Carola Niesler

TGF-beta's delay skeletal muscle progenitor cell differentiation in an isoform-independent manner.

Satellite cells are a quiescent heterogeneous population of mononuclear stem and progenitor cells which, once activated, differentiate into myotubes and facilitate skeletal muscle repair or growth. The Transforming Growth Factor-beta (TGF-beta) superfamily members are elevated post-injury and their importance in the regulation of myogenesis and wound healing has been demonstrated both in vitro and in vivo. Most studies suggest a negative role for TGF-beta on satellite cell differentiation. However, none have compared the effect of these three isoforms on myogenesis in vitro. This is despite known isoform-specific effects of TGF-beta1, -beta2 and -beta3 on wound repair in other tissues. In the current study we compared the effect of TGF-beta1, -beta2 and -beta3 on proliferation and differentiation of the C2C12 myoblast cell-line. We found that, irrespective of the isoform, TGF-beta increased proliferation of C2C12 cells by changing the cellular localisation of PCNA to promote cell division and prevent cell cycle exit. Concomitantly, TGF-beta1, -beta2 and -beta3 delayed myogenic commitment by increasing MyoD degradation and decreasing myogenin expression. Terminal differentiation, as measured by a decrease in myosin heavy chain (MHC) expression, was also delayed. These results demonstrate that TGF-beta promotes proliferation and delays differentiation of C2C12 myoblasts in an isoform-independent manner.

In Vitro myoblast motility models: investigating migration dynamics for the study of skeletal muscle repair

Skeletal muscle repair requires the migration of myoblasts (activated satellite cells) both to the injury site and then within the wound to facilitate cellular alignment in preparation for differentiation, fusion and eventual healing. Along this journey, the cells encounter a range of soluble and extracellular matrix factors which regulate their movement and ultimately determine how successful the repair process will be. Sub-optimal migration can lead to a number of scenarios, including reduced myoblast numbers entering the wound, poor alignment and insufficient differentiation to correctly repair the damage. It is therefore critical that all aspects of myoblast migration are understood, particularly in response to the changing growth and matrix factor profile prevalent following skeletal muscle injury. Since 1962, when Boyden first introduced his chemotactic chamber, numerous in vitro migration assays have been developed to mimic the wound more closely. These have increased in complexity to account for the complex micro-environment found in vivo during muscle repair and include a range of modified cell exclusion, chemotactic and three-dimensional assays. This review describes and discusses these advances and highlights the importance they have in expanding our understanding of myoblast migration dynamics.

Potential myogenic stem cell populations: sources, plasticity, and application for cardiac repair.

The ability of unspecialized stem cells to differentiate into mature, specialized cell types has made them attractive as potential agents for enhanced tissue repair and regenerative medicine. This is especially true of diseases and disorders for which no or only partially effective treatments are currently available. Recently, increased focus has been placed on the regenerative potential of satellite cells (myogenic precursor cells found in the adult skeletal muscle) in various muscular disorders, such as dystrophy and myocardial injury following ischemia. Animal studies and clinical trials are in progress using satellite cells as cellular candidates; however, this early rollout in the clinical setting has deflected attention from the potential of other less specialized, but potentially more maliable, stem cell sources. Published data is still lacking on the best methods for identification, isolation, and further expansion or nuclear manipulation of these cells in vitro. Also, although differentiation capacity has been proven in terms of protein expression patterns characteristic of myogenesis, proof of contractile and energetic compatibility between graft and host is more difficult to establish. In this regard, although future animal model studies will be invaluable, they must be designed with short- and long-term functional outcomes in mind. This review moves beyond initial excitement regarding the acknowledged potential of cell therapy and provides a realistic exposition of the themes and specific issues that should be considered in current experimental research study designs.

Dose-dependent modulation of myogenesis by HGF: implications for c-Met expression and downstream signalling pathways

Abstract Hepatocyte growth factor (HGF) regulates satellite cell activation, proliferation, and differentiation. We analyzed the dose-dependent effects of HGF on myogenesis. Murine C2C12 and human donor-derived skeletal muscle myoblasts were treated with 0, 2, or 10 ng/ml HGF followed by assessment of proliferation and differentiation. HGF (2 ng/ml) significantly promoted cell division, but reduced myogenic commitment and fusion. Conversely, 10 ng/ml HGF reduced proliferative capability, but increased differentiation. c-Met expression analysis revealed significantly decreased expression in differentiating cells cultured with 2 ng/ml HGF, but increased expression in proliferating cells with 10 ng/ml HGF. Mitogen-activated protein kinase (MAPKs: ERK, JNK, or p38K) and phosphatidylinositol-3-kinase (PI3K) inhibition abrogated the HGF-stimulated increase in cell number. Interestingly, PI3K and p38 kinase facilitated the negative effect of HGF on proliferation, while ERK inhibition abrogated the HGF-mediated decrease in differentiation. Dose-dependent effects of HGF are mediated by changes in c-Met expression and downstream MAPK and PI3K signalling.

Satellite cell count, VO2max, and p38 MAPK in inactive to moderately active young men

Satellite cells (SCs) are responsible for muscle repair following strenuous exercise or injury. SC responses to intervention have been studied, but most studies do not discuss or take into account the substantial variability in SC number among young individuals. We hypothesized that an active lifestyle reflected in higher VO2max may be associated with greater SC number. As training alters basal p38‐mitogen‐activated protein kinase (MAPK) activity, which is associated with SC proliferation, SC count may also correlate with this stress signaling kinase. Muscle biopsies from vastus lateralis of eight male participants were analyzed for fiber type, myogenin, and p38/phospho‐p38 MAPK using SDS‐PAGE and Western blotting. Immunofluorescence was used to detect Pax7+ SCs. Two weeks following the biopsy, subjects underwent an incremental treadmill test to determine VO2max. A strong positive correlation (P = 0.0087) was found between the number of Pax7+ nuclei and VO2max. Pax7+ cell number correlated negatively with phospho‐p38/p38 MAPK (P = 0.0006), but had no correlation with fiber type or myogenin. SC number is proportional to VO2max, and hence it can be postulated that higher levels of physical activity activate SC proliferation but not fusion, underlining the relevance of exercise in stimulating SC pool size even without injury.

ROCK‐2 Is Associated With Focal Adhesion Maturation During Myoblast Migration

Satellite cell migration is critical for skeletal muscle growth and regeneration. Controlled cell migration is dependent on the formation of mature focal adhesions between the cell and the underlying extracellular matrix (ECM). These cell–ECM interactions trigger the activation of signalling events such as the Rho/ROCK pathway. We have previously identified a specific role for ROCK‐2 during myoblast migration. In this study we report that ROCK inhibition with Y‐27632 increases C2C12 myoblast velocity, but at the expense of directional migration. In response to Y‐27632 an increased number of smaller focal adhesions were distributed across adhesion sites that in turn were clearly larger than sites in untreated cells, suggesting a reduction in focal adhesion maturation. We also confirm ROCK‐2 localisation to the focal adhesion sites in migrating myoblasts and demonstrate a change in the distribution of these ROCK‐2 containing adhesions in response to Y‐27632. Taken together, our observations provide further proof that ROCK‐2 regulates directional myoblast migration through focal adhesion formation and maturation. J. Cell. Biochem. 115: 1299–1307, 2014.

MMP-14 in skeletal muscle repair

MMP-14 (also known as MT1-MMP) is a membrane-bound collagenase and member of the Matrix Metalloprotease (MMP) family known to target a broad range of extracellular matrix (ECM) proteins. Remodelling of the ECM is of particular importance following skeletal muscle injury involving myofiber necrosis, when satellite cells are activated to facilitate myogenesis and regeneration. Myogenesis (broadly encompassed by the processes of satellite cell activation, proliferation, migration, differentiation and fusion) requires the myoblast to move either on or through a changing milieu of ECM components. The ECM composition, and especially the degree of fibrosis, influences ability of satellite cells to mediate a successful regenerative program. As a result, MMP activity is central to this regeneration; its activity increases following skeletal muscle injury, while inhibition of MMP reduces regeneration in this tissue. Besides its direct effect on matrix invasion, MMP-14 itself can affect this regeneration via activation of other MMPs (MMP-2, -9 and -13) as well as cytokines, chemokines and growth factors. Indeed recent research suggests that MMP-14 is necessary for the migration of human myoblasts into a collagen I matrix. Here we provide a current review on MMP-14 in the context of its role as a critical mediator of skeletal muscle regeneration.

Satellite cell pool expansion is affected by skeletal muscle characteristics

Introduction: We investigated changes in satellite cell (SC) pool size after an acute bout of strenuous exercise and evaluated the influence of baseline SC count and fiber type. Methods: Participants completed a downhill running (DHR) intervention (5 × 8 min, 2‐min rest; 80% VO2max; −10% gradient). Muscle biopsies were taken 7 days before VO2max and 7–9 days after the DHR intervention. Delayed‐onset muscle soreness (DOMS) and creatine kinase activity (CK) were measured on days 1, 2, 7, and 9 post‐DHR. SCs were identified by Pax7 and laminin staining. Relative distribution of MHC isoforms was determined by electrophoresis. Results: DOMS and CK peaked on day 1 post‐DHR (P < 0.01). The SC pool increased (26%) after DHR (P = 0.005). SCs/total myonuclei after recovery correlated with baseline SCs (r = 0.979, P = 0.003) and VO2max (r = 0.956, P = 0.011), whereas change in SC pool (Pax7+ cells/total myonuclei: recovery minus baseline) tended to correlate with percent MHC II (r = 0.848; P = 0.06). Conclusion: Interindividual physiological characteristics affect SC pool expansion after a single bout of DHR and are influenced by VO2max. Muscle Nerve, 2013

Simple silicone chamber system for in vitro three-dimensional skeletal muscle tissue formation

Bioengineering skeletal muscle often requires customized equipment and intricate casting techniques. One of the major hurdles when initially trying to establish in vitro tissue engineered muscle constructs is the lack of consistency across published methodology. Although this diversity allows for specialization according to specific research goals, lack of standardization hampers comparative efforts. Differences in cell type, number and density, variability in matrix and scaffold usage as well as inconsistency in the distance between and type of adhesion posts complicates initial establishment of the technique with confidence. We describe an inexpensive, but readily adaptable silicone chamber system for the generation of skeletal muscle constructs that can readily be standardized and used to elucidate myoblast behavior in a three-dimensional space. Muscle generation, regeneration and adaptation can also be investigated in this model, which is more advanced than differentiated myotubes.

Simultaneous isolation of enriched myoblasts and fibroblasts for migration analysis within a novel co-culture assay.

Skeletal muscle injury elicits the activation of satellite cells and their migration to the wound area for subsequent terminal differentiation and tissue integration. However, interstitial fibroblasts recruited to the site of injury promote deposition of fibrotic tissue, which hampers myoblast-mediated muscle regeneration. Currently, analysis of myoblast migration in vitro can be accomplished using chemotactic, cell-exclusion, or wound healing assays. Yet, to investigate cell motility following skeletal muscle damage more accurately, migration assays need to better simulate the repair process. Here we present a protocol for the simultaneous isolation of myoblasts and fibroblasts from the same muscle tissue, ensuring the consistent generation of enriched, purified, and matched cell populations at a low passage number. We then describe a wound assay that uses a novel approach to the co-culture of myoblasts and fibroblasts to mimic the injured environment more closely than other established methods. Using this assay, we demonstrate that fibroblasts are able to increase myoblast migration significantly, validating our new in vitro method. As the observed effect on migration is most likely mediated by secreted factors, our assay could easily be extended to include antibody-based protein analysis of secreted factors in animal or human systems.