Cultured myf5 null and myoD null muscle precursor cells display distinct growth defects

Myf-5 and MyoD are the two muscle regulatory factors expressed from the myoblast stage to maintain the identity and to promote the subsequent differentiation of muscle precursor cells. To get insight into their role we have studied the capacity to proliferate and to differentiate of myf-5 and myoD null myoblasts in primary cultures and in the subsequent passages. Our results indicate that myf-5 null myoblasts differ from wild type (wt) myoblasts in that they undergo precocious differentiation: they become myogenin- and troponin T-positive and fail to incorporate bromodeoxyuridine (BrdU) under culture conditions and at a time when wt cells are not yet differentiated and continue to proliferate. In primary cultures of myoD null cells, up to 60% of the cells were scored as myoblasts on the basis of the expression of myf-5. These myoD-deficient myoblasts, unlike myoD-expressing cells, were poorly differentiating and displayed a severe growth defect that led to their elimination from the cultures: within a few passages myoblasts were absent from myoD-deficient cultures, which mostly consisted of senescent cells. That a null mutation in either gene reduces the proliferative potential of cultured myoblasts raises the possibility that Myf-5 and MyoD serve proliferation of muscle precursor cells.     

In vitro differentiation of myoblasts from skeletal muscle of rainbow trout

Substrata, plating densities and tissue culture media were compared for their effects on the proliferation and differentiation of myoblasts from skeletal muscle of rainbow trout. Mononuclear cells were isolated from the lateralis muscle of 4–11-month-old trout and plated on to glass coverslips coated with fibronectin, laminin or Matrigel. Cell proliferation was estimated by determining the density of nuclei on successive days in culture, and myoblast differentiation was detected by immunostaining cultures with the myosin-specific monoclonal antibody MF20. Mononuclear cell proliferation was highest for cells cultured on fibronectin or laminin and lowest for cells cultured on Matrigel, but the total number of nuclei in myosin-positive cells did not differ between substrata. The percentage of nuclei in myosin-positive myocytes and myotubes was significantly higher for cells cultured on Matrigel. The proportion of cells adhering to Matrigel and undergoing differentiation increased with plating density. Of three media tested, Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640 (RPMI), Leibovitz's L-15 (L-15) supplemented with 1 or 10% fetal bovine serum (FBS), a significantly greater proportion of the myoblasts differentiated when cells were cultured in L-15+ 10% FBS. These results suggest that culturing trout muscle-derived cells on a substratum of Matrigel at a high density and maintaining cells in L-15+ 10% FBS provide the conditions that maximize the proportion of cells that actively synthesize muscle myosin and facilitate trout myoblast differentiation in vitro .     

Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E₂ (PGE₂), a bioactive lipid mediator in various physiological or pathological conditions. PGE₂ is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE₂ signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE₂ or specific agonists. All four PGE₂ receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE₂ and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE₂ (17-PT PGE₂) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE₂ signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.     

Hypoxia promotes satellite cell self-renewal and enhances the efficiency of myoblast transplantation

Microenvironmental oxygen (O(2)) regulates stem cell activity, and a hypoxic niche with low oxygen levels has been reported in multiple stem cell types. Satellite cells are muscle-resident stem cells that maintain the homeostasis and mediate the regeneration of skeletal muscles. We demonstrate here that hypoxic culture conditions favor the quiescence of satellite cell-derived primary myoblasts by upregulating Pax7, a key regulator of satellite cell self-renewal, and downregulating MyoD and myogenin. During myoblast division, hypoxia promotes asymmetric self-renewal divisions and inhibits asymmetric differentiation divisions without affecting the overall rate of proliferation. Mechanistic studies reveal that hypoxia activates the Notch signaling pathway, which subsequently represses the expression of miR-1 and miR-206 through canonical Hes/Hey proteins, leading to increased levels of Pax7. More importantly, hypoxia conditioning enhances the efficiency of myoblast transplantation and the self-renewal of implanted cells. Given the robust effects of hypoxia on maintaining the quiescence and promoting the self-renewal of cultured myoblasts, we predict that oxygen levels in the satellite cell niche play a central role in precisely balancing quiescence versus activation, and self-renewal versus differentiation, in muscle stem cells in vivo.     

Stem cell antigen-1 regulates the tempo of muscle repair through effects on proliferation of alpha7 integrin-expressing myoblasts

Skeletal muscle repair occurs through a programmed series of events including myogenic precursor activation, myoblast proliferation, and differentiation into new myofibers. We previously identified a role for Stem cell antigen-1 (Sca-1) in myoblast proliferation and differentiation in vitro. We demonstrated that blocking Sca-1 expression resulted in sustained myoblast cell division. Others have since demonstrated that Sca-1-null myoblasts display a similar phenotype when cultured ex vivo. To test the importance of Sca-1 during myogenesis in vivo, we employed a myonecrotic injury model in Sca-1(-/-) and Sca-1(+/+) mice. Our results demonstrate that Sca-1(-/-) myoblasts exhibit a hyperproliferative response consisting of prolonged and accelerated cell division in response to injury. This leads to delayed myogenic differentiation and muscle repair. These data provide the first in vivo evidence for Sca-1 as a regulator of myoblast proliferation during muscle regeneration. These studies also suggest that the balance between myogenic precursor proliferation and differentiation is critical to normal muscle repair.     

Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation

The role of integrins in muscle differentiation was addressed by ectopic expression of integrin alpha subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human alpha5 subunit or the chicken alpha6 subunit produced contrasting phenotypes. The alpha5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the alpha6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail alpha6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic alpha subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric alpha subunits, alpha5ex/6cyto and alpha6ex/5cyto, produced phenotypes opposite to those observed with ectopic alpha5 or alpha6 expression. Myoblasts that express alpha5ex/6cyto show decreased proliferation while differentiation is partially restored. In contrast, the alpha6ex/5cyto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human alpha5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in alpha5 regulation of differentiation. Ectopic alpha5 and alpha6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human alpha5 subunit differentiate only in the absence of serum while differentiation of untransfected and alpha6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to alpha5-transfected myoblasts results in unique responses that differ from their effects on untransfected cells. Both bFGF or TGFbeta inhibit the serum-free differentiation of alpha5- transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-beta inhibits it. Insulin or TGF-alpha promote proliferation and differentiation of alpha5-transfected myoblasts; however, insulin alters myotube morphology. TGF-alpha or PDGF-BB enhance muscle alpha-actinin organization into myofibrils, which is impaired in differentiated alpha5 cultures. With the exception of TGF- alpha, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic alpha5 expression only in the presence of bFGF and insulin while TGF-alpha and TGF-beta promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin alpha subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the alpha subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells reside.     

Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System

Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.     

Ornithine decarboxylase is upregulated by the androgen receptor in skeletal muscle and regulates myoblast proliferation

The aim of this study is to determine if the Odc1 gene, which encodes ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is directly regulated by the androgen receptor (AR) in skeletal muscle myoblasts and if Odc1 regulates myoblast proliferation and differentiation. We previously showed that expression of Odc1 is decreased in muscle from AR knockout male mice. In this study, we show in vivo that Odc1 expression is also decreased >60% in muscle from male muscle-specific AR knockout mice. In normal muscle homeostasis, Odc1 expression is regulated by age and sex, reflecting testosterone levels, as muscle of adult male mice expresses high levels of Odc1 compared with age-matched females and younger males. In vitro, expression of Odc1 is 10- and 1.5-fold higher in proliferating mouse C(2)C(12) and human skeletal muscle myoblasts, respectively, than in differentiated myotubes. Dihydrotestosterone increases Odc1 levels 2.7- and 1.6-fold in skeletal muscle cell myoblasts after 12 and 24 h of treatment, respectively. Inhibition of ODC activity in C(2)C(12) myoblasts by α-difluoromethylornithine decreases myoblast number by 40% and 66% following 48 and 72 h of treatment, respectively. In contrast, overexpression of Odc1 in C(2)C(12) myoblasts results in a 27% increase in cell number vs. control when cells are grown under differentiation conditions for 96 h. This prolonged proliferation is associated with delayed differentiation, with reduced expression of the differentiation markers myogenin and Myf6 in Odc1-overexpressing cells. In conclusion, androgens act via the AR to upregulate Odc1 in skeletal muscle myoblasts, and Odc1 promotes myoblast proliferation and delays differentiation.     

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 10⁶ cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 10³ cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.     

Myogenic differentiation in permanent clonal mouse myoblast cell lines: Regulation by macromolecular growth factors in the culture medium

A permanent clonal cell line of mouse myoblasts (MM14) has been used to study the transition from proliferation to terminal differentiation. Results indicate that the transition is strictly dependent on the culture medium environment. Evidence from clonal density cultures suggests that (1) specific macromolecular mitogenic components of the culture medium stimulate mouse myoblast proliferation and prevent differentiation, (2) mouse myoblasts eliminate mitogenic activity from the culture medium before differentiating, and (3) lowered activity of specific mitogens stops mouse myoblast proliferation and triggers the program of terminal differentiation leading to the elaboration of muscle specific gene products and formation of myotubes. Evidence for the regulatory role of specific mitogens is the stimulation of proliferation and delay of differentiation by the addition of nanomolar concentrations of fibroblast growth factor to mitogen-depleted, differentiation-promoting, culture medium, whereas the addition of other purified mitogens has no effect. The results support and extend evidence from other muscle culture systems that stimulation of proliferation delays myoblast differentiation, and they provide an experimental basis for controlling the synchronous differentiation of pure populations of clonally derived mouse myoblasts.     

Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype

Skeletal myoblasts from fetal muscle respond adversely to fibronectin and laminin substrata: when primary mouse skeletal myoblasts are plated onto laminin, more myosin and desmin-positive myoblasts (myo+ cells) develop than on plates coated with fibronectin or collagen. In clonal cultures virtually all cells differentiate into postmitotic, fusion-capable myo + myoblasts on laminin after 3 days. In contrast, on fibronectin, the majority of the cells becomes myosin- and desmin-negative, partially due to proliferation of undifferentiated myoblast precursor cells, partially due to dedifferentiation or modulation of myoblasts into fibroblast-like myo- cells. Loss of the myogenic phenotype on fibronectin was also observed in cloned mouse myoblasts and in cultures of a differentiating mouse satellite cell line, MM14Dy, confirming that the appearance of desmin-negative cells is a result of myoblast modulation and not due simply to overgrowth by muscle fibroblasts. In the light of other effects of laminin on myoblasts, such as the stimulation of migration, differentiation and proliferation, our findings are consistent with the notion that laminin and fibronectin may be counteracting factors in the control of muscle differentiation.     

Atmospheric Oxygen Tension Slows Myoblast Proliferation via Mitochondrial Activation

Background

Mitochondrial activity inhibits proliferation and is required for differentiation of myoblasts. Myoblast proliferation is also inhibited by the ∼20% oxygen level used in standard tissue culture. We hypothesize that mitochondrial activity would be greater at hyperoxia (20% O₂) relative to more physiological oxygen (5% O₂).

Methodology/Principal Findings

Murine primary myoblasts from isolated myofibres and conditionally immortalized H-2K myoblasts were cultured at 5% and 20% oxygen. Proliferation, assayed by cell counts, EdU labeling, and CFSE dilution, was slower at 20% oxygen. Expression of MyoD in primary myoblasts was delayed at 20% oxygen, but myogenicity, as measured by fusion index, was slightly higher. FACS-based measurement of mitochondrial activity indicators and luminometric measurement of ATP levels revealed that mitochondria exhibited greater membrane potential and higher levels of Reactive Oxygen Species (ROS) at 20% oxygen with concomitant elevation of intracellular ATP. Mitochondrial mass was unaffected. Low concentrations of CCCP, a respiratory chain uncoupler, and Oligomycin A, an ATP synthase inhibitor, each increased the rate of myoblast proliferation. ROS were investigated as a potential mechanism of mitochondrial retrograde signaling, but scavenging of ROS levels by N-acetyl-cysteine (NAC) or α-Phenyl-N-tert-butylnitrone (PBN) did not rescue the suppressed rate of cell division in hyperoxic conditions, suggesting other pathways. Primary myoblasts from older mice showed a slower proliferation than those from younger adult mice at 20% oxygen but no difference at 5% oxygen.

Conclusions/Significance

These results implicate mitochondrial regulation as a mechanistic explanation for myoblast response to oxygen tension. The rescue of proliferation rate in myoblasts of aged mice by 5% oxygen suggests a major artefactual component to age-related decline of satellite cell proliferation in standard tissue culture at 20% oxygen. It lends weight to the idea that these age-related changes result at least in part from environmental factors rather than characteristics intrinsic to the satellite cell.     

An alternate protocol for establishment of primary caprine fetal myoblast cell culture: an in vitro model for muscle growth study

Cultured myoblasts have been used extensively as an in vitro model in understanding the underlying mechanisms of myogenesis. Various protocols for establishing a pure myoblast culture have been reported which involve the use of special procedures like flow cytometry and density gradient centrifugation. In goat, only a few protocols for establishing a myogenic cell culture are available and these protocols use adult muscle tissues which often does not yield sufficient numbers of precursor cells with adequate proliferative capacity. Considering the disadvantages of adult myoblasts, we are proposing an alternate protocol using caprine fetus which does not require any special procedures. In the present study, more than 90–95% fetal-derived cell populations had the typical spindle to polyhedral shape of myoblast cell and stained positive for desmin, hence confirming their myogenic origin. These cells attained the maximum confluency as early as 3–4 d against 3 wk by adult myoblasts indicating a better growth potential. Further, quantitative real-time PCR analysis revealed a higher expression (p?<?0.01) of myogenic regulatory factors (i.e., myogenic determination factor 1, myogenic factor 5, and myogenin) and myostatin (MSTN) in the fetal as compared to the adult myoblasts. Consequently, higher proliferation and differentiation ability along with higher abundance of myogenic markers and MSTN make the fetal myoblasts a better in vitro model.     

Expression and functional roles of angiopoietin-2 in skeletal muscles

Background

Angiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2) are angiogenesis factors that modulate endothelial cell differentiation, survival and stability. Recent studies have suggested that skeletal muscle precursor cells constitutively express ANGPT1 and adhere to recombinant ANGPT1 and ANGPT2 proteins. It remains unclear whether or not they also express ANGPT2, or if ANGPT2 regulates the myogenesis program of muscle precursors. In this study, ANGPT2 regulatory factors and the effects of ANGPT2 on proliferation, migration, differentiation and survival were identified in cultured primary skeletal myoblasts. The cellular networks involved in the actions of ANGPT2 on skeletal muscle cells were also analyzed.

Methodology/Principal Findings

Primary skeletal myoblasts were isolated from human and mouse muscles. Skeletal myoblast survival, proliferation, migration and differentiation were measured in-vitro in response to recombinant ANGPT2 protein and to enhanced ANGPT2 expression delivered with adenoviruses. Real-time PCR and ELISA measurements revealed the presence of constitutive ANGPT2 expression in these cells. This expression increased significantly during myoblast differentiation into myotubes. In human myoblasts, ANGPT2 expression was induced by H₂O₂, but not by TNFα, IL1β or IL6. ANGPT2 significantly enhanced myoblast differentiation and survival, but had no influence on proliferation or migration. ANGPT2-induced survival was mediated through activation of the ERK1/2 and PI-3 kinase/AKT pathways. Microarray analysis revealed that ANGPT2 upregulates genes involved in the regulation of cell survival, protein synthesis, glucose uptake and free fatty oxidation.

Conclusion/Significance

Skeletal muscle precursors constitutively express ANGPT2 and this expression is upregulated during differentiation into myotubes. Reactive oxygen species exert a strong stimulatory influence on muscle ANGPT2 expression while pro-inflammatory cytokines do not. ANGPT2 promotes skeletal myoblast survival and differentiation. These results suggest that muscle-derived ANGPT2 production may play a positive role in skeletal muscle fiber repair.     

Insulin-like growth factors (IGF-I and IGF-II) inhibit C2 skeletal myoblast differentiation and enhance TNF alpha-induced apoptosis

IGF-I and IGF-II are thought to be unique in their ability to promote muscle cell differentiation. Murine C2 myoblasts differentiate when placed into low serum media (LSM), accompanied by increased IGF-II and IGF binding protein-5 (IGFBP-5) production. Addition of 20 ng/ml TNF alpha on transfer into LSM blocked differentiation, IGF-II and IGFBP-5 secretion and induced apoptosis. We, therefore, wished to assess whether IGFs could protect against the effects of TNF alpha. Neither inhibition of differentiation or induction of apoptosis was rescued by co-incubation with IGF-I or IGF-II. A lower dose of TNF alpha (1 ng/ml) while not inducing apoptosis still inhibited myoblast differentiation by 56% +/- 12, (P < 0.001), indicating that induction of apoptosis is not the sole mechanism by which TNF alpha inhibits myoblast differentiation. Addition of IGF-I or IGF-II alone reduced differentiation by 49% +/- 15 and 33% +/- 20, respectively, (P < 0.001), although neither induced apoptosis. For muscle cells to differentiate, they must arrest in G0. We established that addition of IGF-I, IGF-II or TNF alpha to the myoblasts promoted proliferation. The myoblasts could not exit the cell cycle as efficiently as controls and differentiation was thus reduced. Unexpectedly, co-incubation of IGF-I or IGF-II with 1 ng/ml TNF alpha enhanced the inhibition of differentiation and induced apoptosis. In the absence of apoptosis we show an association between IGF-induced inhibition of differentiation and increased IGFBP-5 secretion. These results indicate that the effects of the IGFs on muscle may depend on the cytokine environment. In the absence of TNF alpha, the IGFs delay differentiation and promote myoblast proliferation whereas in the presence of TNF alpha the IGFs induce apoptosis.     

Triiodothyronine influences quail myoblast proliferation and differentiation*

The influence of triiodothyronine (T3) on avian myoblast proliferation and differentiation was studied in secondary cultures using plating densities of 2500 and 7000 cells/cm². Culture media were depleted of T3 (control myoblasts) and increasing amounts were then added to concentrations of 0.6, 3 and 15 nM T3 (treated myoblasts). Independent of the cell density, T3 induced a dose-related decrease in myoblast proliferation measured by cell number, doubling time and ³H-thymidine incorporation. However, with the lower plating density, this influence was delayed, occurring only after the third day of culture for 0.6 nM T3-treated myoblasts and simultaneous with the onset of myosin heavy chain accumulation. Moreover, when myoblasts were exposed to BrdU for 48 h, the T3 growth inhibitory effect disappeared, thus showing that this effect was clearly linked to differentiation. In addition, we have shown that T3 induced an early fusion of myoblasts: 65% of the maximal value of the fusion index was reached on day 3 in the T3-treated cells in comparison to 25% in the control myoblasts. This hormone also enhanced accumulation of muscle-specific proteins (connectin, acetylcholine receptors, myosin heavy chain), tested by cytoimmunofluorescence, ELISA, binding experiments and Western blot. All these results show that T3 increased myoblast differentiation through a pathway including myoblast withdrawal from the cell cycle. The influence of T3 could partly explain its previously reported positive effect on the number of muscle fibers.     

Blunting effect of hypoxia on the proliferation and differentiation of human primary and rat L6 myoblasts is not counteracted by Epo

Objectives: The aim of this study was to evaluate whether hypoxia and/or erythropoietin would be able to modulate proliferation/differentiation processes of rat and human myoblasts. Materials and methods: Rat L6 and primary human myoblasts were grown in 21% or 1% O₂ in the presence or absence of recombinant human erythropoietin (RhEpo). Presence of erythropoietin receptors (EpoR) was assayed using RT‐PCR and Western blotting techniques. Cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. Cell differentiation was analysed by determining myogenic fusion index using antibodies against the myosin heavy chain. Expression of myogenin and myosin heavy chain (MHC) proteins were evaluated using the Western blotting technique. Results: After 96 h culture in growth medium for 2.5 and 9 h, doubling time of L6 and human primary myoblasts respectively, had increased in 1% O₂ conditions (P < 0.01). Kinetics of culture showed alteration in proliferation at 72 h in L6 myoblast cultures and at 4 days in human primary myoblasts. The myogenic fusion index had reduced by 30% in L6 myoblasts and by 20% in human myoblasts (P < 0.01). Expression of myogenin and MHC had reduced by around 50%. Despite presence of EpoR mRNA and protein, RhEpo did not counteract the effects of hypoxia either in L6 cells or in human myoblasts. Conclusions: The data show that exposure to hypoxic conditions (1% O₂) of rat and human myoblasts altered their proliferation and differentiation processes. They also show that Epo is not an efficient growth factor to counteract this deleterious effect.