of these cells in the repair process depends on their precise mobilisation to the site of the injury. RESULTS: In this study, we showed that stromal-derived factor-1 (Sdf-1) impacts on the mobilisation of CXC chemokine receptor (Cxcr)4-positive cells and improves skeletal muscle regeneration. Analysis of isolated and in vitro cultured satellite cells showed that Sdf-1 did not influence myoblasts proliferation and expression of myogenic regulatory transcription factors but induced migration of the myoblasts in Cxcr4-dependent ways. This phenomenon was also associated with the increased activity of crucial extracellular matrix modifiers, i.e. metalloproteases Mmp-2 and Mmp-9. CONCLUSIONS: Thus, positive impact of Sdf-1 on muscle regeneration is related to the mobilisation of endogenous cells, that is satellite cells and myoblasts, as well as non-muscle stem cells, expressing Cxcr4 and CD34.

Skeletal muscles exhibit great plasticity and an ability to reconstruct in response to injury. However, the repair process is often inefficient and hindered by the development of fibrosis. We explored the possibility that during muscle repair, the different regeneration ability of the fast (extensor digitorum longus; EDL) and slow twitch (Soleus) muscles depends on the differential expression of metalloproteinases (MMP-9 and MMP-2) involved in the remodeling of the extracellular matrix. Our results show that MMP-9 and MMP-2 are present in the intact muscle and are up-regulated after crush-induced muscle injury. The expression and the activity of these two enzymes depend on the type of muscle and the phase of muscle regeneration. In the regenerating Soleus muscle, elevated levels of MMP-9 occurred during the myolysis and reconstruction phase. In contrast, regenerating EDL muscles exhibited decreased MMP-9 levels during myolysis and increased MMP-2 activity at the reconstruction phase. Moreover, satellite cells (mononuclear myoblasts) derived from Soleus and EDL muscles showed no differences in localization or activity of MMP-9 and MMP-2 during proliferation and differentiation in vitro. MMP-9 activity was present during all stages of myoblast differentiation, whereas MMP-2 activity reached its highest level during myoblast fusion. We conclude that MMPs are involved in muscle repair, and that fast and slow twitch muscles exhibit different patterns of MMP-9 and MMP-2 activity.

When injured by crushing, the repair of the slow-twitch soleus rat muscle, unlike the fast-twitch EDL, is associated with fibrosis. As TGFbeta1, whose activity can be controlled by glycosaminoglycans (GAG), plays a major role in fibrosis, we hypothesized that levels of TGFbeta1 and GAG contents could account for this differential quality of regeneration. Here we show that the regeneration of the soleus was accompanied by elevated and more sustained TGFbeta1 level than in the EDL. Neutralization of TGFbeta1 effects by antibodies to TGFbeta1 or its receptor TGFbeta-R1 improved muscle repair, especially of the soleus muscle, increased in vitro growth of myoblasts, and accelerated their differentiation. These processes were accompanied by alterations of GAG contents. These results indicate that the control of TGFbeta1 activity is important to improve regeneration of injured muscle and accelerate myoblast differentiation, in part through changes in GAG composition of muscle cell environment.

The regeneration of skeletal muscles relies on the function of satellite cells that are quiescent myogenic precursors associated with adult skeletal muscle fibers. Upon injury, the satellite cells are activated, divide extensively, and differentiate into new myofibers. These events are accompanied by the remodeling of the surrounding extracellular matrix, which is mediated by variety of factors, including matrix metalloproteinases (MMPs). Regeneration of certain type of muscles, such as Soleus slow twitch muscle, is often inefficient and hindered by the development of fibrosis. Here, we studied the effect of inhibition of MMP-9 and MMP-2 activity on the Soleus muscle regeneration in vivo and on the in vitro differentiation of myoblasts derived from this muscle. Using in situ and in-gel zymography, we tested the activity of these two MMPs in vivo, during regeneration of the muscle, and in vitro, during differentiation of the myoblasts. We also analyzed the histology of regenerating muscles and morphology of differentiating myoblasts. All these analyses showed that treatment with doxycycline and anti-MMP-9, but not MMP-2 antibody, significantly improved Soleus muscle regeneration and ameliorated development of excessive fibrosis, as well as delayed myoblast proliferation and differentiation in vitro.