Myogenic cells of regenerating adult chicken muscle can fuse into myotubes after a single cell division in vivo

Autoradiographic studies were carried out on regenerating muscles of adult chickens. Three different muscles of hens were injured, and tritiated thymidine (1 microCi/g) was injected at various times after injury to label replicating muscle precursors. Detailed comparisons of grain counts over premitotic nuclei in samples removed one hour after injection of tritiated thymidine, and of postmitotic myotube nuclei in samples removed 10 days after injury (when labeled precursors had fused to form myotubes), revealed how many times some labeled precursors had divided before fusing into myotubes. DNA synthesis in muscle precursors was initiated 30 h after injury. Grain counts of myotube nuclei indicated that many muscle precursors labeled at the onset of myogenic cell proliferation had divided only once, or twice, before fusing into myotubes. The relationship of these in vivo results to the cell lineage model of myogenesis is discussed.     

Myogenic cells of regenerating adult chicken muscle can fuse into myotubes after a single cell division in vivo

Autoradiographic studies were carried out on regenerating muscles of adult chickens. Three different muscles of hens were injured, and tritiated thymidine (1 μCi/g) was injected at various times after injury to label replicating muscle precursors. Detailed comparisons of grain counts over premitotic nuclei in samples removed one hour after injection of tritiated thymidine, and of postmitotic myotube nuclei in samples removed 10 days after injury (when labeled precursors had fused to form myotubes), revealed how many times some labeled precursors had divided before fusing into myotubes. DNA synthesis in muscle precursors was initiated 30 h after injury. Grain counts of myotube nuclei indicated that many muscle precursors labeled at the onset of myogenic cell proliferation had divided only once, or twice, before fusing into myotubes. The relationship of these in vivo results to the cell lineage model of myogenesis is discussed.     

Reutilisation of tritiated thymidine in studies of regenerating skeletal muscle

Summary Two different aspects of tritiated thymidine (³H-Tdr) reutilisation in skeletal muscle were examined. Injection of a high dose (7 Ci/g) of ³H-Tdr into mice prior to crush injury of skeletal muscle resulted in heavy labelling (grain counts) of myotube nuclei 9 d later. In contrast, myotube nuclei were essentially unlabelled when a low dose (1 Ci/g) of ³H-Tdr was injected at similar times with respect to injury. It was concluded that labelling seen after the high dose was due to reutilisation of ³H-Tdr. (Such ³H-Tdr reutilisation can account for the results of Sloper et al. (1970) which previously supported the concept of a circulating muscle precursor cell.) When replicating muscle precursors were labelled directly with ³H-Tdr 48 h after injury, the percentages of labelled myotube nuclei and the distribution of nuclear grain counts were similar with either high or low dose.We also investigated whether the light labelling seen in regenerated myotube nuclei after 9 d, when ³H-Tdr had been injected before the onset of myogenesis (as found by McGeachie and Grounds 1987), was due to ³H-Tdr reutilisation or, alternatively, to proliferation of local cells in the wound which subsequently gave rise to muscle precursors. Labelling of myotube nuclei was compared in mice injected with ³H-Tdr either 2 h before, or 2 h after injury. In another experiment, mice were injected 12 h after injury and lesions sampled 1, 12 or 36 h later, to see whether local cells were replicating 12 h after injury, and what labelled cells subsequently entered to wound. No difference was found in myotube labelling between mice injected before or after injury, and no cells replicating locally in the wound at 12 h after injury were observed. The results clearly show that the light labelling was due to ³H-Tdr reutilisation.     

A comparison of muscle precursor replication in crush-injured skeletal muscle of Swiss and BALBc mice

Summary Muscle precursor replication in Swiss mice, in which muscle regeneration is exceptionally vigorous, was compared with previous data for regeneration in BALBc mice. The tibialis anterior muscles of 23 male and 15 female inbred Swiss SJL/J mice were crush injured, and tritiated thymidine injected into mice at various times after injury to label replicating muscle precursors. Lesion samples were taken 10 days after injury, processed for autoradiography, and grain counts of myotube nuclei analysed. Muscle regeneration was more vigorous in male compared with female Swiss mice, and in both was strikingly greater than that in BALBc mice in which there was extensive fibrous connective tissue throughout the lesions. Autoradiographic analysis showed that muscle precursor replication started at 24 hours in Swiss mice, 6 hours earlier than the onset at 30 hours in BALBc mice. Muscle precursor replication appeared to be more active 96 hours after injury in female Swiss compared with male BALBc and male Swiss mice respectively, although numbers of precursor cells replicating at other times were similar. It is not known whether the slight difference in onset of muscle precursor replication can alone account for the more complete muscle regeneration seen in Swiss mice. Similar studies were carried out in 11 male and 10 female F₁ hybrid (SJL/J x BALBc) mice. Analysis of labelled myotube nuclei showed that muscle precursors did not synthesise DNA prior to 30 hours after injury, and regeneration resembled that of the parental BALBc strain.     

An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats

Summary Satellite cells were traced autoradiographically during the regeneration of skeletal muscle in young Sprague-Dawley rats. Approximately 31% of the satellite cells in uninjured muscles appeared labelled after three injections of tritiated thymidine; none of the myonuclei were labelled in the same muscles. Four to six days after transplanting the radioactive muscles to non-radioactive littermates, regenerating myotube nuclei in the host appeared labelled. Thus, this study confirms that satellite cells in young rats can differentiate into multinucleated myotubes following muscle injury.Supported by NIH grant No. 5 S01-RR05356-13I wish to acknowledge the excellent technical assistance of Ms. Amy Erisman     

Multinucleation during myogenesis of the myotome of Xenopus laevis: a qualitative study

Ultrastructural studies of myogenesis in the myotome of Xenopus laevis reveal that the myotubes developed by stage 33/34 have peripheral myofibrils but are still uninucleate with a single large nucleus. By stage 45, the cytoplasm of the muscle cells is filled with myofibrils and there are many small peripheral nuclei, resulting in multinucleate muscle fibres. With the electron microscope, we have examined myotomes from stages 33/34 to 59 of development and some stages were also investigated by autoradiography. There was no evidence from autoradiographic studies for DNA synthesis in muscle cells, and the increase in the number of myonuclei was accompanied by a decrease in their size. Satellite cells were not seen at the myotube stage but were first seen after the cells had become multinucleate, with many small nuclei close together forming rows. Constrictions were frequently observed in the large single nuclei. It is concluded that division of the myonuclei by amitosis is mainly responsible for the multinucleation that occurs during development of the myotome muscle in Xenopus laevis.     

Initiation and duration of muscle precursor replication after mild and severe injury to skeletal muscle of mice

Summary We test the proposal (McGeachie and Grounds 1985) that myogenesis following severe (crush) injury is prolonged compared with minor (cut) injury. Forty-four mice were injured with a cut and a crush lesion on different legs, and tritiated thymidine was injected at various times after injury (0 to 120 h), samples of regenerated muscle were taken 9d after injury and autoradiography was used to determine the initiation of muscle precursor replication, and duration of proliferation after the two different injuries.In both lesions replication of potential myoblasts was initiated 30 h after injury. Myogenesis was essentially completed in cut lesions by 96 h after injury, although the peak was finished by 60 h. In contrast, significant muscle precursor replication in crush lesions was still occurring 96 h after injury, and myogenesis was almost finished by 120 h. The pronounced difference in duration of myogenesis in different lesions strongly supports the original proposalThe extended duration of myogenesis in crush lesions, in conjunction with tritiated thymidine reutilisation, appears to account for conflicting experimental results in support of the concept of a circulating muscle precursor cell.     

Myonuclear birthdates distinguish the origins of primary and secondary myotubes in embryonic mammalian skeletal muscles

Myotubes were isolated from enzymically disaggregated embryonic muscles and examined with light microscopy. Primary myotubes were seen as classic myotubes with chains of central nuclei within a tube of myofilaments, whereas secondary myotubes had a smaller diameter and more widely spaced nuclei. Primary myotubes could also be distinguished from secondary myotubes by their specific reaction with two monoclonal antibodies (MAbs) against adult slow myosin heavy chain (MHC). Myonuclei were birth dated with [3H]thymidine autoradiography or with 2-bromo-5'-deoxyuridine (BrdU) detected with a commercial monoclonal antibody. After a single pulse of label during the 1-2 day period when primary myotubes were forming, some primary myotubes had many myonuclei labelled, usually in adjacent groups, while in others no nuclei were labelled. If a pulse of label was administered after this time labelled myonuclei appeared in most secondary myotubes, while primary myotubes received few new nuclei. Labelled and unlabelled myonuclei were not grouped in the secondary myotubes, but were randomly interspersed. We conclude that primary myotubes form by a nearly synchronous fusion of myoblasts with similar birthdates. In contrast, secondary myotubes form in a progressive fashion, myoblasts with asynchronous birthdates fusing laterally with secondary myotubes at random positions along their length. These later-differentiating myoblasts do not fuse with primary myotubes, despite being closely apposed to their surface. Furthermore, they do not generally fuse with each other, as secondary myotube formation is initiated only in the region of the primary myotube endplate.     

Enhanced susceptibility of DNA-synthesizing nuclei of epithelial cells of the intestine to acid hydrolysis

Summary Albino mice were injected intraperitoneally with tritiated thymidine and killed 1/2 and 30 h later. Pieces of ileum were excised and fixed. Tissue sections were hydrolyzed with 5 N HCl at 21° C for 1/2, 1, 2, 3, 4 and 6 h, some sections remained unhydrolyzed. Both the hydrolyzed and nonhydrolyzed sections were autoradiographed. Grain counts per labelled nucleus of either cryptal (DNA-synthesizing) or villous (DNA-nonsynthesizing) cells nonhydrolyzed and hydrolyzed for various time intervals were recorded.The results indicate, that the grain count of nonhydrolyzed, labelled nuclei from cryptal cells was by 1.49 higher than that of villous cells demonstrating the rate of grain counts diminution caused by cell divisions.Hydrolysis caused a diminution of grain count of cryptal cells by approximately 15% higher than that of the grain count of villous cells.Financial support was partially obtained from grant MR II.1.We wish to thank Dr. W. Sawicki for his supervision, advice and encouragement throughout this work     

Nuclear movement during myotube formation is microtubule and dynein dependent and is regulated by Cdc42, Par6 and Par3

Cells actively position their nucleus within the cytoplasm. One striking example is observed during skeletal myogenesis. Differentiated myoblasts fuse to form a multinucleated myotube with nuclei positioned in the centre of the syncytium by an unknown mechanism. Here, we describe that the nucleus of a myoblast moves rapidly after fusion towards the central myotube nuclei. This movement is driven by microtubules and dynein/dynactin complex, and requires Cdc42, Par6 and Par3. We found that Par6β and dynactin accumulate at the nuclear envelope of differentiated myoblasts and myotubes, and this accumulation is dependent on Par6 and Par3 proteins but not on microtubules. These results suggest a mechanism where nuclear movement after fusion is driven by microtubules that emanate from one nucleus that are pulled by dynein/dynactin complex anchored to the nuclear envelope of another nucleus.     

Muscle tissue regeneration of the lymph hearts in adult Rana temporaria frogs. A study by light and electron microscopy autoradiography methods

The regeneration response of adult frog lymph heart muscle tissue was studied from 2 to 3 weeks after mechanical injury. High resolution autoradiographic studies showed that regenerative necrotic zones have many actively proliferating mononuclear cells deprived of cytoplasmic myofilaments. Some of them have numerous free ribosomes, so they might be identified as myoblasts. On the 13th day after injury newly-formed myotubes with chains of myonuclei and pictures of active sarcomerogenesis were observed. On the other hand, the surviving muscle fibers of the perinecrotic zone were rich in myonuclei at their growing ends. In the vicinity of nuclei, accumulation of a mass of non-differentiated cytoplasm rich in free ribosomes and polysomes, rough endoplasmic reticulum, Golgi apparatus, and centrioles are seen. Tritiated thymidine pulse-labeling showed that only rare myonuclei of the perinecrotic zone muscle fibers were labeled, whereas numerous non-differentiated cells of granulation tissue and myosatellites incorporated thymidine. The number of labeled myonuclei markedly increased 96 hours after 3HTdr administration. These data evidence that the myoblastic mechanism is predominant in the regeneration of adult frog lymph heart muscle tissue. It is necessary to emphasize that during the lymph heart muscle tissue reparative myogenesis some of the perinecrotic myonuclei are able to synthesize DNA and to divide mitotically, which distinguishes this type of muscle from skeletal muscle tissue of vertebrates.     

Synthesis of Mitochondrial DNA Precursors during Myogenesis,an Analysis in Purified C2C12 Myotubes

During myogenesis, myoblasts fuse into multinucleated myotubes that acquire the contractile fibrils and accessory structures typical of striated skeletal muscle fibers. To support the high energy requirements of muscle contraction, myogenesis entails an increase in mitochondrial (mt) mass with stimulation of mtDNA synthesis and consumption of DNA precursors (dNTPs). Myotubes are quiescent cells and as such down-regulate dNTP production despite a high demand for dNTPs. Although myogenesis has been studied extensively, changes in dNTP metabolism have not been examined specifically. In differentiating cultures of C2C12 myoblasts and purified myotubes, we analyzed expression and activities of enzymes of dNTP biosynthesis, dNTP pools, and the expansion of mtDNA. Myotubes exibited pronounced post-mitotic modifications of dNTP synthesis with a particularly marked down-regulation of de novo thymidylate synthesis. Expression profiling revealed the same pattern of enzyme down-regulation in adult murine muscles. The mtDNA increased steadily after myoblast fusion, turning over rapidly, as revealed after treatment with ethidium bromide. We individually down-regulated p53R2 ribonucleotide reductase, thymidine kinase 2, and deoxyguanosine kinase by siRNA transfection to examine how a further reduction of these synthetic enzymes impacted myotube development. Silencing of p53R2 had little effect, but silencing of either mt kinase caused 50% mtDNA depletion and an unexpected decrease of all four dNTP pools independently of the kinase specificity. We suggest that during development of myotubes the shortage of even a single dNTP may affect all four pools through dysregulation of ribonucleotide reduction and/or dissipation of the non-limiting dNTPs during unproductive elongation of new DNA chains.     

An autoradiographic study of the role of satellite cells and myonuclei during myogenesis in vitro

Satellite cells and myonuclei of neonatal rat muscles were differentially labeled with 3H-thymidine according to the procedure of Moss and Leblond (1971). Minced muscles fragments containing either labeled satellite cells or labeled myonuclei were cultured until multinucleated myotubes grew out from the explants. Reutilzation of isotope released from degenerating nuclei was competitively inhibited by using a culture medium containing excess (0.32-0.41 mM) cold thymidine. after an 8-10 day growth period, the explants were fixed and prepared for autoradiographic (ARG) examination to determine whether labeled satellite cells or myonuclei had contributed to the myonuclear population of the developing myotubes. Counts were made of the number of labeled myotubes in the explants and compared with the number of labeled satellite cells and myonuclei in samples of the original muscle tissues fixed at the time of explantation. The original muscles showed a mean satellite cell labeling index of 51.7% and gave rise to myotubes with a mean labeling incidence of 40%. In contrast, myonuclear labeling in the original muscle tissues showed no correlation with subsequent myotube labeling. Only 3.4% myotube labeling was found in explants in which over 30% of the original tissue myonuclei had been labeled. Under conditions controlled for isotope reutilization, these observations confirm results of in vivo ARG studies indicating that satellite cells are the only significant source of regenerating myoblasts in injured muscle tissue.     

Immunocytochemical localization of transient DNA strand breaks in differentiating myotubes using in situ nick-translation

We have localized DNA strand breaks during in vitro chicken myogenesis by repairing nicks in nuclei of fixed cell monolayers in situ with biotin-11-dUTP, followed by immunocytochemical detection of incorporated biotin with rabbit anti-biotin and FITC-labeled goat anti-rabbit antibodies. No accumulations of biotin sufficient for immunocytochemical detection were observed in 23-hr cultures of dividing cells. In 33- and 43-hr cultures, biotin was first detected in only 3% of the nuclei, all of which appeared to be in fusing myoblasts or small myotubes. In contrast, cultures of young, highly fused myotubes (56 hr) exhibited 18% biotinylated nuclei; virtually all of these nuclei, most of which were grouped as aggregates, were within myotubes. In older cultures (73 and 94 hr) incorporation of biotin into myotube nuclei markedly decreased, while increases were noted in nuclei of mononuclear cells. These results indicate that extensive single-stranded DNA nicking occurs in nuclei of young myotubes, followed by repair as terminal differentiation ensues.     

Can cells extruded from denervated skeletal muscle become circulating potential myoblasts?

Summary The hypothesis that satellite cells which leave denervated skeletal muscle might become circulating potential myoblasts which could participate in myogenesis in distant sites in the body has been tested.Sixteen mice had one hindlimb denervated and were given 7 daily injections of ³H-thymidine (³H-Tdr). One day later extensor digitorum longus muscle isografts from unlabelled mice were inserted into each hindlimb. As controls, the procedure was repeated in 6 non-denervated labelled mice. Fourteen days after their insertion, isografts in denervated mice contained many labelled myotubes with a labelling index of 55±4% (mean±SEM). In the control isografts in non-denervated mice, 38±4% of myotube nuclei were labelled. The results show that either labelled cells, or ³H-Tdr, had transferred from the host to isografts in both cases. The probability of ³H-Tdr reutilization was demonstrated in regenerating livers of 8 similarly labelled mice, where 34±3% hepatocytes adjacent to crush lesions were labelled after 14 days. This conclusion was reached because only 2–3% of normal hepatocytes incorporate ³H-Tdr under these conditions and this population is inadequate to provide sufficient labelled precursor cells for the large numbers of labelled regenerated hepatocytes. Therefore, it was concluded that ³H-Tdr reutilization is the most likely explanation for labelled myotube nuclei in the muscle isografts (rather than movement of labelled precursor cells), and that additional label for reutilization had been derived from breakdown of labelled cells in denervated muscle. The data do not support the hypothesis of a circulating precursor for skeletal muscle cells.     

ABSENCE OF EFFECT OF MULTIPLE INTRAPERITONEAL INJECTIONS ON CELL PROLIFERATION, AND ESTIMATION OF THE CELL CYCLE TIME IN THE EPITHELIUM OF THE OESOPHAGUS AND FORESTOMACH IN MICE, HAMSTERS AND RATS

Counts of labelled epithelial nuclei in mice given single or multiple injections of tritiated thymidine (to label cells synthesizing deoxyribonucleic acid), either alone or after 24 or 48 hr multiple injections of water, showed that multiple intraperitoneal injections did not disturb normal cell proliferation. The rate of cell proliferation was the same in the epithelium of the oesophagus and forestomach, and in these epithelia there was no difference between mice, hamsters and rats. Cell cycle times were estimated in these epithelia from the number of nuclei labelled in animals given single or multiple injections of tritiated thymidine.