Retarded myogenic cell replication in regenerating skeletal muscles of old mice: an autoradiographic study in young and old BALBc and SJL/J mice

The patterns of skeletal muscle precursor cell replication after crush injury were compared by the use of autoradiographic techniques, in young (4-week-old) and old (39-week-old) BALBc and SJL/J mice. Similar comparisons were made between cut and crush lesions in old BALBc muscle. Muscle precursor cell replication commenced at 18–24 h after injury in both young and old muscles from both strains of mice. In young BALBc muscle the peak of myogenic activity at 60 h was 36 h earlier than in old mice. SJL/J muscle responded more rapidly than did BALBc: in young SJL/J the peak myogenic activity was at 46 h (14 h earlier than in young BALBc muscle), and in old SJL/J muscle the peak activity at 72 h was 24 h earlier than in old BALBc muscle. In all mice (both young and old) myogenic cell replication was substantially reduced by 120 h after injury. A comparison of the timing of muscle precursor cell replication in cut and crush lesions in old BALBc mice revealed a more rapid response in the cut lesion: this difference between the lesions in comparable with data from identical lesion in 6-8-week-old BALBc mice (McGeachie and Grounds 1987). However, the peak of myogenic replication in the older mice in the present study was some 26–36 h later than in the younger 6-8-week-old mice. These experiments show that, whilst muscle precursor cell replication commences at approximately the same time (about 24 h) after injury in young and old mice, the peak level of activity is delayed by some 24–36 h in old mice. In addition, the SJL/J mouse strain responds more rapidly and prolifically to muscle injury than does the BALBc strain.     

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.     

A model of myogenesis in vivo,derived from detailed autoradiographic studies of regenerating skeletal muscle,challenges the concept of quantal mitosis

Summary We have recently shown that myogenesis following severe injury is prolonged compared with minor injury (McGeachie and Grounds 1987). In this previous autoradiographic study 44 mice were injected with tritiated thymidine at various times after muscle injury (0 to 120 h), and samples were taken 9d after injury to determine the percentage of labelled myotube nuclei. In the present study the same experimental data are analysed in detail to reveal how many times labelled muscle precursors divided before fusing to form myotubes.Additional mice were prepared and samples removed 1 h after injection of tritiated thymidine to determine the maximum grain counts of premitotic nuclei. When a labelled premitotic nucleus divides, each of the two daughter nuclei will contain half of the original label. The grain counts of nuclei resulting from sequential divisions of a maximally labelled premitotic nucleus, forms the basis for our detailed analysis which can reveal how many times a muscle precursor has divided after labelling.Nine days after injury the autoradiographic grain counts of labelled myotube nuclei were analysed in detail. The results describe an in vivo model of myogenesis which we use to evaluate quantitatively observations derived from tissue culture studies. The analysis shows that, at the onset of myogenesis in regenerating muscle (30 h after injury), muscle precursors divide only twice before fusing to form myotubes. This observation challenges the concept of quantal mitosis as defined by the tissue culture studies of Quinn et al. (1984, 1985).     

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.     

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.     

Gene expression profile of adhesion and extracellular matrix molecules during early stages of skeletal muscle regeneration

Skeletal muscle regeneration implies the coordination of myogenesis with the recruitment of myeloid cells and extracellular matrix (ECM) remodelling. Currently, there are no specific biomarkers to diagnose the severity and prognosis of muscle lesions. In order to investigate the gene expression profile of extracellular matrix and adhesion molecules, as premises of homo‐ or heterocellular cooperation and milestones for skeletal muscle regeneration, we performed a gene expression analysis for genes involved in cellular cooperation, migration and ECM remodelling in a mouse model of acute crush injury. The results obtained at two early time‐points post‐injury were compared to a GSE5413 data set from two other trauma models. Third day post‐injury, when inflammatory cells invaded, genes associated with cell‐matrix interactions and migration were up‐regulated. After day 5, as myoblast migration and differentiation started, genes for basement membrane constituents were found down‐regulated, whereas genes for ECM molecules, macrophage, myoblast adhesion, and migration receptors were up‐regulated. However, the profile and the induction time varied according to the experimental model, with only few genes being constantly up‐regulated. Gene up‐regulation was higher, delayed and more diverse following more severe trauma. Moreover, one of the most up‐regulated genes was periostin, suggestive for severe muscle damage and unfavourable architecture restoration.     

Skeletal muscle regeneration involves macrophage-myoblast bonding

Regeneration in adult skeletal muscle relies on the activation, proliferation, and fusion of myogenic precursor cells (MPC), mostly resident satellite cells (SC). However, the regulatory mechanism during this process is still under evaluation, with the final aim to manipulate regeneration when the intrinsic mechanism is corrupted. Furthermore, intercellular connections during skeletal muscle regeneration have not been previously thoroughly documented. Our hypothesis was that a direct and close cellular interaction between SC/MPC and invading myeloid cells is a key step to control regeneration. We tested this hypothesis during different steps of skeletal muscle regeneration: (a) the recruitment of activated SC; (b) the differentiation of MPC; (c) myotubes growth, in a mouse model of crush injury. Samples harvested (3 and 5 days) post-injury were screened by light and confocal microscopy. Ultrastructural analysis was performed by conventional transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) followed by 3D modeling of electron tomography (ET) data. This revealed a new type of interaction between macrophages and myogenic cells by direct heterocellular surface apposition over large areas and long linear distances. In the analyzed volume, regions spaced below 20 nm, within molecular range, represented 31% of the macrophage membrane surface and more than 27% of the myotube membrane. The constant interaction throughout all stages of myogenesis suggests a potential new type of regulatory mechanism for the myogenic process. Thus, deciphering structural and molecular mechanisms of SC-macrophage interaction following injury might open promising perspectives for improving muscle healing.     

Regeneration restores some of the altered electrical properties of axotomized bullfrog B-cells

In bullfrog B-type sympathetic neurones axon injury produces substantial changes in somal membrane properties. These include a shortening of action potential afterhyperpolarization (AHP) and an increase in action potential (AP) duration. In the present experiments we compared two injury situations: nerve crush, which was followed by regeneration, and nerve cut, after which regeneration to the original target was prevented, to investigate whether these electrophysiological changes were related to axon regeneration. Both crush and cut injuries produced a similar maximum decrease in AHP duration (to 33 and 30%) by 14 days after axotomy. After nerve crush, AHP duration recovered to within control values by 42 days, while after cut it remained depressed. AHP amplitude decreased to the same extent after nerve crush or cut (to 62 and 58%), but the rate of decrease was slower following crush when compared with cut, and following both types of injury it still remained depressed at 42 and 49 days. Changes in AP duration also took longer to occur following nerve crush, reaching maximal values at 35-42 days, at which time AHP duration had returned to within the normal range. The early reduction in AHP duration and its rapid recovery in regenerating neurones suggests that the current underlying this membrane property is regulated by events associated with axon outgrowth and peripheral reconnection. In contrast, changes in AHP amplitude and AP repolarization appeared to be independent of the occurrence of axon regeneration and remained abnormal at 49 days despite the recovery of AHP duration. These results imply that the electrophysiological changes seen in B-cells following injury are differentially regulated during subsequent regeneration.     

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

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

Uptake of tritiated thymidine in muscle of juvenile carp

In juvenile carp (4.5–6 cm s.l .) labelled myosatellite cell nuclei are found 24 h after injection of tritiated thymidine, but labelled myonuclei after 48 h, indicating that fish myonuclei do not proliferate but originate from undifferentiated myogenic cells. The time pattern accords with the estimated cell-cycle time of adult myogenic cells of carp.     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.     

Undifferentiated cells in the snail myocardium are capable of DNA synthesis and myodifferentiation

Cellular mechanisms of heart-muscle growth in the snail Achatina fulica have been studied using cytophotometry and electron microscopic autoradiography. Cytophotometric DNA measurements showed that the snail cardiomyocytes are mononucleated cells with diploid nuclei. Ultrastructural analysis of the snail myocardium revealed that, in addition to mature myocytes, it contains small roundish undifferentiated cells (UCs) and poorly differentiated muscle cells. EM autoradiography detected silver grains over the nuclei of UCs 2 h after injection of tritiated thymidine ([(3)H]Tdr), while the nuclei of both mature and poorly differentiated myocytes remained unlabeled. In EM autographs of the myocardial tissue fixed 14 days after [(3)H]Tdr administration, labeled myonuclei were evident, which may suggest some myodifferentiation of prelabeled UCs. Many labeled UCs persist for 14 days after a single [(3)H]Tdr injection, suggesting that not all UCs undergo myodifferentiation after passing through the cell cycle, and that those that do not can enter the next cycle. UCs in the snail myocardium presumably provide not only reserve but also stem cells for myocytes. Thus, the heart muscle of the adult snail consists of mononucleated diploid myocytes with blocked proliferative activity and a renewable population of precursor myogenic cells. The results obtained suggest that the growth of this muscle involves a myoblastic mechanism of myogenesis; this mechanism differs from that of vertebrate cardiac muscle growth, which is non-myoblastic-that is, based on proliferation or polyploidization of cardiomyocytes. Evolutionary aspects of cellular mechanisms of the heart-muscle growth are discussed.     

The role of p53 in vivo during skeletal muscle post-natal development and regeneration: studies in p53 knockout mice

The tumour suppressor gene p53 is recognised as a central regulator of the cell cycle and apoptosis. Post-natally, p53 mutations are associated with many cancers and mice lacking p53 are prone to spontaneous tumour formation. The present study examines skeletal muscle formation in post-natal mice lacking p53 using two different models of skeletal muscle regeneration. The level of endogenous myogenic cell proliferation in mature skeletal muscle was examined and the time course of muscle regeneration after whole muscle transplantation or crush injury were compared in p53 (-/-) and control C57Bl/6J adult mice, using desmin and proliferating cell nuclear antigen (PCNA) immunohistochemistry and histological analysis. The pattern of inflammation, myoblast proliferation and myotube formation in regenerating p53 (-/-) skeletal muscles appears normal and similar to those in control C57Bl/6J muscle. These data indicate that p53 is not required for the regulation of myoblast proliferation, differentiation and myotube formation in vivo during myogenesis of adult skeletal muscle.     

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.     

Exercise-induced satellite cell activation in growing and mature skeletal muscle

The time course and extent of satellite cell activation were studied in the soleus (m-SOL) and extensor digitorum longus (m-EDL) muscles of untrained growing and mature rats after a single bout of prolonged eccentric treadmill running. At 24, 48, 72, and 120 h postexercise, satellite cell mitotic activity was quantitated in autoradiographs of whole-fiber segments after injection of [3H]thymidine. Fiber damage and localization of labeled cells were also examined in muscle cross sections. Labeling in growing muscles progressively increased to peak levels (approximately 250% of control) at 72 h postexercise, whereas mature muscles exhibited an earlier peak (approximately 250% of control) at 24 (m-SOL) and 48 (m-EDL) h, followed by a more rapid decline to control levels by 120 h postexercise. In all exercised muscles the calculated satellite cell activation was far greater than required to repair the small number (less than 3.0%) of necrotic fibers identified at the light-microscopic level. These results suggest that satellite cells were activated not only on fibers exhibiting overt necrosis but also on those with lesions not discernible with light microscopy.     

Cytosolic androgen receptor in regenerating rat levator ani muscle.

The development of the cytosolic androgen receptor was studied after degeneration and regeneration of the rat levator ani muscle after a crush lesion. Muscle regeneration appears to recapitulate myogenesis in many respects. It therefore provides a model tissue in sufficiently in large quantity for investigating the ontogenesis of the androgen receptor. The receptor in the cytosol of the normal levator ani muscle has binding characteristics similar to those of the cytosolic receptor in other androgen-sensitive tissues. By day 3 after a crush lesion of the levator ani muscle, androgen binding decreased to 25% of control values. This decrease was followed by a 4-5 fold increase in hormone binding, which attained control values by day 7 after crush. Androgen binding remained stable at the control value up to day 60 after crushing. These results were correlated with the morphological development of the regenerating muscle after crushing. It is concluded that there is little, if any, androgen receptor present in the early myoblastic stages of regeneration; rather, synthesis of the receptor may occur after the fusion of myoblasts and during the differentiation of myotubes into cross-striated muscle fibres.     

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

Background

Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy.

Methods

Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors.

Results

Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors.

Conclusions

In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.     

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.