Malgorzata Zimowska

Distinct patterns of MMP-9 and MMP-2 activity in slow and fast twitch skeletal muscle regeneration in vivo

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.

Sdf-1 (CXCL12) improves skeletal muscle regeneration via the mobilisation of Cxcr4 and CD34 expressing cells

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.

Heparan Sulfate Mimetics Modulate Calpain Activity During Rat Soleus Muscle Regeneration

Skeletal muscle regenerates after injury. Tissue remodelling, which takes place during muscle regeneration, is a complex process involving proteolytic enzymes. It is inferred that micro and milli calpains are involved in the protein turnover and structural adaptation associated with muscle myolysis and reconstruction. Using a whole‐crush injured skeletal muscle, we previously have shown that in vivo muscle treatment with synthetic heparan sulfate mimetics, called RGTAs (for ReGeneraTing Agents), greatly accelerates and improves muscle regeneration after crushing. This effect was particularly striking in the case of the slow muscle Soleus that otherwise would be atrophied. Therefore, we used this regeneration model to study milli and micro calpain expressions in the regenerating Soleus muscle and to address the question of a possible effect of RGTAs treatment on calpain levels. Micro and milli calpain contents increased by about five times to culminate at days 7 and 14 after crushing respectively, thus during the phases of fibre reconstruction and reinnervation. After 64 days of regeneration, muscles still displayed higher levels of both calpains than an intact uninjured muscle. Milli calpain detected by immunocytochemistry was shown in the cytoplasm whereas micro calpain was in both nuclei and cytoplasm in small myofibres but appeared almost exclusively in nuclei of more mature fibres. Interestingly, the treatment of muscles with RGTA highly reduced the increase of both milli and micro calpain contents in Soleus regenerating muscles. These results suggest that the improvement of muscle regeneration induced by RGTA may be partly mediated by minimising the consequences of calpain activity.

Regulation of muscle stem cells activation: the role of growth factors and extracellular matrix.

Vertebrate skeletal muscle is composed of organized multinucleate muscle fibers and also various subpopulations of cells localized in between. Some of them can be considered as the stem cells, however, few of them are able to follow myogenic program. First and most extensively studied so far, are the satellite cells that serve as tissue-specific precursors for muscle growth and repair. They are located between the basal membrane and the sarcolemma of adult muscle myofibers. They remain quiescent but can be activated in response to muscle damage resulted from mechanical injury, stretching, exercise, denervation, or progressing muscle dystrophy. Except the satellite cells also other stem cells could participate in muscle fibers reconstruction. Such cells as pericytes and mesangioblasts, muscle-derived stem cells, including so-called muscle side population, or CD133 expressing cells, were proved to be able to undergo myogenic differentiation in experiments involving their in vitro coculture with myoblasts or transplantation to injured skeletal muscle. In the current review, we will summarize stimuli influencing skeletal muscle stem cells activation, that is, growth factors which are secreted by muscle fibers, satellite cells, inflammatory cells, or released from basal lamina. We will also describe factors present within the skeletal muscle niche which interactions with stem cells lead to their activation, proliferation, asymmetric divisions, migration, and finally differentiation into myotubes, and then terminally differentiated myofibers.

Regulation of Muscle Stem Cells Activation

Vertebrate skeletal muscle is composed of organized multinucleate muscle fibers and also various subpopulations of cells localized in between. Some of them can be considered as the stem cells, however, few of them are able to follow myogenic program. First and most extensively studied so far, are the satellite cells that serve as tissue-specific precursors for muscle growth and repair. They are located between the basal membrane and the sarcolemma of adult muscle myofibers. They remain quiescent but can be activated in response to muscle damage resulted from mechanical injury, stretching, exercise, denervation, or progressing muscle dystrophy. Except the satellite cells also other stem cells could participate in muscle fibers reconstruction. Such cells as pericytes and mesangioblasts, muscle-derived stem cells, including so-called muscle side population, or CD133 expressing cells, were proved to be able to undergo myogenic differentiation in experiments involving their in vitro coculture with myoblasts or transplantation to injured skeletal muscle. In the current review, we will summarize stimuli influencing skeletal muscle stem cells activation, that is, growth factors which are secreted by muscle fibers, satellite cells, inflammatory cells, or released from basal lamina. We will also describe factors present within the skeletal muscle niche which interactions with stem cells lead to their activation, proliferation, asymmetric divisions, migration, and finally differentiation into myotubes, and then terminally differentiated myofibers.

From Planarians to Mammals - the many faces of regeneration

This report presents the history of the involvement of the Department of Cytology in studies of different aspects of regeneration. It can be divided into two major phases; the first focused on the regeneration of Turbellarians and the second on the regeneration of rat skeletal muscles including the differentiation of satellite cells in vitro. Regeneration of Turbellarians was investigated both at the cellular and molecular levels including the role of the protein kinase C (PKC) in this process. Studies on skeletal muscle regeneration initially focused on factors involved in regulation of signal transduction pathways. Next, we explored the influence of growth factors on the modulation of the regeneration process. Another important aspect of our studies was investigating of the distribution and function of different proteins involved in adhesion and fusion of myoblasts. Finally, we are also conducting research on the role of stem cells from other tissues in the regeneration of skeletal muscle.

Nuclear MMP-9 role in the regulation of rat skeletal myoblasts proliferation

Matrix metalloproteinases (MMPs) are the key enzymes responsible for the remodelling of extracellular matrix. Two of them, namely MMP‐2 and MMP‐9 (gelatinases A and B, respectively), are expressed in skeletal muscles and are involved in their regeneration after the injury. Although MMPs are primarily known to act extracellularly, recent studies have shown that some of them are also found within the cell. In this study, we examine intracellular localisation of gelatinases during myoblasts differentiation in vitro, focussing the impact of MMPs inhibition on the myoblasts proliferation and function.

Novel glycosaminoglycan mimetic (RGTA, RGD120) contributes to enhance skeletal muscle satellite cell fusion by increasing intracellular Ca2+ and calpain activity.

Glycosaminoglycans (GAG) are classes of molecules that play an important role in cellular processes. The use of GAG mimetics called regenerating agent (RGTA) represents a tool to investigate the effect of GAG moiety on cellular behavior. A first member of the RGTA family (RG1192), a dextran polymers with defined amounts of sulfate, carboxymethyl, as well as hydrophobic groups (benzylamide), was shown to stimulate skeletal muscle repair after damage and myoblast differentiation. To obtain a comprehensive insight into the mechanism of action of GAG mimetics, we investigated the effect on myoblast differentiation of a novel RGTA, named RGD120, which was devoid of hydrophobic substitution and had ionic charge similar to heparin. Myoblasts isolated from adult rat skeletal muscles and grown in primary cultures were used in this study. We found that chronic treatment with RGD120 increased the growth of adult myoblasts and induced their precocious fusion into myotubes in vitro. It also partially overcame the inhibitory effect of the calpain inhibitor N‐acetyl‐leu‐leu‐norleucinal (ALLN) on these events. Western blot and zymography analyses revealed that milli calpain was slightly increased by RGD120 chronic treatment. In addition, using fluorescent probes (Indo‐1 and Boc‐leu‐met‐MAC), we demonstrated that RGD120 added to prefusing myoblast cultures accelerates myoblast fusion into myotubes, induced an increase of cytosolic free calcium concentration, and concomitantly an increase of intracellular calpain protease activity. Altogether, these results suggested that the efficiency of RGD120 in stimulating myogenesis might be in part explained through its effect on calcium mobilization as well as on the calpain amount and activity.

The role of TGF-β1 during skeletal muscle regeneration

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.

Inflammatory response during slow- and fast-twitch muscle regeneration.

Introduction: Skeletal muscles are characterized by their unique ability to regenerate. Injury of a so‐called fast‐twitch muscle, extensor digitorum longus (EDL), results in efficient regeneration and reconstruction of the functional tissue. In contrast, slow‐twitch muscle (soleus) fails to properly reconstruct and develops fibrosis. This study focuses on soleus and EDL muscle regeneration and associated inflammation. Methods: We determined differences in the activity of neutrophils and M1 and M2 macrophages using flow cytometry and differences in the levels of proinflammatory cytokines using Western blotting and immunolocalization at different times after muscle injury. Results: Soleus muscle repair is accompanied by increased and prolonged inflammation, as compared to EDL. The proinflammatory cytokine profile is different in the soleus and ED muscles. Conclusions: Muscle repair efficiency differs by muscle fiber type. The inflammatory response affects the repair efficiency of slow‐ and fast‐twitch muscles. Muscle Nerve 55: 400–409, 2017