Upregulation of HARP during in vitro myogenesis and rat soleus muscle regeneration

Heparin affin regulatory peptide (HARP) is a heparin binding growth factor that belongs to a family of molecule whose biological function in myogenesis has been suspected without formal demonstration. In the present study, we investigated the expression and the distribution of HARP and its mRNA during soleus muscle regeneration using a crushed-induced regeneration model and also during differentiation of muscle satellite cells in primary cultures. We show that HARP mRNA and protein expression are increased during the regeneration process with a peak at day 5 after muscle crushing when new myotubes are formed. In situ hybridization and immunohistochemical studies showed that activated myoblasts expressed HARP at day two after crushing. Five days after muscle lesion, HARP is localised in newly formed myotubes as well as in prefused activated myoblasts. In regenerated myofibers, 15 days after crushing, expression of HARP was reduced. In vitro experiments using primary cultures of rat satellite cells indicated that HARP expression level increased during the differentiation process and peaked on fusion of myoblasts into myotubes. This is the first study demonstrating the presence of HARP in fusing myogenic cells suggests that this growth factor could play a function in myogenic differentiation.     

Muscle satellite cells from dystrophic (mdx) mice have elevated levels of heparan sulphate proteoglycan receptors for fibroblast growth factor

Skeletal muscle has the remarkable capacity to regenerate new muscle fibres in the event of injury or disease. This capacity lies in the satellite cells, which are myogenic stem cells residing in adult muscle. While the signals that activate satellite cells to divide in vivo are not fully understood, satellite cells grown in culture respond to the mitogenic action of fibroblast growth factor (FGF). Satellite cells from the dystrophic mdx mouse are more sensitive to FGF in culture than satellite cells from normal mice. In this study we investigated the basis for this heightened sensitivity of mdx satellite cells to FGF by measuring the number and affinity of protein and heparan sulphate proteoglycan (HSPG) receptors for FGF. We found that HSPG receptors were elevated over four-fold in the mdx cells compared with cells from normal animals. We supported this observation by measuring the synthesis of heparan sulphate (HS) and chondroitin sulphate (CS) by satellite cells in culture. Mdx satellite cells synthesized approximately ten times more of these sulphated glycosaminoglycans (GAGs) than did normal cells. For muscle fibroblasts, however, we found no significant difference in the number or affinity of protein or HSPG receptors, or in the amount of sulphated GAGs synthesized, between normal and mdx cells. We propose that the increase in FGF HSPG receptors is the basis for the heightened response of mdx satellite cells to FGF in culture and may reflect exposure of the cells to growth factors in the degenerating mdx muscleThis revised version was published online in July 2005 with corrections to the Cover Date.     

The myogenic factor Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification

The myogenic factor Myf5 defines the onset of myogenesis in mammals during development. Mice lacking both Myf5 and MyoD fail to form myoblasts and are characterized by a complete absence of skeletal muscle at birth. To investigate the function of Myf5 in adult skeletal muscle, we generated Myf5 and mdx compound mutants, which are characterized by constant regeneration. Double mutant mice show an increase of dystrophic changes in the musculature, although these mice were viable and the degree of myopathy was modest. Myf5 mutant muscles show a small decrease in the number of muscle satellite cells, which was within the range of physiological variations. We also observed a significant delay in the regeneration of Myf5 deficient skeletal muscles after injury. Interestingly, Myf5 deficient skeletal muscles were able to even out this flaw during the course of regeneration, generating intact muscles 4 weeks after injury. Although we did not detect a striking reduction of MyoD positive activated myoblasts or of Myf5-LacZ positive cells in regenerating muscles, a clear decrease in the proliferation rate of satellite cell-derived myoblasts was apparent in satellite cell-derived cultures. The reduction of the proliferation rate of Myf5 mutant myoblasts was also reflected by a delayed transition from proliferation to differentiation, resulting in a reduced number of myotube nuclei after 6 and 7 days of culture. We reason that Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification. Disclosure of potential conflicts of interest is found at the end of this article.     

Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice

 It is commonly accepted, that regenerative capacity of striated muscle is confined to skeletal muscle by activation of satellite cells that normally reside quiescent between the plasmalemma and the basement membrane of muscle fibers. Muscular dystrophies are characterized by repetitive cycles of de- and regeneration of skeletal muscle fibers and by the frequent involvement of the cardiac muscle. Since during the longstanding course of muscular dystrophies there is a permanent demand of myogenic progenitors we hypothesized that this may necessitate a recruitment of additional myogenic precursors from an undifferentiated, permanently renewed cell pool, such as bone marrow (BM) cells. To this end normal and dystrophic (mdx) female mice received bone marrow transplantation (BMT) from normal congenic male donor mice. After 70 days, histological sections of skeletal and cardiac muscle from BMT mice were probed for the donor-derived Y chromosomes. In normal BMT recipients, no Y chromosome-containing myonuclei were detected, either in skeletal or in cardiac muscle. However, in all samples from dystrophic mdx skeletal muscles Y chromosome-specific signals were detected within muscle fiber nuclei, which additionally were found to express the myoregulatory proteins myogenin and myf-5. Moreover, in the hearts of BMT-mdx mice single cardiomyocytes with donor derived nuclei were identified, indicating, that even cardiac muscle cells are able to regenerate by recruitment of circulating BM-derived progenitors. Our findings suggest that further characterization and identification of the BM cells capable of undergoing myogenic differentiation may have an outstanding impact on therapeutic strategies for diseases of skeletal and cardiac muscle. Accepted: 27 October 1998     

Satellite cells express distinct patterns of myogenic proteins in immature skeletal muscle.

Satellite cells are the myogenic cells lying between the myofiber sarcolemma and basal lamina. The objective of this study was to determine the expression patterns of MyoD, myogenin, and Pax7 within the satellite cell population in the growing rat soleus and extensor digitorum longus (EDL) muscles. Secondly, the expression of the myogenic markers was also studied within the interstitial cell compartment and myonuclei. It was discovered that the soleus contained a higher number of Pax7, MyoD, or myogenin-positive nuclei compared with the EDL. Similarly, myogenin was expressed at a lower level in the myonuclei of the soleus compared with the EDL, and myogenin was expressed at a higher level in the interstitial compartment of the soleus compared with the EDL. When interstitial nuclei, myonuclei, and double-labeled nuclei were used in the estimate of the satellite cell population, it was discovered that approximately of 13% of the myofibers in a transverse section of the soleus muscle and 4.1% of EDL myofibers exhibit a labeled satellite cell nucleus. Overall, results from this study suggest that expression patterns of these markers vary predictably among muscles with different growth dynamics and phenotypic characteristics.     

Embryonic myogenesis pathways in muscle regeneration.

Embryonic myogenesis involves the staged induction of myogenic regulatory factors and positional cues that dictate cell determination, proliferation, and differentiation into adult muscle. Muscle is able to regenerate after damage, and muscle regeneration is generally thought to recapitulate myogenesis during embryogenesis. There has been considerable progress in the delineation of myogenesis pathways during embryogenesis, but it is not known whether the same signaling pathways are relevant to muscle regeneration in adults. Here, we defined the subset of embryogenesis pathways induced in muscle regeneration using a 27 time-point in vivo muscle regeneration series. The embryonic Wnt (Wnt1, 3a, 7a, 11), Shh pathway, and the BMP (BMP2, 4, 7) pathway were not induced during muscle regeneration. Moreover, antagonists of Wnt signaling, sFRP1, sFRP2, and sFRP4 (secreted frizzled-related proteins) were significantly up-regulated, suggesting active inhibition of the Wnt pathway. The pro-differentiation FGFR4 pathway was transiently expressed at day 3, commensurate with expression of MyoD, Myogenin, Myf5, and Pax7. Protein verification studies showed fibroblast growth factor receptor 4 (FGFR4) protein to be strongly expressed in differentiating myoblasts and newly formed myotubes. We present evidence that FGF6 is likely the key ligand for FGFR4 during muscle regeneration, and further suggest that FGF6 is released from necrotic myofibers where it is then sequestered by basal laminae. We also confirmed activation of Notch1 in the regenerating muscle. Finally, known MyoD coactivators (MEF2A, p/CIP, TCF12) and repressors (Twist, Id2) were strongly induced at appropriate time points. Taken together, our results suggest that embryonic positional signals (Wnt, Shh, and BMP) are not induced in postnatal muscle regeneration, whereas cell-autonomous factors (Pax7, MRFs, FGFR4) involving muscle precursor proliferation and differentiation are recapitulated by muscle regeneration.     

Placental perivascular cells for human muscle regeneration

Perivascular multipotent mesenchymal progenitors exist in a variety of tissues, including the placenta. Here, we suggest that the abundant vasculature present in the human placenta can serve as a source of myogenic cells to regenerate skeletal muscle. Chorionic villi dissected from the mid-gestation human placenta were first transplanted intact into the gastrocnemius muscles of SCID/mdx mice, where they participated in muscle regeneration by producing myofibers expressing human dystrophin and spectrin. In vitro-cultured placental villi released rapidly adhering and migratory CD146+CD34?CD45?CD56? cells of putative perivascular origin that expressed mesenchymal stem cell markers. CD146+CD34?CD45?CD56? perivascular cells isolated and purified from the placental villi by flow cytometry were indeed highly myogenic in culture, and generated dystrophin-positive myofibers, and they promoted angiogenesis after transplantation into SCID/mdx mouse muscles. These observations confirm the existence of mesenchymal progenitor cells within the walls of human blood vessels, and suggest that the richly vascularized human placenta is an abundant source of perivascular myogenic cells able to migrate within dystrophic muscle and regenerate myofibers.     

Cell-lineage regulated myogenesis for dystrophin replacement: a novel therapeutic approach for treatment of muscular dystrophy

Duchenne muscular dystrophy (DMD) is characterized in skeletal muscle by cycles of myofiber necrosis and regeneration leading to loss of muscle fibers and replacement with fibrotic connective and adipose tissue. The ongoing activation and recruitment of muscle satellite cells for myofiber regeneration results in loss of regenerative capacity in part due to proliferative senescence. We explored a method whereby new myoblasts could be generated in dystrophic muscles by transplantation of primary fibroblasts engineered to express a micro-dystrophin/enhanced green fluorescent protein (muDys/eGFP) fusion gene together with a tamoxifen-inducible form of the myogenic regulator MyoD [MyoD-ER(T)]. Fibroblasts isolated from mdx(4cv) mice, a mouse model for DMD, were efficiently transduced with lentiviral vectors expressing muDys/eGFP and MyoD-ER(T) and underwent myogenic conversion when exposed to tamoxifen. These cells could also be induced to differentiate into muDys/eGFP-expressing myocytes and myotubes. Transplantation of transduced mdx(4cv) fibroblasts into mdx(4cv) muscles enabled tamoxifen-dependent regeneration of myofibers that express muDys. This lineage control method therefore allows replenishment of myogenic stem cells using autologous fibroblasts carrying an exogenous dystrophin gene. This strategy carries several potential advantages over conventional myoblast transplantation methods including: (i) the relative simplicity of culturing fibroblasts compared with myoblasts, (ii) a readily available cell source and ease of expansion and (iii) the ability to induce MyoD gene expression in vivo via administration of a medication. Our study provides a proof of concept for a novel gene/stem cell therapy technique and opens another potential therapeutic approach for degenerative muscle disorders.     

Expression of mRNA for specific fibroblast growth factors associates with that of the myogenic markers MyoD and proliferating cell nuclear antigen in regenerating and overloaded rat plantaris muscle

Aim: To examine the relations between specific fibroblast growth factors (FGFs) and satellite cell activation during muscle regeneration and hypertrophy in vivo, we measured mRNA expression of FGFs and myogenic markers in rat plantaris muscle after bupivacaine administration and synergist ablation. Methods: mRNA levels for MyoD, myogenin, proliferating cell nuclear antigen (PCNA), p21, M‐cadherin, Pax7, FGF‐1, FGF‐2, FGF‐3, FGF‐4, FGF‐5, FGF‐6, FGF‐7, FGF‐8 and hepatocyte growth factor (HGF) were measured continually for up to 72 h after bupivacaine administration and synergist ablation. FGF‐5, FGF‐7 and HGF proteins were immunostained at 72 h after bupivacaine administration. Results: MyoD and PCNA mRNAs started increasing 24 h after bupivacaine administration. Myogenin, p21, M‐cadherin and Pax7 mRNAs started to increase after 48 and 72 h. After synergist ablation, MyoD, PCNA, M‐cadherin and Pax7 mRNAs had increased at 24 and 48 h, and myogenin and p21 mRNAs at 12 and 24 h. FGF‐1, FGF‐7 and HGF mRNAs after the treatments started to increase at the same time as MyoD and PCNA mRNAs. FGF‐5 was expressed at the same time as MyoD and PCNA mRNAs after bupivacaine administration but did not after the ablation. FGF‐2, FGF‐3, FGF‐4, FGF‐6 and FGF‐8 mRNAs were not associated with the expression of the myogenic markers. FGF‐7 and HGF proteins were expressed in immature muscle fibre nuclei and the extracellular matrix, but FGF‐5 protein was preferentially expressed in extracellular matrix. Conclusion: These results indicate that FGF‐1, FGF‐7 and HGF are associated with specific myogenic marker expression during muscle regeneration and hypertrophy.     

A role for FGF-6 in skeletal muscle regeneration

Fibroblast growth factor-6 (FGF-6) belongs to a family of cytokines that control cell proliferation, cell differentiation, and morphogenetic events. Individual FGFs are either expressed widely or in a restricted pattern during embryonic, fetal, and adult life. FGF-6 exhibits a restricted expression profile predominantly in the myogenic lineage. Important functions in wound healing and tissue regeneration have been proposed for various FGFs in the past, although data from knockout mice have not supported this view. We have inactivated the FGF-6 gene in mice to investigate the role of FGF-6 in skeletal muscle development and regeneration. Wild-type mice up-regulate FGF-6 after skeletal muscle injuries and completely restore experimentally damaged skeletal muscle. In contrast, FGF-6(−/−) mutant mice show a severe regeneration defect with fibrosis and myotube degeneration. The number of MyoD- and Myogenin-expressing activated satellite cells after injury were significantly reduced in mutants. This reduction was not caused by a reduced pool of quiescent satellite cells but presumably by a lack of activation or proliferation. Interbreeding of FGF-6(−/−) mutants with mdx mice leads to striking dystrophic changes in skeletal muscles of double homozygous mice characterized by myotube degeneration, the presence of large amounts of mononuclear cells, and deposition of collagen. RNA analysis revealed an up-regulation of MyoD mRNA in mdx but not in FGF-6(−/−)/mdx double mutant mice. We conclude that FGF-6 is a critical component of the muscle regeneration machinery in mammals, possibly by stimulating or activating satellite cells.     

The identification of myogenic cells in skeletal muscle, with emphasis on the use of tritiated thymidine autoradiography and desmin antibodies

The identification of myogenic precursor cells (mpc) is a key factor in determining the early events in the myogenesis and regeneration of skeletal muscle. Although satellite cells have long been established as the providers of myoblastic cells, very little is really known (apart from their anatomical location in relation to muscle fibres and their ability to migrate) about the precise role of satellite cells in myogenesis. Numerous techniques for labelling mpc have been devised, but none of these has proven to be completely reliable in firmly establishing the origin of myogenic cells. The use of tritiated thymidine to label DNA in proliferating mpc (which are not specifically distinguishable at the time) and the subsequent location of their labelled progeny in myotube nuclei has revealed a great deal of data on the timing of myogenesis, but not about the nature of mpc themselves. DNA synthesis can also be detected by antibodies to the thymidine analogue, bromodeoxyuridine, and also by antibody staining for proliferating nuclear cell antigen. Like tritiated thymidine, these other markers are not specific for muscle but are general markers for DNA synthesis. In situ hybridisation of various muscle-specific genetic markers and their products has been informative, as has immunolabelling of myogenin, MyoD1 and desmin. Desmin labelling has been particularly instructive in identifying mpc because it is one of the first muscle-specific proteins to be produced in mpc. This review covers some of the techniques mentioned above and their usefulness in determining the early events in myogenesis.     

Impact of static magnetic fields on human myoblast cell cultures

Treatment of skeletal muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of functional muscle tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal muscle tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal muscle α1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human satellite cultures. SMF alone enhances the maturation of human satellite cells treated with GM, but not satellite cells that were additionally stimulated with serum cessation. Therefore, further investigations are necessary before consideration of SMF for skeletal muscle tissue engineering approaches.     

Myosatellite cells,growth, and regeneration in murine dystrophic muscle: A quantitative study

Patterns of growth and regeneration in 2-, 4-, 8-, and 17-week-old murine dystrophic (129 ReJ dy/dy) extensor digitorum longus muscles have been determined. Necrosis and myofiber loss, hypertrophy, and regeneration result in a reduced population of myofibers whose diameter distribution is more extensive than that found in the extensor digitorum longus muscles of age-matched normal mice. At the onset of dystrophic symptoms (2 weeks postnatal), the ratio of myosatellite cell nuclei to the total sublaminal nuclear population (myonuclei + myosatellite cells) is similar to that found in 2-week-old control muscles. The frequency of finding myosatellite cells decreases with age in both control and dystrophic muscles. Myosatellite cells account for 11%, 6%, 5%, and 3% of the total sublaminal nuclear population in control muscle and 12%, 8%, 6%, and 5% of the total sublaminal nuclear population in dystrophic muscle at 2, 4, 8, and 17 weeks, respectively. No preferential association of myosatellite cells with myofibers of a particular diameter is found in control muscle or in the two youngest dystrophic groups. At 8 and 17 weeks, myosatellite cells are less frequently encountered on small-diameter, regenerating myofibers of dystrophic muscle, and they are preferentially associated with large diameter, hypertrophied myofibers. The labeling index of myosatellite cells decreases with age in both normal and dystrophic muscle. At all ages the myosatellite cell labeling index is higher in dystrophic muscle (23%, 7%, 5%, and 2% at 2, 4, 8, and 17 weeks, respectively) than in normal muscle (5%, < 1% at 2 and 4 weeks, respectively), with no labeled myosatellite cells being found in 8- and 17-week-old normal muscles. It is suggested that the magnitude of the regenerative response of dystrophic murine muscle decreases with age and that this factor may be responsible for the inability of the regenerative response of dystrophic muscle to keep pace with the rapid muscle deterioration.     

A population of myogenic stem cells that survives skeletal muscle aging

Age-related decline in integrity and function of differentiated adult tissues is widely attributed to reduction in number or regenerative potential of resident stem cells. The satellite cell, resident beneath the basal lamina of skeletal muscle myofibers, is the principal myogenic stem cell. Here we have explored the capacity of satellite cells within aged mouse muscle to regenerate skeletal muscle and to self-renew using isolated myofibers in tissue culture and in vivo. Satellite cells expressing Pax7 were depleted from aged muscles, and when aged myofibers were placed in culture, satellite cell myogenic progression resulted in apoptosis and fewer total differentiated progeny. However, a minority of cultured aged satellite cells generated large clusters of progeny containing both differentiated cells and new cells of a quiescent satellite-cell-like phenotype characteristic of self-renewal. Parallel in vivo engraftment assays showed that, despite the reduction in Pax7(+) cells, the satellite cell population associated with individual aged myofibers could regenerate muscle and self-renew as effectively as the larger population of satellite cells associated with young myofibers. We conclude that a minority of satellite cells is responsible for adult muscle regeneration, and that these stem cells survive the effects of aging to retain their intrinsic potential throughout life. Thus, the effectiveness of stem-cell-mediated muscle regeneration is determined by both extrinsic environmental influences and diversity in intrinsic potential of the stem cells themselves.