SPARC Interacts with Actin in Skeletal Muscle in Vitro and in Vivo.

The cytoskeleton is an integral part of skeletal muscle structure, and reorganization of the cytoskeleton occurs during various modes of remodeling. We previously found that the extracellular matrix protein secreted protein acidic and rich in cysteine (SPARC) is up-regulated and expressed intracellularly in developing muscle, during regeneration and in myopathies, which together suggests that SPARC might serve a specific role within muscle cells. Using co-immunoprecipitation combined with mass spectrometry and verified by staining for direct protein-protein interaction, we find that SPARC binds to actin. This interaction is present in regenerating myofibers of patients with Duchenne muscular dystrophy, polymyositis, and compartment syndrome. Analysis of the α-, ß-, and γ-actin isoforms in SPARC knockout myoblasts reveals a changed expression pattern with dominance of γ-actin. In SPARC knockout mice, we performed an injury study to investigate whether lack of SPARC would compromise the ability to repair muscle. We report that these mice develop normal skeletal muscle with retained ability to regenerate. However, when we subject muscle from SPARC-deficient mice to an in vitro fatigue stimulation protocol, we find a defective force recovery. Therefore, SPARC appears to be an important modulator of the actin cytoskeleton, implicating maintenance of muscular function. This direct interaction with actin suggests a new role of SPARC during tissue remodeling.

A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene.

Adult stem cell quiescence is critical to ensure regeneration while minimizing tumorigenesis. Epigenetic regulation contributes to cell cycle control and differentiation, but few regulators of the chromatin state in quiescent cells are known. Here we report that the tumor suppressor PRDM2/RIZ, an H3K9 methyltransferase, is enriched in quiescent muscle stem cells in vivo and controls reversible quiescence in cultured myoblasts. We find that PRDM2 associates with >4400 promoters in G0 myoblasts, 55% of which are also marked with H3K9me2 and enriched for myogenic, cell cycle and developmental regulators. Knockdown of PRDM2 alters histone methylation at key promoters such as Myogenin and CyclinA2 (CCNA2), and subverts the quiescence program via global de-repression of myogenesis, and hyper-repression of the cell cycle. Further, PRDM2 acts upstream of the repressive PRC2 complex in G0. We identify a novel G0-specific bivalent chromatin domain in the CCNA2 locus. PRDM2 protein interacts with the PRC2 protein EZH2 and regulates its association with the bivalent domain in the CCNA2 gene. Our results suggest that induction of PRDM2 in G0 ensures that two antagonistic programs-myogenesis and the cell cycle-while stalled, are poised for reactivation. Together, these results indicate that epigenetic regulation by PRDM2 preserves key functions of the quiescent state, with implications for stem cell self-renewal.

The effect of low intensity shockwave treatment (Li-SWT) on human myoblasts and mouse skeletal muscle.

BACKGROUND: Transplanting myogenic cells and scaffolds for tissue engineering in skeletal muscle have shown inconsistent results. One of the limiting factors is neovascularization at the recipient site. Low intensity shockwave therapy (Li-SWT) has been linked to increased tissue regeneration and vascularization, both integral to survival and integration of transplanted cells. This study was conducted to demonstrate the response of myoblasts and skeletal muscle to Li-SWT. METHOD: Primary isolated human myoblasts and explants were treated with low intensity shockwaves and subsequently cell viability, proliferation and differentiation were tested. Cardiotoxin induced injury was created in tibialis anterior muscles of 28 mice, and two days later, the lesions were treated with 500 impulses of Li-SWT on one of the legs. The treatment was repeated every third day of the period and ended on day 14 after cardiotoxin injection.. The animals were followed up and documented up to 21 days after cardiotoxin injury. RESULTS: Li-SWT had no significant effect on cell death, proliferation, differentiation and migration, the explants however showed decreased adhesion. In the animal experiments, qPCR studies revealed a significantly increased expression of apoptotic, angiogenic and myogenic genes; expression of Bax, Bcl2, Casp3, eNOS, Pax7, Myf5 and Met was increased in the early phase of regeneration in the Li-SWT treated hind limbs. Furthermore, a late accumulative angiogenic effect was demonstrated in the Li-SWT treated limbs by a significantly increased expression of Angpt1, eNOS, iNOS, Vegfa, and Pecam1. CONCLUSION: Treatment was associated with an early upregulation in expression of selected apoptotic, pro-inflammatory, angiogenic and satellite cell activating genes after muscle injury. It also showed a late incremental effect on expression of pro-angiogenic genes. However, we found no changes in the number of PAX7 positive cells or blood vessel density in Li-SWT treated and control muscle. Furthermore, Li-SWT in the selected doses did not decrease survival, proliferation or differentiation of myoblasts in vitro.

Secreted protein acidic and rich in cysteine (SPARC) in human skeletal muscle.

Secreted protein acidic and rich in cysteine (SPARC)/osteonectin is expressed in different tissues during remodeling and repair, suggesting a function in regeneration. Several gene expression studies indicated that SPARC was expressed in response to muscle damage. Studies on myoblasts further indicated a function of SPARC in skeletal muscle. We therefore found it of interest to study SPARC expression in human skeletal muscle during development and in biopsies from Duchenne and Becker muscular dystrophy and congenital muscular dystrophy, congenital myopathy, inclusion body myositis, and polymyositis patients to analyze SPARC expression in a selected range of inherited and idiopathic muscle wasting diseases. SPARC-positive cells were observed both in fetal and neonatal muscle, and in addition, fetal myofibers were observed to express SPARC at the age of 15-16 weeks. SPARC protein was detected in the majority of analyzed muscle biopsies (23 of 24), mainly in mononuclear cells of which few were pax7 positive. Myotubes and regenerating myofibers also expressed SPARC. The expression-degree seemed to reflect the severity of the lesion. In accordance with these in vivo findings, primary human-derived satellite cells were found to express SPARC both during proliferation and differentiation in vitro. In conclusion, this study shows SPARC expression both during muscle development and in regenerating muscle. The expression is detected both in satellite cells/myoblasts and in myotubes and muscle fibers, indicating a role for SPARC in the skeletal muscle compartment.

Injectable scaffold materials differ in their cell instructive effects on primary human myoblasts.

Scaffolds are materials used for delivery of cells for regeneration of tissues. They support three-dimensional organization and improve cell survival. For the repair of small skeletal muscles, injections of small volumes of cells are attractive, and injectable scaffolds for delivery of cells offer a minimally invasive technique. In this study, we examined in vitro the cell instructive effects of three types of injectable scaffolds, fibrin, alginate, and poly(lactic-co-glycolic acid)-based microparticles on primary human myoblasts. The myoblast morphology and progression in the myogenic program differed, depending on the type of scaffold material. In alginate gel, the cells obtained a round morphology, they ceased to proliferate, and entered quiescence. In the fibrin gels, differentiation was promoted, and myotubes were observed within a few days in culture, while poly(lactic-co-glycolic acid)-based microparticles supported prolonged proliferation. Myoblasts released from the alginate and fibrin gels were studied, and cells released from these scaffolds had retained the ability to proliferate and differentiate. Thus, the study shows that human myogenic cells combined with injectable scaffold materials are guided into different states depending on the choice of scaffold. This opens for in vivo experiments, including testing of the significance of the cell state on regeneration potential of primary human myoblasts.

Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest.

OBJECTIVES: Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts. MATERIALS AND METHODS: Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot. RESULTS AND CONCLUSIONS: We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFß, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and ß-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts.

A novel in vitro model for studying quiescence and activation of primary isolated human myoblasts.

Skeletal muscle stem cells, satellite cells, are normally quiescent but become activated upon muscle injury. Recruitment of resident satellite cells may be a useful strategy for treatment of muscle disorders, but little is known about gene expression in quiescent human satellite cells or the mechanisms involved in their early activation. We have developed a method to induce quiescence in purified primary human myoblasts isolated from healthy individuals. Analysis of the resting state showed absence of BrdU incorporation and lack of KI67 expression, as well as the extended kinetics during synchronous reactivation into the cell cycle, confirming arrest in the G0 phase. Reactivation studies showed that the majority (>95%) of the G0 arrested cells were able to re-enter the cell cycle, confirming reversibility of arrest. Furthermore, a panel of important myogenic factors showed expression patterns similar to those reported for mouse satellite cells in G0, reactivated and differentiated cultures, supporting the applicability of the human model. In addition, gene expression profiling showed that a large number of genes (4598) were differentially expressed in cells activated from G0 compared to long term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis of quiescence and reactivation of human myoblasts.

Delta-like 1 homolog (dlk1): a marker for rhabdomyosarcomas implicated in skeletal muscle regeneration.

Dlk1, a member of the Epidermal Growth Factor family, is expressed in multiple tissues during development, and has been detected in carcinomas and neuroendocrine tumors. Dlk1 is paternally expressed and belongs to a group of imprinted genes associated with rhabdomyosarcomas but not with other primitive childhood tumors to date. Here, we investigate the possible roles of Dlk1 in skeletal muscle tumor formation. We analyzed tumors of different mesenchymal origin for expression of Dlk1 and various myogenic markers and found that Dlk1 was present consistently in myogenic tumors. The coincident observation of Dlk1 with a highly proliferative state in myogenic tumors led us to subsequently investigate the involvement of Dlk1 in the control of the adult myogenic programme. We performed an injury study in Dlk1 transgenic mice, ectopically expressing ovine Dlk1 (membrane bound C2 variant) under control of the myosin light chain promotor, and detected an early, enhanced formation of myotubes in Dlk1 transgenic mice. We then stably transfected the mouse myoblast cell line, C2C12, with full-length Dlk1 (soluble A variant) and detected an inhibition of myotube formation, which could be reversed by adding Dlk1 antibody to the culture supernatant. These results suggest that Dlk1 is involved in controlling the myogenic programme and that the various splice forms may exert different effects. Interestingly, both in the Dlk1 transgenic mice and the DLK1-C2C12 cells, we detected reduced myostatin expression, suggesting that the effect of Dlk1 on the myogenic programme might involve the myostatin signaling pathway. In support of a relationship between Dlk1 and myostatin we detected reciprocal expression of these two transcripts during different cell cycle stages of human myoblasts. Together our results suggest that Dlk1 is a candidate marker for skeletal muscle tumors and might be involved directly in skeletal muscle tumor formation through a modulatory effect on the myogenic programme.