Myoblast Morphology Research Articles

Fluid shear stress-induced mechanotransduction in myoblasts: Does it depend on the glycocalyx?

Muscle stem cells (MuSCs) are involved in muscle maintenance and regeneration. Mechanically loaded MuSCs within their native niche undergo tensile and shear deformations, but how MuSCs sense mechanical stimuli and translate these into biochemical signals regulating function and fate is still poorly understood. We aimed to investigate whether the glycocalyx is involved in the MuSC mechanoresponse, and whether MuSC morphology affects mechanical loading-induced pressure, shear stress, and fluid velocity distribution. FSS-induced deformation of active proliferating MuSCs (myoblasts) with intact or degraded glycocalyx was assessed by live-cell imaging. Glycocalyx-degradation did not significantly affect nitric oxide production, but reduced FSS-induced myoblast deformation and modulated gene expression. Finite-element analysis revealed that the distribution of FSS-induced pressure, shear stress, and fluid velocity on myoblasts was non-uniform, and the magnitude depended on myoblast morphology and apex-height. In conclusion, our results suggest that the glycocalyx does not play a role in NO production in myoblasts but might impact mechanotransduction and gene expression, which needs further investigation. Future studies will unravel the underlying mechanism by which the glycocalyx affects FSS-induced myoblast deformation, which might be related to increased drag forces. Moreover, MuSCs with varying apex-height experience different levels of FSS-induced pressure, shear stress, and fluid velocity, suggesting differential responsiveness to fluid shear forces.

One-Step Synthesis of Gelatin-Conjugated Supramolecular Hydrogels for Dynamic Regulation of Adhesion Contact and Morphology of Myoblasts

Hydrogels possessing fine-adjustable and switchable elasticity emulate the mechanical microenvironments of biological cells, which are known to change dynamically during development and disease progression. In this study, a supramolecular hydrogel conjugated with gelatin side chains was synthesized. By systematically screening the molar fraction of supramolecular host/guest cross-linkers, Young’s modulus of the substrate was fine-adjusted to the level for myoblasts, E ≈ 10 kPa. C2C12 myoblasts reproducibly and firmly adhered to the gelatin-conjugated hydrogel via focal adhesion contacts consisting of integrin clusters, whereas only a few cells adhered to the gel without gelatin side chains. The elasticity of the gelatin-conjugated hydrogel was switchable to desired levels by simply adding and removing free guest molecules in appropriate concentrations without interfering with cell viability. Immunofluorescence confocal microscopy images of fixed cells confirmed the adaptation of focal adhesions and remodeling of actin cytoskeletons on the gelatin-conjugated hydrogel. Time-lapse phase-contrast images demonstrated the dynamic response of the cells, manifested in their morphology, to an abrupt change in the substrate elasticity. Gelatin-conjugated hydrogels with switchable elasticity enable the direct and reversible mechanical stimulation of cells in one step without tedious surface functionalization with adhesion ligands.

Fusion of Normoxic- and Hypoxic-Preconditioned Myoblasts Leads to Increased Hypertrophy.

Injuries, high altitude, and endurance exercise lead to hypoxic conditions in skeletal muscle and sometimes to hypoxia-induced local tissue damage. Thus, regenerative myoblasts/satellite cells are exposed to different levels and durations of partial oxygen pressure depending on the spatial distance from the blood vessels. To date, it is unclear how hypoxia affects myoblasts proliferation, differentiation, and particularly fusion with normoxic myoblasts. To study this, we investigated how 21% and 2% oxygen affects C2C12 myoblast morphology, proliferation, and myogenic differentiation and evaluated the fusion of normoxic- or hypoxic-preconditioned C2C12 cells in 21% or 2% oxygen in vitro. Out data show that the long-term hypoxic culture condition does not affect the proliferation of C2C12 cells but leads to rounder cells and reduced myotube formation when compared with myoblasts exposed to normoxia. However, when normoxic- and hypoxic-preconditioned myoblasts were differentiated together, the resultant myotubes were significantly larger than the control myotubes. Whole transcriptome sequencing analysis revealed several novel candidate genes that are differentially regulated during the differentiation under normoxia and hypoxia in mixed culture conditions and may thus be involved in the increase in myotube size. Taken together, oxygen-dependent adaption and interaction of myoblasts may represent a novel approach for the development of innovative therapeutic targets.

Platelet-poor plasma of athletes is a potent inducer of myogenic differentiation of C2C12 myoblasts

Introduction. Blood products, i.e. platelet rich plasma (PRP), leukocyte-poor plasma (PRP) and platelet poor plasma (PPP), have previously been used to improve muscle regeneration. In this study, six months? frozen-stored PPP of individuals who practiced different types of physical exercise was analysed; it could steer mouse C2C12 myoblast cells towards proliferation, migration and myogenic differentiation, and it could affect the morphology/shape of myotubes. Materials and Methods. PPP of male Olympic weightlifters, football players and professional folk dancers, aged 15-19, was collected 12 h post-training and stored for 6 months at -20?C. C2C12 cell proliferation was assessed by MTT test, motility by scratch assay, myogenic differentiation by myotube formation and gelatinase activity by gel-zymography. Results and Conclusions. PPP induced proliferation and migration of C2C12 cells. Proliferative capacity was as follows: weightlifters > dancers > football players; mean migratory capacity was: weightlifters = dancers > football players. PPP induced formation of myotubes; significant inter-individual variations were detected: PPP from weightlifters induced formation of round myotubes, and PPP from football players and dancers induced formation of elongated myotubes. The mean myotube area was as follows: football players > dancers > weightlifters. PPP gelatinolytic activity was observed; it was negatively correlated with C2C12 myoblast proliferation. These results provide general but distinct evidence that PPP of individuals practicing certain types of exercise can specifically modify myoblast morphology/function. This is significant for explaining physiological responses and adaptations to exercise. In conclusion, long-term, frozen-stored PPP preserves its potential to modify myoblast morphology and function.

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.

Involvement of unconventional myosin VI in myoblast function and myotube formation.

The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873–885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.

Temperature-Responsive Poly(ε-caprolactone) Cell Culture Platform with Dynamically Tunable Nano-Roughness and Elasticity for Control of Myoblast Morphology

We developed a dynamic cell culture platform with dynamically tunable nano-roughness and elasticity. Temperature-responsive poly(ɛ-caprolactone) (PCL) films were successfully prepared by crosslinking linear and tetra-branched PCL macromonomers. By optimizing the mixing ratios, the crystal-amorphous transition temperature (Tm) of the crosslinked film was adjusted to the biological relevant temperature (~33 °C). While the crosslinked films are relatively stiff (50 MPa) below the Tm, they suddenly become soft (1 MPa) above the Tm. Correspondingly, roughness of the surface was decreased from 63.4–12.4 nm. It is noted that the surface wettability was independent of temperature. To investigate the role of dynamic surface roughness and elasticity on cell adhesion, cells were seeded on PCL films at 32 °C. Interestingly, spread myoblasts on the film became rounded when temperature was suddenly increased to 37 °C, while significant changes in cell morphology were not observed for fibroblasts. These results indicate that cells can sense dynamic changes in the surrounding environment but the sensitivity depends on cell types.

The Physical Interaction of Myoblasts with the Microenvironment during Remodeling of the Cytoarchitecture

Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts) require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility). Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ∼13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min) of large scale remodeling and physiological stress, which is essential to their in-vivo functionality.

Abstract 74: Naturally Occurring Human α1a-Adrenergic Receptor Genetic Variant Induces Transformation of Cardiomyoblasts to Fibroblast-Like Phenotype

Hypertensive heart disease is a progressive condition leading to myocardial remodeling, alterations in cardiomyocytes and fibrosis. Three α1 adrenergic receptor (AR) subtypes are expressed in human heart (α1a, α1b, α1d). Recently, we reported that human α1aAR with the naturally occurring G247R SNP (247R), originally identified in a hypertensive patient, constitutively couples to EGFR-transactivation pathway in fibroblasts and leads to hyperproliferation. We hypothesized that expression of 247R in cardiomyoblasts plays an important role in their remodeling. We demonstrate that constitutive expression of 247R in H9C2 cardiomyoblasts changes the morphology of myoblasts to fibroblast-like cells (transformed, tr247R); higher 247R expression results in earlier onset of transformation. Expression of α1aAR-WT does not affect cell morphology. Tr247R cells exhibit 2-3 fold increased constitutive proliferation compared to control or WT cells as determined by cell counts and thymidine incorporation assays; pERK is upregulated in tr247R cells as determined by Western blot analyses. Treatment with AraC, a DNA replication inhibitor, eliminates the 247R cells but not the H9C2 myoblasts. Expression of cell-specific markers is being evaluated in tr247R and control cells. RT-PCR and zymograms revealed increased MMP2 mRNA and protein levels in tr247R cells compared to control or WT cells and are comparable to MMP2 levels in fibroblasts. Increased proliferation of tr247R cells is reduced by EGFR specific inhibitor AG1478 but not general MMP inhibitor GM6001. Agonist stimulation of tr247R results in hypertrophy as determined by leucine incorporation. Interestingly, hypertrophy but not cell proliferation is inhibited by prazosin, an α1aAR inverse agonist, suggesting that increased proliferation and hypertrophy observed in transformed cells are two independent pathways triggered by 247R. Our data demonstrate that expression of α1aAR genetic variant triggers alterations in cardiomyoblasts via distinct signaling pathways leading to hyperproliferation, transformation of the cells to fibroblast-like phenotype and hypertrophy. Consequently, these changes may lead to fibrosis in human heart, extracellular matrix remodeling and heart failure.

Myoblast morphology and organization on biochemically micro-patterned hydrogel coatings under cyclic mechanical strain

Mechanical forces and geometric constraints play critical roles in determining cell functionality and tissue development. Novel experimental methods are essential to explore the underlying biological mechanisms of cell response. We present a versatile method to culture cells on adhesive micro-patterned substrates while applying long-term cyclic tensile strain (CTS). A polydimethysiloxane (PDMS) mold is coated with a cell repulsive NCO-sP(EO- stat-PO) hydrogel which in turn is covalently patterned by fibronectin using micro-contact printing. This results in two-dimensional, highly selective cell-adhesive micro-patterns. The substrates allow application of CTS to adherent cells for more than 4 days under cell culture conditions without unspecific adhesion. The applicability of our system is demonstrated by studying the adaptive response of C2C12 skeletal myoblasts seeded on fibronectin lines with different orientations relative to the strain direction. After application of CTS (amplitude of 7%, frequency of 0.5 Hz) we find that actin fiber organization is dominantly controlled by CTS. Nuclei shape is predominantly affected by the constraint of the adhesive lines, resulting in significant elongation. Morphologically, myotube formation was incomplete after 4 days of culture, but actin striations were observed exclusively on the 45° line patterns subjected to CTS, the direction of maximum shear strain.

CSX/Nkx2.5 Modulates Differentiation of Skeletal Myoblasts and Promotes Differentiation into Neuronal Cells in Vitro

CSX/Nkx2.5 transcription factor plays a pivotal role in cardiac development; however, its role in development and differentiation of other organs has not been investigated. In this study, we used C2C12 myoblasts and human fetal primary myoblasts to investigate the function of Nkx2.5 in skeletal myogenesis. The expression levels of Nkx2.5 decreased as C2C12 myoblasts elongated and fused to form myotubes. The expression of human NKX2.5 in C2C12 myoblasts inhibited myocyte differentiation and myotube formation, and up-regulated Gata4 and Tbx5 expression. The expression of NKX2.5 in terminally differentiated C2C12 myotubes resulted in a change in morphology and breakdown into smaller myotubes. Furthermore, overexpression of NKX2.5 in C2C12 cells and primary cultures of human fetal myoblasts led to differentiation of myoblasts into neuron-like cells and expression of neuronal markers. This study sheds light on the previously unknown non-cardiac functions of Nkx2.5 transcription factor.

380. Dedifferentiation of Primary Murine Myoblasts Induced by Ectopic Expression of Msx1

Top of pageAbstract Ectopic expression of msx1 was shown to induce dedifferentiation of multinucleated myotubes derived from myogenic cell line (C2C12) to mononucleated cells and these dedifferentiated cells displayed stem cell like pluripotency (Odelberg et al, Cell 103: 1099, 2000). We hypothesize that if the same phenomenon exists in primary cells, using this method, we can generate pluripotent cells from myoblasts or fibroblasts which are easily accessible and can be propagated in culture. In the present study, we transduced purified primary myoblasts isolated from muscles of adult mice with a retroviral vector that expresses msx1 gene regulated by a Tat off system and a neomyocin resistant gene expressing from 5′ LTR of retrovirus or a control vector that contains the same elements except the msx1 sequence reversed. In the presence of 6μg/ml doxycycline (an analog of Tetracycline, transduced myoblasts were selected with 400μg/ml G418 for 14 days. RT- PCR confirmed the expression of msx1 in clonal myoblasts of experimental group after withdrawal of doxycycline. To assess whether or not ectopic expression of msx1 can induce dedifferentiation of these primary myoblasts, first the myoblasts from experimental and control groups were forced differentiation by serum withdrawal and then the formed myotubes were placed into 96 well culture plates with one myotube per well. One to five days after withdrawal of doxycycline, 7.5% of multinucleated myotubes in experimental group cleaved into mononucleated cells or smaller myotubes. In contrast, none of myotubes in the control group cleaved. These mononucleated cells are capable of proliferating and the cells cleaved from single myotube were capable of propagating into multimillions of cells without signs of senescence. Two clones of these cells were characterized for their stem cell like properties. RT-PCR revealed that these cells expressed Oct 4, a marker of embryonic and mesenchymal stem cells, and NST, a marker of mesenchymal stem cells. In contrast, there was no detectable level of both markers in control group and uninfected primary myoblasts. When adding LIF into the culture, the growth rate of these dedifferentiated cells was increased but not the rate of control group and uninfected primary myoblasts. Immunochemistry showed that a fraction of these dedifferentiated cells were MyoD (a myogenic marker) negative while the whole population of myoblasts are MyoD positive. Most of these dedifferentiated cells had typical myoblast morphology but some of the cells appeared to have tendency to form ES cell-like clumps. After suppression of msx1 expression, these cells were cultured in adipogenic, osteogenic, chondrogenic or myogenic media to assess their pluripotency. In the osteogenic culture condition, some of the cells expressed alkaline phosphatase (a marker for osteogenesis). In the myogenic culture condition, all of the cells differentiated into myotubes. But we haven't detected the signals of adipogenesis and chondrogenesis in the present culture conditions. Thus, our data demonstrated that ectopic expression of msx1 could induce a subpopulation of primary mouse myoblast dedifferentiation. The further characterization of these dedifferentiated cells and development of methods to isolate them from main population are in the process.

Myoblast structure affects subsequent skeletal myotube morphology and sarcomere assembly

Skeletal myogenesis is a precise procedure marked by specific changes in muscle cell morphology and cytoarchitecture. Cessation of proliferation by skeletal muscle precursor cells (myoblasts) coincides with the induction of fusion to form multinucleated myotubes and the initiation of differentiation, the process through which sarcomeres are formed. Concurrently, there is a distinct upregulation in expression of muscle-specific isoforms and an extreme downregulation of non-muscle-specific cytoskeletal isoforms. The sarcomere is the contractile unit of the cell and is comprised of a number of different proteins aggregated and aligned in very ordered arrays along the myotube. It is this rigorously controlled alignment that gives striated muscle its characteristic “striped” appearance. Previous studies, conducted predominantly in cardiac muscle, propose models for the development of the sarcomere that attribute little of the differentiative process to the myoblast morphology and cytoskeletal arrangement. In this study, perturbation of myoblast morphology and cytoskeletal arrangement by transfection with nonmuscle actin genes in the mouse skeletal muscle cell line C2 resulted in myotubes of both varied morphology and sarcomeric structure. The results presented herein not only provide novel insights into the formation of the sarcomere in skeletal muscle, but also suggest a role for myoblast morphology and cytoskeletal structure in the subsequent differentiation of the myotube.

Human myoblast culture as muscle stem cells in medical and biological studies

The method for obtaining human myoblast culture has been modified to consider the specific histological localization of the satellite cells as well as their growth properties; the cultivation conditions have been selected to grow up to 150000 cells/cm2. At high densities, the cells remain mononuclear and preserve their typical myoblast morphology as well as the capacity for fusion and the formation of myotubes. By contrast to fibroblasts, up to 80% of the cells in the myoblast culture were positive in the acid phosphatase test, which indicates their stem nature. The obtained myoblast cultures were used in the clinical tests of cell-mediated gene therapy of Duchenne's muscular dystrophy as well as in the bioassay for the effects of biologically active compounds.

β1 Integrin Binds the 16-kDa Subunit of Vacuolar H+-ATPase at a Site Important for Human Papillomavirus E5 and Platelet-derived Growth Factor Signaling

Integrins mediate adhesive interactions between cells and the extracellular matrix, and play a role in cell migration, proliferation, differentiation, cytoskeletal organization, and signal transduction. We have identified an interaction between the beta(1) integrin and the 16-kDa subunit of vacuolar H(+)-ATPase (16K). This interaction was first isolated in a yeast two-hybrid screen and confirmed by coimmunoprecipitation and in in vitro binding assays using bacterially expressed proteins. Immunofluorescent studies performed in L6 myoblasts expressing both native and epitope-tagged 16K demonstrate co-localization with beta(1) integrin in focal adhesions. Deletion of the fourth of four transmembrane helices in 16K results in loss of interaction with beta(1) integrin in vitro and in the two-hybrid system, and less prominent staining in focal adhesions. This helix is also required for ligand-independent activation of platelet-derived growth factor-beta receptor signaling by the human papillomavirus E5 oncoprotein. Overexpression of 16K or expression of 16K lacking this helix alters the morphology of myoblasts and fibroblasts, suggesting that the interaction of 16K with integrins could be important for cell growth control. We also discuss the possible role 16K might play in integrin movement.

Impact of alpha-skeletal actin but not alpha-cardiac actin on myoblast morphology.

Muscle differentiation involves a profound change in cell cytoarchitecture. This is accompanied by extensive isoform replacement in which the major non-muscle isoforms of the actin filament system are replaced by their muscle isoform counterparts. We have tested whether the sequential expression of the actin isoforms is functionally significant by precociously expressing the two striated muscle actins (alpha-skeletal and alpha-cardiac) in mouse myoblasts. The human alpha-skeletal and alpha-cardiac actin genes were transfected into mouse C2 myoblasts and clones expressing the human genes at the highest level were identified. Expression of the human alpha-skeletal actin gene was low with the highest mRNA level found to be 4% of that in adult human skeletal muscle. Clones expressing alpha-cardiac actin accumulated the mRNA up to 13% of the level of alpha-skeletal actin in adult human skeletal muscle. Despite the low level of alpha-skeletal actin expression, myoblasts transfected with this gene displayed a profound decrease in cell spreading. In contrast, alpha-cardiac actin had no impact on cell spreading. Neither alpha-skeletal actin nor alpha-cardiac actin had any impact on the total actin protein pool nor on the levels of the high molecular weight tropomyosins. The organisation of actin and tropomyosin into stress fibres was similar between transfected and control cells. We conclude that precocious expression of alpha-skeletal actin, but not alpha-cardiac actin, compromises myoblast morphology but not the ability of the cell to assemble stress-fibre-like structures.

Noncoding regions of the gamma-actin gene influence the impact of the gene on myoblast morphology.

We have addressed the question of whether two highly conserved noncoding regions of the gamma-actin gene are of functional importance. Human gamma-actin gene constructs deleted for either the entire 3' untranslated region (UTR) and 3' flank or intron III sequences were transfected into mouse myoblasts and the resulting clones were analyzed for cell morphology and actin protein expression. Transfectants carrying the gamma-actin gene deleted for the 3' end (gamma 22) exhibited numerous long pseudopods and increased surface area. In contrast, transfectants expressing the gamma-actin gene deleted for intron III (gamma 156) were rounded with blebs over the cell surface and showed decreased surface area. The relative expression of beta- to gamma-actin protein decreased for both transfectant types. The total actin protein levels remained constant for the gamma 22 cells but decreased for the gamma 156 cells. The results indicate that alterations to transfectant cell morphology can be influenced by the presence or absence of different noncoding regions in the transfected gamma-actin gene. The mechanisms by which noncoding regions of the gamma-actin gene influence the impact of the gene are unknown. Nevertheless, these noncoding regions are isoform specific and highly conserved in evolution. We propose that the functional significance of the different actin isoforms may involve the properties of these noncoding regions in addition to the differences in protein sequence.

Actions of transforming growth factor-beta on muscle cells.

It has recently been reported by three laboratories that transforming growth factor-beta (TGF-beta) is a potent and reversible inhibitor of differentiation in myogenic cells. To improve our understanding of this inhibition, we investigated the effects of TGF-beta on several other processes in L6 myoblasts, with emphasis on actions of the insulin-like hormones (which stimulate myoblast differentiation). We found that TGF-beta had no effect on the binding of insulin-like growth factors (IGFs) to their receptors on the cell surface, and it had little or no effect on some actions of the IGFs. There was essentially no change in the suppression of proteolysis or the stimulation of cell proliferation by IGFs when TGF-beta was also added to the medium. However, there was an effect of TGF-beta on another process stimulated by the IGFs; TGF-beta was an equally active and more potent stimulator of amino acid uptake than was IGF-I, and the stimulation was additive beyond the maximal response attained with IGF-I, suggesting that the two act by different mechanisms. TGF-beta had significant effects on myoblast morphology, causing the formation of abundant stress fibers containing cytoplasmic (but not myofibrillar) actin. Addition of TGF-beta at various times after initiation of differentiation demonstrated that TGF-beta inhibits an early process in differentiation. Thus it appears that the interactions of TGF-beta and the IGFs in myoblasts are complex; in some instances the effects of IGFs are inhibited and in others they are mimicked or are unaffected. It is clear that TGF-beta does not act by simply interfering with IGF binding or blocking early steps in its action on myoblasts.

A fine-structural analysis of normal and modulated cells in myogenic cultures

It has formerly been reported that muscle and nonmuscle progenitor cells in clonal culture could be distinguished from one another on the basis of their distinctive morphologies. In light of recent studies critical of this interpretation, this investigation was concerned with identifying whether one could truly discriminate between myoblasts and fibroblast-like cells. The results reveal that not only is cell morphology a reliable criterion, but myoblasts and fibroblasts can also be distinguished on the basis of their fine-structural characteristics. Cytologically, spindle-shaped myoblasts exhibit an unusually dense concentration of free ribosomes and polysomes, a Golgi of variable dimensions, and a paucity of rough endoplasmic reticulum (RER). Flattened, stellate fibroblasts show an extensive elaboration of RER, multiple Golgi complexes, actomyosin stress fibers, and dense accumulations of intermediate (10 nm) filaments. A third cell type, the mesenchyme-like cell, exhibits the morphology of the fibroblast and has a myoblast-like cytology. The morphological and ultrastructural characteristics of myoblasts are dependent upon culture conditions. In less than optimum culture environments, myoblast morphology, cytology, and differentiation can be reversibly altered or “modulated.” Myoblast modulations induced by both bromodeoxyuridine and by culture conditions which simulate an ischemic environment are analyzed. Myoblast modulation and its implication to muscular dystrophy are discussed.