Slow muscle myoblasts differentiating in vitro synthesize both slow and fast myosin light chains

Whether fast and slow skeletal muscles of the embryo develop from cells of a common origin or from two separate cellular origins is not known. Recent evidence suggests that prior to innervation all muscles of the embryo are of one type, the fast type, i.e., all synthesize fast but not slow myosin light chains. Innervation has been thought to play the central role in the shift of a fast to a slow muscle. Experiments reported here demonstrate that myoblasts from slow muscle regions of the embryo when isolated in tissue culture differentiate into myotubes which synthesize both fast and slow myosin light chains, and that innervation is not required to initiate slow myosin light-chain synthesis.     

Activation of myosin synthesis in fusing and mononucleated myoblasts

The synthesis of the heavy chain subunit of myosin has been studied in breast muscle myoblasts from embryos of the Japanese quail, Coturnix coturnix japonica, during differentiation of these cells in culture. Specifically, these experiments were done to examine the roles of myoblast fusion and the regulation of myoblast cell division in the control of myosin heavy chain synthesis.The rates of myosin heavy chain synthesis have been quantitated in cultures of fusing myoblasts by measurement of the incorporation of radioactive leucine and valine precursors into myosin heavy chain, and simultaneous determination of the intracellular specific activities of these radioactive amino acids. These measurements demonstrate that, prior to fusion, dividing myoblasts synthesize little, if any, myosin heavy chain, but that during the period of myoblast fusion, myosin heavy chain synthesis becomes activated at least 50 to 100-fold. Myosin heavy chain synthesis was also measured in mononucleated myoblasts inhibited from fusing by the presence of EGTA in the culture medium. These experiments demonstrate that myosin synthesis can be activated in mononucleated myoblasts to reach rates similar to those attained in fused myoblasts. This activation occurs under conditions in which EGTA-inhibited myoblasts were induced to withdraw from the cell division cycle by reducing the concentrations of the serum and embryo extract components of the culture medium or by prior “conditioning” of standard growth medium.These experiments, therefore, establish that the activation of myosin synthesis in breast muscle myoblasts does not require fusion, but indicate that activation is co-ordinated with the withdrawal of myoblasts from the cell division cycle.     

Myoblast differentiation in vitro: morphological differentiation of mononucleated myoblasts.

Phospholipase C from Clostridium perfringens has been shown previously to inhibit the fusion of cultured chick myoblasts without affecting recognition or cell cycle parameters. In this paper we report that the mononucleated myoblasts, in phospholipase C, synthesize thick and thin filaments and organize them into myofibrils, and that T-tubules and sarcoplasmic reticulum differentiate and join in morphologically typical junctions. The structurally differentiated myoblasts can then fuse with one another to form myotubes. We conclude that cell fusion is not necessary for muscle differentiation.     

The dissipative contribution of myosin II in the cytoskeleton dynamics of myoblasts

We have determined the microrheological response of the actin meshwork for individual cells. We applied oscillating forces with an optical tweezer to a micrometric bead specifically bound to the actin meshwork of C2 myoblasts, and measured the amplitude and phase shift of the induced cell deformation. For a non-perturbed single cell, we have shown that the elastic and loss moduli G and G behave as power laws f and f of the frequency f (0.01<f <50 Hz), and being in the range 0.15–0.35. This demonstrates that the dissipation mechanisms in a single cell involve a broad and continuous distribution of relaxation times. After adding blebbistatin, an inhibitor of myosin II activity, the exponent of G decreases to about 0.10, and G becomes roughly constant for 0.01<f<10 Hz. The actin meshwork appears less rigid and less dissipative than in the control experiment. This is consistent with an inhibition of ATPase and reduction of the gliding mobility of myosin II on actin filaments. In this frequency range, the actomyosin activity appears as an essential mechanism allowing the cell to adapt to an external mechanical stress.     

Human platelet myosin. II. In vitro assembly and structure of myosin filaments

We have used electron microscopy and solubility measurements to investigate the assembly and structure of purified human platelet myosin and myosin rod into filaments. In buffers with ionic strengths of less than 0.3 M, platelet myosin forms filaments which are remarkable for their small size, being only 320 nm long and 10-11 nm wide in the center of the bare zone. The dimensions of these filaments are not affected greatly by variation of the pH between 7 and 8, variation of the ionic strength between 0.05 and 0.2 M, the presence or absence of 1 mM Mg++ or ATP, or variation of the myosin concentration between 0.05 and 0.7 mg/ml. In 1 mM Ca++ and at pH 6.5 the filaments grow slightly larger. More than 90% of purified platelet myosin molecules assemble into filaments in 0.1 M KC1 at pH 7. Purified preparations of the tail fragment of platelet myosin also form filaments. These filaments are slightly larger than myosin filaments formed under the same conditions, indicating that the size of the myosin filaments may be influenced by some interaction between the head and tail portions of myosin molecules. Calculations based on the size and shape of the myosin filaments, the dimensions of the myosin molecule and analysis of the bare zone reveal that the synthetic platelet myosin filaments consists of 28 myosin molecules arranged in a bipolar array with the heads of two myosin molecules projecting from the backbone of the filament at 14-15 nm intervals. The heads appear to be loosely attached to the backbone by a flexible portion of the myosin tail. Given the concentration of myosin in platelets and the number of myosin molecules per filament, very few of these thin myosin filaments should be present in a thin section of a platelet, even if all of the myosin molecules are aggregated into filaments.     

Inhibition of non-muscle myosin II leads to G0/G1 arrest of Wharton's jelly-derived mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSCs) have remarkable clinical potential for cell-based therapy. Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) from umbilical cord share unique properties with both embryonic and adult stem cells. MSCs are found at low frequency in vivo, and their successful therapeutic application depends on rapid and efficient large-scale expansion in vitro. Non-muscle myosin II (NMII) has pivotal roles in different cellular activities, such as cell division, migration and differentiation. We performed this study to understand the role of NMII in proliferation and cell cycle progression in WJ-MSCs.MethodsWJ-MSCs were cultured in the presence of blebbistatin, and cell cycle analysis was performed using flow cytometry, proliferation kinetics, senescence assay and gene expression profile using polymerase chain reaction array.ResultsWhen cultured in the presence of blebbistatin, an inhibitor of NMII adenosine triphosphatase activity, WJ-MSCs exhibited dose-dependent reduction in proliferative potential along with increase in cell size and induction of early senescence. Inhibition of NMII activity also affected cell cycle progression in WJ-MSCs and led to an increase in the percentage of cells in G₀/G₁ phase with a corresponding reduction in the percentage of cells in G₂/M phase. Blebbistatin-induced G₀/G₁ arrest of WJ-MSCs was further associated with up-regulation of cell cycle inhibitory genes CDKN1A, CDKN2A and CDKN2B and down-regulation of numerous genes related to progression through S and M phases of the cell cycle.ConclusionsOur study demonstrates that inhibition of NMII activity in WJ-MSCs leads to G₀/G₁ arrest and alteration in the expression levels of certain key cell cycle-related genes.     

In differentiating prefusion myoblasts connexin43 gap junction coupling is upregulated before myoblast alignment then reduced in post-mitotic cells

Previously we have shown that during in vivo muscle regeneration differentiating rat primary myoblasts transiently upregulate connexin43 (Cx43) gap junctions and leave cell cycle synchronously. Here, we studied the temporal regulation of Cx expression in relation to functional dye coupling in allogenic primary myoblast cultures using western blotting, immuno-confocal microscopy and dye transfer assays. As in vivo, Cx43 was the only Cx isotype out of Cx26, 32, 37, 40, 43 and 45 found in cultured rat myoblasts by immunostaining. Cultured myoblasts showed similar temporal regulation of Cx43 expression and phenotypic maturation to those regenerating in vivo. Cx43 protein was progressively upregulated in prefusion myoblasts, first by the cytoplasmic assembly in sparse myoblast meshworks and then in cell membrane particles in aligned cells. Dye injection using either Lucifer Yellow alone, Cascade Blue with a non-junction permeant FITC-dextran revealed an extensive gap junction coupling between the sparse interacting myoblasts and a reduced communication between the aligned, but still prefused cells. The aligned myoblasts, uniformly upregulate p21^waf1/cip1 and p27^kip1 cell cycle control proteins. Taken together, in prefusion myoblasts less membrane-bound Cx43 was found to mediate substantially more efficient dye coupling in the growing cell fraction than those in the aligned post-mitotic myoblasts. These and our in vivo results in early muscle differentiation are consistent with the role of Cx43 gap junctions in synchronizing cell cycle control of myoblasts to make them competent for a coordinated syncytial fusion.     

Analysis of expression of heavy myosin chains during in vitro differentiation of satellite cells and myoblasts derived from rat skeletal muscles

Differentiation of cultured myogenic progenitor cells (satellite cells and mononucleated myoblasts) derived from hindlimb muscles of rat embryos and newborn animals was studied. Immunocytochemical methods and PCR analysis revealed expression of heavy myosin chains at the earliest stages of myogenesis (in mononucleated myoblasts). Expression of the gene encoding the embryonic form of myosin and a low level of expression of the gene encoding perinatal myosin in cultured progenitor cells derived from embryonic muscles was detected by PCR. Cells derived from muscles of newborn animals also expressed these two myosin forms, though at a lower level. The progenitor cells derived from muscles of rat embryos and newborn animals were found to express myosin 2a, which is characteristic of fast-twitch definitive muscle fibers.     

The duration of the terminal G1 of fusing myoblasts

We found earlier that the initiation of fusion in cultures of embryonic myoblasts is accompanied by a marked protraction of G₁, that phase of the cycle to which fusion is restricted. To test the relationship of this increase in G₁ to the appearance of multinucleated cells we have compared the distribution of the length of G₁ in cycling myoblasts (at both prefusion and fusion stages) to the G₁ preceding fusion. Our data indicate that myoblasts spend a minimum of 4 hr in G₁ before fusing. Although 87.5% of cycling myoblasts in fusion-stage clones satisfy this minimum, only 17.5% of the myoblasts at prefusion stages would be competent by this criterion. Based on these percentages, the probability of contact between competent cells is 54-fold greater at fusion than at prefusion stages suggesting that fusion is initiated when a large enough fraction of the cycling myoblasts spends more than 4 hr in G₁. The fact that the graph of terminal G₁s exhibits two distinct peaks suggests that the distribution may be bimodal, the modal value of the first peak being close to the modal value of those myoblasts in fusing cultures which reenter S. Bimodality is confirmed by a transitional probability plot of the data, which may also indicate that, when G₁ exceeds 11 hr the probability of fusing increases. These data are compatible with the concept that myoblasts (of the first mode, at least) fuse in an indeterminate state from which they can, alternatively, reenter S. This may also be true of two-thirds of the myoblasts of the second mode the terminal G₁s of which fall within the limits of G₁ of fusion-stage cycling myoblasts.     

Cell-free translation of mammalian myosin heavy-chain messenger ribonucleic acid from growing and fused-L6E9 myoblasts.

An mRNA-dependent reticulocyte cell-free protein synthesizing system very efficient in the translation of myosin heavy-chain mRNA from a rat myogenic cell line is described. This system exhibits a high degree of fidelity with regard to the spectrum and relative proportion of the different proteins synthesized from a sample of cytoplasmic RNA as compared to the proteins synthesized in vivo by the cells from which the RNA is prepared. The main feature of this system is the use of a K+ and Cl- concentration similar to those of the reticulocyte cytoplasm. Using this system, myosin heavy chain, identified by low-salt precipitation, electrophoretic mobility, and partial peptide analysis, represents 17% of the total protein synthesis when cytoplasmic RNA from well-fused L6E9 cells is used. Furthermore, when RNA preparations from growing myoblasts, that when analyzed in other cell-free translational systems seem not to contain any myosin heavy-chain mRNA, are tested in the system reported here, they are proven to contain high amounts of translatable myosin heavy-chain mRNA.     

Alignment of myoblasts on ultrafine gratings inhibits fusion in vitro

During development, skeletal muscle precursor cells fuse to form multi-nucleated myotubes. However, it is unclear how this fusion is regulated such that linear myotubes are produced. In a previous study, we found that linear arrays of myoblasts cultured on micropatterns of laminin fused to form linear myotubes of a constant diameter, independent of the width of the laminin track. This suggested that a mechanism exists to prevent myoblasts from fusing laterally [Exp. Cell Res. 230 (1997) 275]. In this study, we have investigated this further by culturing myoblasts on ultrafine grooved surfaces previously shown to align fibroblasts and epithelial cells. We found that all the individual myoblasts were highly aligned along the groove axis, and time-lapse recordings showed that motility was mostly restricted to a direction parallel to the grooves. In contrast to the previous study, however, there was a strong tendency for early differentiating cells to form aggregates either at an angle of approximately 45 degrees or perpendicular to the groove axis. Nevertheless, we rarely saw myotubes formed at those angles, supporting our earlier idea that the ability of cells to fuse laterally is prohibited. Our data strongly suggest that myoblasts are most likely to fuse in an end-to-end configuration, and it is this that enables them to form linear, rather than irregular myotubes.     

Detection of myosin heavy chain mRNA during myogenesis in tissue culture by in vitro and in situ hybridization

A cDNA copy of purified chick embryonic skeletal myosin heavy chain mRNA (MHC mRNA) distinguished between myogenic and nonmyogenic cells compared by in vitro and in situ hybridization. The majority of cells in replicating mononucleate myogenic cell cultures contained no detectable MHC mRNA. Among the earliest cells to contain MHC mRNA were cells engaged in mitosis. A relatively large amount of MHC mRNA was found in postmitotic monucleate cells and myotubes, and we observed nucleolar localization of MHC mRNA in these cells.