Changes in the basement membrane zone components during skeletal muscle fiber degeneration and regeneration

The basement membrane of skeletal muscle fibers is believed to persist unchanged during myofiber degeneration and act as a tubular structure within which the regeneration of new myofibers occurs. In the present study we describe macromolecular changes in the basement membrane zone during muscle degeneration and regeneration, as monitored by immunofluorescence using specific antibodies against types IV and V collagen, laminin, and heparan sulfate proteoglycan and by the binding of concanavalin A (Con A). Skeletal muscle regeneration was induced by autotransplantation of the extensor digitorum longus muscle in rats. After this procedure, the myofibers degenerate; this is followed by myosatellite cell activation, proliferation, and fusion, resulting in the formation of new myotubes that mature into myofibers. In normal muscle, the distribution of types IV and V collagen, laminin, heparan sulfate proteoglycan, and Con A binding was seen in the pericellular basement membrane region. In autotransplanted muscle, the various components of the basement membrane zone disappeared, leaving behind some unidentifiable component that still bound Con A. Around the regenerated myotubes a new basement membrane (zone) reappeared, which persisted during maturation of the regenerating muscle. The distribution of various basement membrane components in the regenerated myofibers was similar to that seen in the normal muscle. Based on our present and previous study (Gulati, A.K., A.H. Reddi, and A.A. Zalewski, 1982, Anat. Rec. 204:175-183), it appears that some of the original basement membrane zone components disappear during myofiber degeneration and initial regeneration. As a new basement membrane develops, its components reappear and persist in the mature myofibers. We conclude that skeletal muscle fiber basement membrane (zone) is not a static structure as previously thought, but rather that its components change quite rapidly during myofiber degeneration and regeneration.     

Role of matrix metalloproteinases in skeletal muscle

Matrix metalloproteases (MMPs) are key regulatory molecules in the formation, remodeling, and degradation of extracellular matrix (ECM) components in both physiological and pathological processes in many tissues. In skeletal muscle, MMPs play an important role in the homeostasis and maintenance of myofiber functional integrity by breaking down ECM and regulating skeletal muscle cell migration, differentiation and regeneration. Skeletal muscle satellite cells, a group of quiescent stem cells located between the basement membrane and the plasmalemma of myofibers, are responsible for lifelong maintenance and repairing, which can be activated and as a result migrate underneath the basement membrane to promote regeneration at the injured site. MMPs are able to degrade ECM components, thereby facilitating satellite cell migration and differentiation. This current review will focus on the critical roles of MMPs in skeletal muscle injury and repair, which include satellite cell activation with migration and differentiation. The effect of MMPs on muscle regeneration and fibrous scar tissue formation, as well as therapeutic insights for the future will be explored.     

Role of matrix metalloproteinases in skeletal muscle: Migration,differentiation, regeneration and fibrosis

Matrix metalloproteases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of extracellular matrix (ECM) components in both physiological and pathological processes in many tissues. In skeletal muscle, MMPs play an important role in the homeostasis and maintenance of myofiber functional integrity by breaking down ECM and regulating skeletal muscle cell migration, differentiation and regeneration. Skeletal muscle satellite cells, a group of quiescent stem cells located between the basement membrane and the plasmalemma of myofibers, are responsible for lifelong maintenance and repairing, which can be activated and as a result migrate underneath the basement membrane to promote regeneration at the injured site. MMPs are able to degrade ECM components, thereby facilitating satellite cell migration and differentiation. This current review will focus on the critical roles of MMPs in skeletal muscle injury and repair, which include satellite cell activation with migration and differentiation. The effect of MMPs on muscle regeneration and fibrous scar tissue formation, as well as therapeutic insights for the future will be explored.Key words: matrix metalloproteinases, skeletal muscle satellite cells, migration, differentiation, regeneration, fibrosis     

Hindlimb suspension reduces muscle regeneration

Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cell mitotic activity. However, the effect of a weightless environment on the satellite cell population during muscle repair remains unknown. Muscle injury was induced in rat soleus muscles using the myotoxic snake venom, notexin. Rats were placed into hindlimb-suspended or weightbearing groups for 10 days following injury. Cellular proliferation during regeneration was evaluated using 5-bromo-2′-deoxyuridine (BrdU) immunohistochemistry and image analysis. Hindlimb suspension reduced (P<0.05) regenerated muscle mass, regenerated myofiber diameter, uninjured muscle mass, and uninjured myofiber diameter compared to weightbearing rats. Hindlimb suspension reduced (P<0.05) BrdU labeling in uninjured soleus muscles compared to weightbearing muscles. However, hindlimb suspension did not abolish muscle regeneration because myofibers formed in the injured soleus muscles of hindlimb-suspended rats, and BrdU labeling was equivalent (P>0.10) on myofiber segments isolated from the soleus muscles of hindlimb-suspended and weightbearing rats following injury. Thus, hindlimb suspension (weightlessness) does not suppress satellite cell mitotic activity in regenerating muscles before myofiber formation, but reduces growth of the newly formed myofibers. Accepted: 11 December 1997     

Hyperbaric oxygen improves contractile function of regenerating rat skeletal muscle after myotoxic injury.

There is growing interest in hyperbaric oxygen (HBO) as an adjunctive treatment for muscle injuries. This experiment tested the hypothesis that periodic inhalation of HBO hastens the functional recovery and myofiber regeneration of skeletal muscle after myotoxic injury. Injection of the rat extensor digitorum longus (EDL) muscle with bupivacaine hydrochloride causes muscle degeneration. After injection, rats breathed air with or without periodic HBO [100% O(2) at either 2 or 3 atmospheres absolute (ATA)]. In vitro maximum isometric tetanic force of injured EDL muscles and regenerating myofiber size were unchanged between 2 ATA HBO-treated and untreated rats at 14 days postinjury but were approximately 11 and approximately 19% greater, respectively, in HBO-treated rats at 25 days postinjury. Maximum isometric tetanic force of injured muscles was approximately 27% greater, and regenerating myofibers were approximately 41% larger, in 3 ATA HBO-treated rats compared with untreated rats at 14 days postinjury. These findings demonstrate that periodic HBO inhalation increases maximum force-producing capacity and enhances myofiber growth in regenerating skeletal muscle after myotoxic injury with greater effect at 3 than at 2 ATA.     

A new function for odorant receptors

Myofibers with an abnormal branching cytoarchitecture are commonly found in various neuromuscular diseases as well as after severe muscle injury. These aberrant myofibers are fragile and muscles containing a high percentage of these myofibers are weaker and more prone to injury. To date the mechanisms and molecules regulating myofiber branching have been obscure. Recent work analyzing the role of mouse odorant receptor 23 (MOR23) in muscle regeneration revealed that MOR23 is necessary for proper skeletal muscle regeneration in mice as loss of MOR23 leads to increased myofiber branching. Further studies demonstrated that MOR23 expression is induced when muscle cells were extensively fusing and plays an important role in controlling cell migration and adhesion. These data demonstrate a novel role for an odorant receptor in tissue repair and identify the first molecule with a functional role in myofiber branching.     

Role of damage and management in muscle hypertrophy: Different behaviors of muscle stem cells in regeneration and hypertrophy

Skeletal muscle is a dynamic tissue with two unique abilities; one is its excellent regenerative ability, due to the activity of skeletal muscle–resident stem cells named muscle satellite cells (MuSCs); and the other is the adaptation of myofiber size in response to external stimulation, intrinsic factors, or physical activity, which is known as plasticity. Low physical activity and some disease conditions lead to the reduction of myofiber size, called atrophy, whereas hypertrophy refers to the increase in myofiber size induced by high physical activity or anabolic hormones/drugs. MuSCs are essential for generating new myofibers during regeneration and the increase in new myonuclei during hypertrophy; however, there has been little investigation of the molecular mechanisms underlying MuSC activation, proliferation, and differentiation during hypertrophy compared to those of regeneration. One reason is that ‘degenerative damage’ to myofibers during muscle injury or upon hypertrophy (especially overloaded muscle) is believed to trigger similar activation/proliferation of MuSCs. However, evidence suggests that degenerative damage of myofibers is not necessary for MuSC activation/proliferation during hypertrophy. When considering MuSC-based therapy for atrophy, including sarcopenia, it will be indispensable to elucidate MuSC behaviors in muscles that exhibit non-degenerative damage, because degenerated myofibers are not present in the atrophied muscles. In this review, we summarize recent findings concerning the relationship between MuSCs and hypertrophy, and discuss what remains to be discovered to inform the development and application of relevant treatments for muscle atrophy.     

Mouse gastrocnemius muscle regeneration after mechanical or cardiotoxin injury

The goal of our study was to compare the skeletal muscle regeneration induced by two types of injury: either crushing, that causes muscle degeneration as a result of mechanical devastation of myofibers, or the injection of a cardiotoxin that is a myotoxic agent causing myolysis of myofibers leading to muscle degeneration. Regenerating muscles were analyzed at selected intervals, until the 14th day following the injury. We analyzed their weight and morphology. We also studied the expression of different myosin heavy chain isoforms as a molecular marker of the regeneration progress. Histological analysis revealed that inflammatory response and myotube formation in crushed muscles was delayed compared to cardiotoxin-injected ones. Moreover, the expression of myosin heavy chain isoforms was observed earlier in cardiotoxin-injured versus crushed muscles. We conclude that the dynamics of skeletal muscle regeneration depends on the method of injury.     

Chondroitin Sulfate Is a Crucial Determinant for Skeletal Muscle Development/Regeneration and Improvement of Muscular Dystrophies

Skeletal muscle formation and regeneration require myoblast fusion to form multinucleated myotubes or myofibers, yet their molecular regulation remains incompletely understood. We show here that the levels of extra- and/or pericellular chondroitin sulfate (CS) chains in differentiating C2C12 myoblast culture are dramatically diminished at the stage of extensive syncytial myotube formation. Forced down-regulation of CS, but not of hyaluronan, levels enhanced myogenic differentiation in vitro. This characteristic CS reduction seems to occur through a cell-autonomous mechanism that involves HYAL1, a known catabolic enzyme for hyaluronan and CS. In vivo injection of a bacterial CS-degrading enzyme boosted myofiber regeneration in a mouse cardiotoxin-induced injury model and ameliorated dystrophic pathology in mdx muscles. Our data suggest that the control of CS abundance is a promising new therapeutic approach for the treatment of skeletal muscle injury and progressive muscular dystrophies.     

A new function of a previously isolated compound that stimulates activation and differentiation of myogenic precursor cells leading to efficient myofiber regeneration and muscle repair

Muscle repair following severe injury is slow and incomplete due to the limited regenerative capacity of muscles comprising the function. In this study, one pure compound structurally corresponding to triterpenoid, which can directly induce the activation, proliferation and maturation of quiescent satellite cells into myocytes in vitro, was isolated from Geum japonicum. The potential effect of this compound on myogenesis was further tested in repair of severe muscle injury. It was found that this compound could significantly stimulate the regenerative potential of the damaged muscle resulting in regeneration of myotubes and myotube bundles time-dependently replacing the damaged muscle tissues. This compound-mediated active regeneration of new myofibers repairing damaged muscles was probably due to its direct action on activation and proliferation of quiescent myogenic precursor cells and enhancement of their maturation into regenerating myotubes, as was demonstrated in our primary myogenic precursor cells culture experiments. The up-regulated expression of endogenous phospho-Akt1 in compound-treated myogenic precursor cells may also contribute to the process of myofiber regeneration and muscle repair probably via promoting myogenic cell survival capacity.     

Necdin mediates skeletal muscle regeneration by promoting myoblast survival and differentiation

Regeneration of muscle fibers that are lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. An important cell type involved in muscle regeneration is the satellite cell. Necdin is a protein expressed in satellite cell-derived myogenic precursors during perinatal growth. However, its function in myogenesis is not known. We compare transgenic mice that overexpress necdin in skeletal muscle with both wild-type and necdin null mice. After muscle injury the necdin null mice show a considerable defect in muscle healing, whereas mice that overexpress necdin show a substantial increase in myofiber regeneration. We also find that in muscle, necdin increases myogenin expression, accelerates differentiation, and counteracts myoblast apoptosis. Collectively, these data clarify the function and mechanism of necdin in skeletal muscle and show the importance of necdin in muscle regeneration.     

In situ real-time imaging of the satellite cells in rat intact and injured soleus muscles using quantum dots

The recruitment of satellite cells, which are located between the basement membrane and the plasma membrane in myofibers, is required for myofiber repair after muscle injury or disease. In particular, satellite cell migration has been focused on as a satellite cell response to muscle injury because satellite cell motility has been revealed in cell culture. On the other hand, in situ, it is poorly understood how satellite cell migration is involved in muscle regeneration after injury because in situ it has been technically very difficult to visualize living satellite cells localized within skeletal muscle. In the present study, using quantum dots conjugated to anti-M-cadherin antibody, we attempted the visualization of satellite cells in both intact and injured skeletal muscle of rat in situ. As a result, the present study is the first to demonstrate in situ real-time imaging of satellite cells localized within the skeletal muscle. Moreover, it was indicated that satellite cell migration toward an injured site was induced in injured muscle while spatiotemporal change in satellite cells did not occur in intact muscle. Thus, it was suggested that the satellite cell migration may play important roles in the regulation of muscle regeneration after injury. Moreover, the new method used in the present study will be a useful tool to develop satellite cell-based therapies for muscle injury or disease.     

Normal myoblast fusion requires myoferlin

Muscle growth occurs during embryonic development and continues in adult life as regeneration. During embryonic muscle growth and regeneration in mature muscle, singly nucleated myoblasts fuse to each other to form myotubes. In muscle growth, singly nucleated myoblasts can also fuse to existing large, syncytial myofibers as a mechanism of increasing muscle mass without increasing myofiber number. Myoblast fusion requires the alignment and fusion of two apposed lipid bilayers. The repair of muscle plasma membrane disruptions also relies on the fusion of two apposed lipid bilayers. The protein dysferlin, the product of the Limb Girdle Muscular Dystrophy type 2 locus, has been shown to be necessary for efficient, calcium-sensitive, membrane resealing. We now show that the related protein myoferlin is highly expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to myotubes. Like dysferlin, we found that myoferlin binds phospholipids in a calcium-sensitive manner that requires the first C2A domain. We generated mice with a null allele of myoferlin. Myoferlin null myoblasts undergo initial fusion events, but they form large myotubes less efficiently in vitro, consistent with a defect in a later stage of myogenesis. In vivo, myoferlin null mice have smaller muscles than controls do, and myoferlin null muscle lacks large diameter myofibers. Additionally, myoferlin null muscle does not regenerate as well as wild-type muscle does, and instead displays a dystrophic phenotype. These data support a role for myoferlin in the maturation of myotubes and the formation of large myotubes that arise from the fusion of myoblasts to multinucleate myotubes.     

Growth factor supplemented matrigel improves ectopic skeletal muscle formation--a cell therapy approach

Following damage to skeletal muscle, satellite cells become activated, migrate towards the injured area, proliferate, and fuse with each other to form myotubes which finally mature into myofibers. We tested a new approach to muscle regeneration by incorporating myoblasts, with or without the exogenous growth factors bFGF or HGF, into three-dimensional gels of reconstituted basement membrane (matrigel). In vitro, bFGF and HGF induced C2C12 myoblast proliferation and migration and were synergistic when used together. In vivo, C2C12 or primary i28 myoblasts were injected subcutaneously together with matrigel and growth factors in the flanks of nude mice. The inclusion of either bFGF or HGF increased the vascularization of the gels. Gels supplemented with bFGF showed myogenesis accompanied by massive mesenchymal cell recruitment and poor organization of the fascicles. Samples containing HGF showed delayed differentiation with respect to controls or bFGF, with increased myoblast proliferation and a significantly higher numbers of cells in myotubes at later time points. HGF samples showed limited mesenchymal cell infiltration and relatively good organization of fascicles. The use of both bFGF and HGF together showed increased numbers of nuclei in myotubes, but with bFGF-mediated fibroblast recruitment dominating. These studies suggest that an appropriate combination of basement membrane components and growth factors could represent a possible approach to enhance survival dispersion, proliferation, and differentiation of myogenic cells during muscle regeneration and/or myoblast transplantation. This model will help develop cell therapy of muscle diseases and open the future to gene therapy approaches.     

An immunofluorescent study of the distribution of fibronectin and laminin during limb regeneration in the adult newt

Fibronectin and laminin are two extracellular glycoproteins which are involved in various processes of cellular development and differentiation. The present investigation describes changes in their distribution during regeneration of the newt forelimb, as determined by indirect immunofluorescence. The distribution of fibronectin and laminin was similar in normal limb tissue components. These glycoproteins were localized in the pericellular region of the myofibers corresponding to its basement membrane; the perineurium and endoneurium of the nerves; and the basement membranes of blood vessels, skin epithelium, and dermal glands. The cytoplasm of myofibers, axons, skin epithelium, and bone matrix lacked fluorescence for both glycoproteins. After limb amputation in the regenerating blastema, extensive presence of fibronectin, but not laminin, was seen in and around the undifferentiated blastemal cells. Increased fluorescence for fibronectin was also seen during blastema growth, blastemal cell aggregation, and early stages of redifferentiation. As redifferentiation continued, staining for fibronectin slowly disappeared from the cartilage matrix and the myoblast fusion zone. Laminin was first observed around the regenerated myotubes; this was followed by the appearance of fibronectin suggesting a sequential formation of these two components of the new myotube basement membrane. In the regenerated limb, the distribution of laminin and fibronectin was similar to that seen in normal limb. Based on the distribution pattern of these glycoproteins, it is concluded that fibronectin may play an important role in blastemal cell aggregation, cell alignment, and initiation of redifferentiation. After redifferentiation, both laminin and fibronectin may be important in the determination of the architecture of the regenerated limb.     

Myofiber degeneration/regeneration is induced in the cachectic ApcMin/+ mouse.

Cachexia is characterized as an inflammatory state induced by the cancer environment, which is accompanied by the loss of muscle and fat mass. Well-investigated mechanisms of cachexia include the suppression of myofiber protein synthesis and the induction of the protein degradation. However, it is not well characterized whether chronic inflammation during cachexia induces myofiber degeneration, which contributes to muscle mass loss and decreased functional capacity. The purpose of this study was to determine whether Apc(Min/+) mice, which demonstrate a chronic systemic inflammatory state due to an intestinal tumor burden, undergo cachexia and whether the myofibers exhibit signs of degeneration and/or regeneration. Six-month-old female Apc(Min/+) body weight decreased 21% compared with C57BL/6 mice and was not the result of blunted growth. Apc(Min/+) gastrocnemius muscle was reduced 45%, and soleus mean fiber cross-sectional area decreased 24% vs. C57BL/6 mice. Soleus muscle morphology demonstrated pathology of myofibers undergoing degeneration and/or regeneration. These data demonstrate that the Apc(Min/+) mouse becomes cachectic by 6 mo of age and that skeletal muscle degeneration and regeneration may be related to the muscle loss.