Creatine kinase and aldolase isoenzyme transitions in cultures of chick skeletal muscle cells

Changes in the isoenzyme patterns and activities of the two enzymes creatine kinase (CPK) and fructose diphosphate aldolase have been followed during the course of differentiation of chick skeletal muscle cells in vitro. The characteristic isoenzyme transitions of both of these enzymes known to occur in developing muscle in situ can be demonstrated in extracts of cultured myogenic cells by cellulose polyacetate electrophoresis followed by specific enzymatic staining: MM-CPK replaces the embryonic BB-CPK, while aldolase isoenzymes containing A subunits replace the C-containing forms which predominate at earlier stages. The specific activities of both enzymes increase during in vitro differentiation. Although the major part of these concomitant changes occurs after myoblast fusion has reached a maximum level, analysis of their timing relative to the process of fusion indicates that the increases in the activities of both enzymes, as well as the accumulation of nuclei within myotubes, proceed exponentially from the beginning of the second day in culture. Fusion and enzyme accumulation are unaffected by addition of dibutyryl cyclic AMP (1 × 10^?4M) to the medium. In calcium-deficient medium, or in media containing 5-bromodeoxyuridine (BrdUrd) at concentrations from 0.2 to 7 × 10^?5M, fusion is almost completely blocked, while cell viability is maintained. The CPK and aldolase isoenzyme transitions fail to occur normally in both fusion-preventing media. This blockage of the normal differentiative changes is, however, less complete in the calcium-deficient cultures, which, in contrast to the BrdUrd containing cultures, contained a number of long bipolar cells thought to be able to differentiate without fusion. These results are interpreted as indicating that for most, but possibly not for all, myogenic cells in typical primary muscle cell cultures, fusion is a prerequisite for the parallel differentiative changes in CPK and aldolase isoenzymes. The possibility is discussed that a “cluster” of proteins, including CPK and aldolase, may be coordinately regulated during myogenesis.     

Differentiation in cultures derived from embryonic chicken muscle: II. Phosphorylase histochemistry and fluorescent antibody staining for creatine kinase and aldolase

The presence or absence of five proteins (glycogen phosphorylase, aldolase A, aldolase C, creatine kinase M, creatine kinase B) in the various classes of cells found in primary cultures derived from embryonic chick breast muscle was investigated using cytological staining methods. Histochemical staining for phosphorylase and indirect fluorescent antibody staining for aldolase A and C as well as for creatine kinases M and B showed the following: All five proteins were found in the many myotubes present in standard medium cultures and in the very few myotubes found in cultures containing 5-bromodeoxyuridine (10^?5M). The elongated bipolar cells prevented from fusing in medium containing EGTA also contain all five proteins. The flattened myogenic cells that predominate in the 5-bromodeoxyuridine-treated cultures contain no phosphorylase or creatine kinase M, though many of them contain creatine kinase B and aldolases A and C. These results are interpreted as indicating that: (1) phosphorylase and creatine kinase M, but not aldolase A, are suitable all-or-none markers for terminal muscle differentiation; (2) the small amounts of creatine kinase M detected in electrophoreses of 5-bromodeoxyruridine-treated cultures can be accounted for by the few myotubes present and are not due to “protodifferentiation” of large numbers of cells; (3) proteins typical of differentiated muscle are produced only in cells that have passed through the last step in myogenesis that is susceptible to 5-bromodeoxyuridine inhibition, and (4) if fusion is blocked by reducing the concentration of calcium ions, accumulation of characteristic muscle proteins can continue in those cells that have initiated terminal differentiation.     

Differential effects of calcium ion concentration on cell fusion,cell division and creatine kinase activity in muscle cell cultures

Cell fusion, cell number, soluble cell protein and creatine kinase activity have been measured simultaneously in chick muscle cell cultures exposed to various calcium ion concentrations for various periods of time, by adding either extra calcium chloride or the calcium-chelating agent, EGTA. Up to 0.75 mM EGTA cell fusion is not inhibited, but the specific activity of creatine kinase is reduced by 20–50%. Between 0.75 and 1.7 mM EGTA, cell fusion is gradually abolished and the increase in cell number prevented, but enzyme specific activity actually increases again and returns to control values. Adding extra Ca²⁺ produces small increases in cell fusion and soluble cell protein, but much greater increases in creatine kinase activity. EGTA stimulates thymidine incorporation into DNA at low concentrations and then inhibits again as its concentration is increased further. These effects of EGTA on cell division may be related to its effects on creatine kinase. The implications of these results are discussed in terms of current ideas about the inter-relationships between cell fusion, cell division and the accumulation of muscle proteins during differentiation. In particular they show that cell fusion is not essential for the attainment of normal levels of creatine kinase.     

Multiple effects of interferon on myogenesis in chicken myoblast cultures

Effects of chicken interferon on the differentiation of chicken skeletal muscle in vitro were examined. Continuous treatment of chicken myoblast culture with 200 IU/ml of interferon (10 IU/mg protein) resulted in significant inhibition of cell fusion and subsequent myotube formation. However, treatment of myoblast culture with 2 to 200 IU/ml of interferon increased activities of creatine kinase and myokinase in 4- or 6-day cultured muscle cells in a dose-dependent fashion. The effect of interferon on myokinase was less than on creatine kinase. Three-fold increase in creatine kinase activity induced by interferon was not accompanied by the accelerated transition of creatine kinase isozyme from BB- to MM-type. On the other hand, accumulation of acetylcholinesterase in interferon-treated cells at day 6 was suppressed to nearly half the level of control cells. Rates of actin and myosin synthesis in 4-day cultures estimated by pulse-labelling with [35S]methionine were also suppressed to 85% of control cultures. However, a proportion of 35S-labelled actin and myosin in labelled proteins associated with glycerinated cells was not changed by interferon treatment. These results indicate that partially purified interferon has multiple effects on the process of the myogenic differentiation of chicken myoblast in vitro.     

Recombinant platelet-derived growth factor-BB stimulates growth and inhibits differentiation of rat L6 myoblasts.

We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.     

Characterization of sarcoplasmic reticulum in skinned muscle cultures

The plasma membranes of chick or rat skeletal muscles, grown in cell culture, were made permeable with saponin in a solution lacking calcium. The cells were then supplied with a medium resembling the cytosol and the ATP-dependent Ca2+ sequestration was performed. Based on the low concentration of free Ca2+ in the medium (below 5 microM), the presence of mitochondrial inhibitors and the effect of drugs that interfere with sarcoplasmic reticulum (SR) function, we assume that the measured Ca2+ accumulation expresses SR function on the saponin-treated myotubes. The development of the SR in muscle cultures is augmented as myogenesis proceeds and depends on its occurrence. Whereas creatine kinase activity is elevated immediately following cell fusion, there is a delay of at least 1 day between myoblast fusion and the increase in Ca2+ accumulation in the SR. Thyroxine or triiodothyronine caused an inhibition of Ca2+ accumulation in rat or chick muscle cultures. This inhibition could explain some of the muscle abnormalities caused by excess of thyroid hormones. A comparison was made between a white-type (fast) and heterogeneous muscle, differentiated in cell culture. There was no significant difference in SR function, indicating the important role of innervation in specifying the properties of muscle fiber types.     

Mitochondrial development during myogenesis.

The enzymatic and ultrastructural pattern of mitochondrial differentiation was investigated during myogenesis. Succinate cytochrome C reductase (SCR), a mitochondrial enzyme complex, increased in activity in developing chick thigh muscle in vivo and in vitro. SCR increase in vitro occurred subsequent to myoblast fusion and correlated with the period of increasing creatine phosphokinase (CPK) activity. Fusion-arrest and Ca²⁺-reversal experiments indicated an apparent coordination between CPK and SCR enzymatic increases and fusion. Analysis of SCR activity in fibroblast cultures suggested that the enzymatic increases observed in differentiating muscle cultures reflected myocyte differentiation, rather than fibroblast contamination or a unique property of the tissue culture environment. Morphological transitions in the myogenic mitochondria were temporally correlated with increased SCR activity. During myogenesis, the mitochondria enlarged in length and volume, exhibited an increase in matrix density, oriented in parallel to the long axis of the myofibrils, and contained increased numbers of parallel cristae. Many mitochondria in fusion-inhibited muscle cultures resembled those found in prefusion myoblasts, although mature mitochondria were observed in some fusion-blocked cells. Quantitative stereological analyses of these mitochondrial changes parallel the biochemical data and suggest that ultrastructural and enzymatic changes in the mitochondria are an integral part of myodifferentiation.     

In Vitro effects of thyroxine and insulin on myoblasts from chick embryo skeletal muscle

The role of insulin and l-thyroxine (L-T₄) in stimulating myoblast proliferation and differentiation was investigated in vitro. A superphysiological concentration of insulin or a physiological concentration of L-T₄ was added to cultures of myoblasts from 11-day-old chick embryo thigh muscle, grown in serum-free DM-153 medium. While the addition of insulin resulted in an increase in the total number of cells, in the extent of fusion, and in the creatine phosphokinase (CPK) activity, myotubes changed into globular structures which tended to degenerate rapidly. On the other hand, while the addition of L-T₄ had less effect on myogenesis, myotubes retained their differentiated state longer. Furthermore, the two hormones exhibited synergistic effects. An increase in the initial cell density resulted in an increase in the amount of protein and CPK activity, irrespective of the presence or absence of the hormones. This suggests that the effect of insulin and L-T₄ on myogenesis is not a differentiation-specific effect, but rather an indirect result of cell proliferation.     

5-Bromodeoxyuridine inhibition of differentiation. Kinetics of inhibition and reversal in myoblasts

This paper describes experiments on the kinetics of inhibition of muscle differentiation in vitro in the presence of 5-bromodeoxyuridine (BrdUrd) and the recovery phenomena that occur when such inhibited cells are permitted growth in normal medium. The studies consist of a quantitation of cell fusion in the presence of the analog and during recovery in its absence coupled with simultaneous studies on changes in buoyant density of cellular DNA. We find that if myoblasts are exposed to BrdUrd during the last doubling before cell fusion would normally occur, most cells do not differentiate, but as many as 18% of the cells can fuse in spite of the incorporation of BrdUrd into their nuclei. These nuclei contain approximately the amount of BrdUrd expected for a full round of DNA synthesis. Studies on the rate of recovery of inhibition of cell fusion following one generation in BrdUrd reveal that after one doubling of inhibited cells in the presence of normal medium. fusion reaches about 50% of the control value; after two doublings it reaches 75% of control value; and after 2.5 doublings of reversal, recovery is essentially complete. We find that both the degree of inhibition after approximately one round of BrdUrd incorporation and the rate of cell differentiation after two generations of reversal are consistent with a model which assumes that BrdUrd “sensitivity” resides on single pair of chromosomes and that inhibition occurs in a dominant fashion if approximately 30% or more of the thymidine is replaced by BrdUrd in the readout strand of either chromosome.     

Developmental changes in sulphation of chondroitin sulphate proteoglycan during myogenesis of human muscle cultures

The synthesis and secretion of chondroitin sulphate proteoglycan (CSPG) was examined in human muscle cultures during myogenesis prior to myoblast fusion and following myotube formation. Results from this study demonstrate that the major CSPG secreted into the medium had a Kav of 0.15 on Sephacryl 500 (exclusion limit of 10(7) Da) and contained predominantly unsulphated residues in mononucleated cell cultures but these became increasingly sulphated in postfusion cultures. Fibroblasts synthesised small amounts of a smaller molecular weight CSPG indicating that the Kav 0.15 proteoglycan is solely synthesised by cells of the myogenic lineage. These findings illustrate that sulphation of CSPG is developmentally regulated during myogenesis of human muscle cells grown under differentiating conditions.     

Creatine kinase, myokinase, and acetylcholinesterase activities in muscle-forming primary cultures of mouse teratocarcinoma cells.

Multipotential mouse teratocarcinoma cells in embryoid bodies were explanted on plastic or collagen substrates. Various modes of cell determination, including myogenesis, occurred. The predominant avenue of differentiation soon became myogenesis: many multinucleated myotubes formed and yielded an extensive network of skeletal muscle fibers. The process does not proceed to normal completion, as the fibers have a paucity of striations and are not contractile. Activities of several enzymes ordinarily associated with muscle differentiation were examined. Acetylcholinesterase activity increases, especially during myotube formation, as in normal myogenesis. However, creatine kinase activity rises during myotube formation and then drops abnormally, and myokinase activity fails to increase appreciably. The fetal isozymic form of creatine kinase is expressed in the cultures, although well differentiated solid tumors taken from mice show attainment of the adult muscle isozyme type if skeletal muscle is demonstrably present. The results are consistent with the interpretation that coordinately regulated changes in gene expressions controlling these functions may be required for later stages of myogenesis.     

Calpain and calpastatin in myoblast differentiation and fusion: Effects of inhibitors

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca²⁺-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-β, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-β and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of μ-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.     

MIR-206 regulates connexin43 expression during skeletal muscle development

Skeletal myoblast fusion in vitro requires the expression of connexin43 (Cx43) gap junction channels. However, gap junctions are rapidly downregulated after the initiation of myoblast fusion in vitro and in vivo. In this study we show that this downregulation is accomplished by two related microRNAs, miR-206 and miR-1, that inhibit the expression of Cx43 protein during myoblast differentiation without altering Cx43 mRNA levels. Cx43 mRNA contains two binding sites for miR-206/miR-1 in its 3′-untranslated region, both of which are required for efficient downregulation. While it has been demonstrated before that miR-1 is involved in myogenesis, in this work we show that miR-206 is also upregulated during perinatal skeletal muscle development in mice in vivo and that both miR-1 and miR-206 downregulate Cx43 expression during myoblast fusion in vitro. Proper development of singly innervated muscle fibers requires muscle contraction and NMJ terminal selection and it is hypothesized that prolonged electrical coupling via gap junctions may be detrimental to this process. This work details the mechanism by which initial downregulation of Cx43 occurs during myogenesis and highlights the tight control mechanisms that are utilized for the regulation of gap junctions during differentiation and development.     

Phorbol myristate acetate (PMA) fails to prevent the appearance of markers associated with muscle and liver differentiation in culture

The tumour promoter PMA has been shown to both prevent and induce differentiation of a variety of cell types in culture. The reason for its paradoxical effects is not known. However, it is clear that PMA alters the cell membrane and therefore it is possible that PMA may only be effective in instances where differentiation is accompanied by changes to the cell membrane e.g. myoblast fusion during myogenesis. In this study, its effects on myoblast fusion as well as the appearance of the muscle specific isoenzyme of creatine phosphokinase (M-CPK) which is not fusion dependent is examined. It is shown that M-CPK accumulates in myogenic cultures exposed to PMA although fusion is prevented. PMA is also tested in foetal rat hepatocytes which differentiate and acquire the enzyme tyrosine aminotransferase during culture. There is no evidence which suggests that this change is membrane dependent. The tumour promoter does not prevent the accumulation of tyrosine aminotransferase in cultured foetal rat hepatocytes.     

Differences in the Expression and Distribution of Flotillin-2 in Chick,Mice and Human Muscle Cells

Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.     

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.     

Long-term analysis of differentiation in human myoblasts repopulated with mitochondria harboring mtDNA mutations

Short-term analysis of myogenesis in respiration-deficient myoblasts demonstrated that respiratory chain dysfunction impairs muscle differentiation. To investigate long-term consequences of a deficiency in oxidative phosphorylation on myogenesis, we quantitated myoblast fusion and expression of sarcomeric myosin in respiration-deficient myogenic cybrids. We produced viable myoblasts harboring exclusively mtDNA with large-scale deletions by treating wild-type myoblasts with rhodamine 6G and fusing them with cytoplasts homoplasmic for two different mutated mtDNAs. Recovery of growth in transmitochondrial myoblasts demonstrated that respiratory chain function is not required for recovery of rhodamine 6G-treated cells. Both transmitochondrial respiration-deficient cultures exhibited impaired myoblast fusion. Expression of sarcomeric myosin was also delayed in deficient myoblasts. However, 4 weeks after induction of differentiation, one cell line was able to quantitatively recover its capacity to form postmitotic muscle cells. This indicates that while oxidative phosphorylation is an important source of ATP for muscle development, myoblast differentiation can be supported entirely by glycolysis.     

Detection of myosin in prefusion G0 lizard myoblasts in vitro.

The relationships between withdrawal of myoblasts from the cell cycle, myosin synthesis, and myoblast fusion have been examined in cultures of skeletal muscle derived from the regenerating tail of the lizard Anolis carolinensis. Utilizing both immunocytochemistry and transmission electron microscopy, we have demonstrated the presence of myosin in mononucleated lizard myoblasts which have entered a prefusion G₀ period. A model is presented summarizing our current view of lizard myogenesis in vitro.     

Temperature sensitivity of muscle and neuron differentiation in embryonic cell cultures from the Drosophila mutant, shibire.

The sex-linked temperature-sensitive mutation, shibire^ts1, which causes, at the restrictive temperature, adult paralysis and pleiotropic morphological defects in embryonic, larval, and pupal development, has been shown to exhibit temperature-sensitive inhibition of differentiation in embryonic cultures in vitro. When shi cultures were incubated at 30°C for 24 hr, both muscle and neuron differentiation were inhibited more than 90% compared to control shi cultures incubated at 20°C. Heat shift experiments showed that the temperature-sensitive periods for neuron and muscle differentiation occurred at 11 to 18 and 14 to 16 hr, respectively, where zero time was the initiation of gastrulation in donor embryos. Short heat pulses (4 and 8 hr) which extended into the temperature-sensitive period resulted in moderate inhibition of differentiation; greater inhibition occurred as the duration of the pulses increased. In contrast, heating wild-type Oregon-R cultures at 30°C for 24 hr did not inhibit muscle cell differentiation and inhibited neuron differentiation relatively little. The temperature-sensitive period in shibire for muscle differentiation occurred well after myoblast division, during the period of myocyte elongation, aggregation, and fusion, whereas that for neuron differentiation took place during a period of enzyme synthesis (acetylcholinesterase and choline acetyltransferase) and axon elongation. Thus, the shi temperature-sensitive gene product affects at least two different cell types, in vitro, at different times during differentiation.