Calpain 3 mRNA expression in mice after denervation and during muscle regeneration.

Lack of functional calpain 3 in humans is a cause of limb girdle muscular dystrophy, but the function(s) of calpain 3 remain(s) unknown. Special muscle conditions in which calpain 3 is downregulated could yield valuable clues to the understanding of its function(s). We monitored calpain 3 mRNA amounts by quantitative RT-PCR and compared them with those of alpha-skeletal actin mRNA in mouse leg muscles for different types of denervation and muscle injury. Intact muscle denervation reduced calpain 3 mRNA expression by a factor of 5 to 10, while alpha-skeletal actin mRNA was reduced in a slower and less extensive manner. Muscle injury (denervation-devascularization), which leads to muscle degeneration and regeneration, induced a 20-fold decrease in the mRNA level of both calpain 3 and alpha-skeletal actin. Furthermore, whereas in normal muscle and intact denervated muscle, the full-length transcript is the major calpain 3 mRNA, in injured muscle, isoforms lacking exon 6 are predominant during the early regeneration process. These data suggest that muscle condition determines the specific calpain 3 isoform pattern of expression and that calpain 3 expression is downregulated by denervation.

Skeletal muscle regeneration is not impaired in Fgf6 -/- mutant mice.

FGF6 is a member of the fibroblast growth factor family. The Fgf6 gene is almost exclusively expressed in adult and developing skeletal muscle. We have obtained mice deficient in FGF6 by targeting the Fgf6 gene by homologous recombination. We studied regeneration of adult skeletal muscle in Fgf6 -/- mice derived on a standard inbred background. Muscle degeneration was induced by notexin drug or crush injury. The defect in FGF6 did not modify the kinetics of muscle regeneration. We bred Fgf6 -/- mice with mdx dystrophin deficient mice; Fgf6 -/-:mdx and mdx muscles were similar. Our study suggests that FGF6 does not play a role in muscle regeneration, i.e., in satellite cell proliferation and fusion, or that this role is strictly compensated by other factors, possibly other FGFs.

Loss of calpain 3 proteolytic activity leads to muscular dystrophy and to apoptosis-associated IkappaBalpha/nuclear factor kappaB pathway perturbation in mice.

Calpain 3 is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.

Comparison of perinatal and adult multi-innervation in human laryngeal muscle fibers.

The innervation of human laryngeal myofibers appears distinct from that of skeletal myofibers, because some of them exhibit multiple neuromuscular junctions. We attempted to understand the significance of the multi-innervation phenomenon by comparing intrinsic laryngeal muscles obtained from autopsies of a fetus, a stillbirth, and a 7-month-old infant to muscles from adults. In longitudinal sections (40 to 60 microm thick) the cholinesterase sites and the nerve terminals were stained simultaneously for light microscopy. Multi-innervation reached no more than 23% at 7 months, and this percentage decreased notably with age. Until 7 months, end plates were innervated either by single or by multiple axons (unineuronal and polyneuronal innervation), whereas in adults, the pattern was exclusively unineuronal. Moreover, the structure of neuromuscular junctions in the young was limited to 1 spot of acetylcholinesterase, while in adults the end plate zones increased in size and complex axon terminals were observed. These observations are consistent with a delayed maturation of human laryngeal innervation compared to that of skeletal muscles.

Successful myoblast transplantation in fibrotic muscles: no increased impairment by the connective tissue.

BACKGROUND: Implantation of normal myoblasts may eventually be a treatment for inherited myopathies such as Duchenne muscular dystrophy. METHODS: We report a comparative study of the effectiveness on myoblast implantation: (1) into the muscles of young (2 months) mdx mice nonirradiated and noninjected with notexin (group 1), (2) into muscles of old mdx mice (15 months) nonirradiated and noninjected with notexin (group 2), and (3) into muscles of 5 months mdx mice irradiated 3 months before the transplantation (group 3). Roughly 3 million cells were injected with bFGF in the Tibialis anterior. RESULTS: Although mice of groups 2 and 3 had significantly more (P<0.05) fibrotic tissue in their muscles than those of group 1, the transplantation success was not significantly different among the three groups. CONCLUSION: Therefore these results demonstrated that myoblast transplantation can be successful even when there is abundant fibrosis.

Hindlimb immobilization applied to 21-day-old mdx mice prevents the occurrence of muscle degeneration.

Dystrophin-deficient skeletal muscles of mdx mice undergo their first rounds of degeneration-regeneration at the age of 14-28 days. This feature is thought to result from an increase in motor activity at weaning. In this study, we hypothesize that if the muscle is prevented from contracting, it will avoid the degenerative changes that normally occur. For this purpose, we developed a procedure of mechanical hindlimb immobilization in 3-wk-old mice to restrain soleus (Sol) and extensor digitorum longus (EDL) muscles in the stretched or shortened position. After a 14-day period of immobilization, the striking feature was the low percentage of regenerated (centronucleated) myofibers in Sol and EDL muscles, regardless of the length at which they were fixed, compared with those on the contralateral side (stretched Sol: 8.4 +/- 6.5 vs. 46.6 +/- 10.3%, P = 0.0008; shortened Sol: 1.2 +/- 1.6 vs. 50.4 +/- 16.4%, P = 0.0008; stretched EDL: 05 +/- 0.5 vs. 32.9 +/- 17.5%, P = 0. 002; shortened EDL: 3.3 +/- 3.1 vs. 34.7 +/- 11.1%, P = 0.002). Total numbers of myofibers did not change with immobilization. This study shows that limb immobilization prevents the occurrence of the first round of myofiber necrosis in mdx mice and suggests that muscle contractions play a role in the skeletal muscle degeneration of dystrophin-deficient mdx mouse muscles.

Mouse muscle regeneration: an in vivo 2D 1H magnetic resonance spectroscopy (MRS) study.

Muscle degeneration and regeneration were studied by 2D 1H magnetic resonance spectroscopy (MRS) and histological examination, in an experimental model of muscle injury using a myotoxic snake venom, notexin. The injured muscles produced a very specific MRS signal, corresponding to a tri-unsaturated fatty acid (linolenic acid-like) signal, from day 2 to day 9 after injury. The combination of MRS with histology showed that this signal was associated with a mechanism occurring during myoblast fusion to form myotubes. 2D 1H MRS is thus a useful non-invasive tool for detecting muscle regeneration in vivo.

Apparent normal phenotype of Fgf6-/- mice.

To study the role of the sixth member of the FGF (fibroblast growth factor) family whose expression is restricted to skeletal muscle, we have derived mouse mutants with a homozygous disruption of the Fgf6 gene. The animals are viable, fertile and apparently normal, indicating that FGF6 is not required for vital functions in the laboratory mouse.

Innervation of adult human laryngeal muscle fibers.

The innervation of laryngeal muscle fibers was appraised in adult humans. Sixteen intrinsic laryngeal muscles were dissected during the autopsy of 4 adults (41-71 years old). Longitudinal serial frozen sections, 60 microm thick, of the whole muscles were double-stained for cholinesterase activity and axonal visualization. About 945 endplates per muscle were analysed using light microscopy. The neuromuscular junctions were always scattered throughout the whole muscles. Most of the muscle fibers showed a single neuromuscular junction, but multi-innervated fibers were found in all of the muscles. Their number was highest in interarytenoid muscles (21% of all the fibers). The distance between multiple neuromuscular junctions was most frequently less than 150 microm. Two neuromuscular junctions were frequently displayed, opposite one another, particularly in thyroarytenoid muscles, and this unusual feature seems specific for laryngeal muscles. The innervation of all of the muscle fibers was exclusively found to be unineuronal, with multi-innervated fibers being innervated by a single axon. Distal axonal degeneration occurred with aging, resulting in a loss in the number of multi-innervated muscle fibers.

Selective motor hyperreinnervation using motor rootlet transfer: an experimental study in rat brachial plexus.

Misdirection of sensory fibers into motor pathways is, in part, responsible for the poor results obtained after peripheral nerve repair. After avulsion of the C-5 root in rats, the authors connected a C-4 ventral rootlet to the musculocutaneous nerve by means of a sural nerve graft. In this way, they were able to increase the number of regenerating motor fibers and avoid growth of sensory fibers into the nerve grafts. Functional recovery was evaluated electrophysiologically and histologically. The origin of the axons that reinnervated the nerve graft was analyzed by means of morphological studies including retrograde labeling procedures. Motor neurons survived and regenerated after the rootlet transfer and there was no functional impairment. Many neurons were retrograde labeled in the ventral horn and widespread biceps muscle reinnervation was demonstrated with recovery of nearly normal electrophysiological properties. Motor hyperreinnervation of the musculocutaneous nerve was observed. This high degree of reinnervation in a long (40-mm) graft was attributed to the good chance that a muscle fiber can be reinnervated by a motor fiber when the number of regenerating motor neurons is increased and when competitive sensory fibers are excluded from reinnervation.

Angiogenic and inflammatory responses following skeletal muscle injury are altered by immune neutralization of endogenous basic fibroblast growth factor, insulin-like growth factor-1 and transforming growth factor-beta 1.

Injured skeletal muscle degeneration comprises early microvascular changes and inflammatory cell infiltration, possibly under the control of several growth factors. We have studied the role of basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF1), and transforming growth factor beta-1 (TGF beta 1), by injecting specific anti-growth factor neutralizing antibodies into mouse extensor digitorum longus muscle at the time of injury (denervation and devascularization). Four days later, at the height of damaged myofiber phagocytosis, we assessed quantitatively revascularization, phagocytic activity, and inflammation. The immune neutralization of bFGF reduced the number of capillaries, macrophages and mast cells, and delayed necrotic myofiber phagocytosis. The immune neutralization of IGF1 or TFG beta 1 promoted muscle revascularization, macrophage infiltration and necrotic myofiber phagocytosis. While IGF1 neutralization reduced the number of mast cells and did not modify that of T-cells or neutrophils, TGF beta 1 neutralization increased the number of all of these cells. This study strongly suggests differing roles for bFGF, IGF1 and TFG beta 1 in angiogenic and inflammatory responses during muscle degeneration, apart from their known effects on the behaviour of myogenic cells.

Factors inducing mast cell accumulation in skeletal muscle.

It has been suggested that mast cells contribute to the phenotype of dystrophinopathies, but the mechanisms of their recruitment into the skeletal muscle remain hypothetical. The aim of this study is to quantify the presence of mast cells in muscle during the cellular events of myofibre degeneration and regeneration. For this purpose, we compare the mast cell profile in dystrophin-deficient mdx mice in which muscles exhibit spontaneous cycles of degeneration-regeneration from 3 weeks of age, with that in Swiss mice in which muscles were injured either by ischaemia or by notexin injection. Notexin is an A2-type phospholipase that rapidly disrupts myofibre plasma membranes, while ischaemia results in a slower process of degeneration. Both lesions are followed by a successful regeneration. In intact muscles, mast cell counts (mean +/- SEM/mm2) range from 1.8 +/- 1 to 4.3 +/- 1.6. The injection of notexin is far more potent in recruiting mast cells into damaged muscle than is ischaemia (118.5 +/- 13.0 vs 12.3 +/- 1.8/mm2). Thus we conclude that the early disruption of the myofibre membrane could elicit mast cell accumulation in skeletal muscle. This may explain the elevated number of mast cells observed in mdx muscles, as dystrophin deficiency is though to induce myofibre membrane leakage. On the other hand, mast cells are more numerous in muscles of young and adult mdx mice that are allowed to regenerate, than in muscles of older animals in which there is little regeneration and fibrosis develops. In injured muscles, the peak of mast cell number is at the onset of regeneration (by day 3 after notexin injection, and by day 11 after ischaemia), rather than during the phase of myofibre necrosis. Therefore, we suggest that the mast cells, through the effects of released mediators, could contribute to muscle regeneration.

Basic fibroblast growth factor promotes in vivo muscle regeneration in murine muscular dystrophy.

Due to the lack of dystrophin, a subsarcolemmal protein, mdx mutant mice undergo spontaneous rounds of myofiber necrosis-regeneration from the age of weaning. Muscle regeneration is likely to be controlled by basic fibroblast growth factor (bFGF) which is detectable in regenerating areas of mdx muscles. Moreover, the proliferation of mdx satellite cells seems to be particularly sensitive to bFGF in culture. We injected various concentrations of bFGF in the tibialis anterior muscle of 4-week-old mice at the time of the first round of muscle necrosis-regeneration, and we evaluated the in vivo effects of bFGF on mdx muscle regeneration by quantitative histology. Seven days after bFGF injection, the number of regenerated myofibers was significantly increased proportionally to the injected bFGF concentration. This effect was due to an enhanced replication of muscle satellite cells as shown by labeling the proliferating cells with 5-bromo-2'-deoxyuridine (BrdU). These results may provide a possibility of improving dystrophin-deficient muscle regeneration by increasing the availability of bFGF.

Morphological and functional study of extensor digitorum longus muscle regeneration after iterative crush lesions in mdx mouse.

The regenerative capacity of mdx Extensor Digitorum Longus (EDL) muscle after iterative muscle crush injuries was examined and compared with that of age-matched control C57BL/10 mice. Muscle crush injuries were performed at 8 weeks and repeated at 12 and 16 weeks. Contralateral non-crushed EDLs from mdx and C57BL/10 mice were used as internal controls for histopathology, histoenzymology, morphometry and for the study of the contractile properties. Morphological examinations were performed at 12, 16 and 20 weeks, respectively one month after a single, a second or a third crush. Contractile properties were studied at 12 to 20 weeks. By 20 weeks, no difference in the number of fibres with internal nuclei could be observed between crushed EDL from both strains, and non-crushed mdx EDL; the area and the diameter of crushed EDL from mdx mice were, respectively, 1.5- and 1.2-fold higher than the ones from crushed EDL from C57BL/10 strain. By 20 weeks, diameter distribution of crushed EDL muscles from C57BL/10 mice were shifted towards smaller fibre diameter, whereas in mdx mice, diameter distribution of crushed EDL muscles paralleled that of non-crushed EDL muscles. By 20 weeks, crushed mdx and C57BL/10 EDL muscles produced 77 and 47% of normalized tetanus tension respectively of non-crushed mdx and C57BL/10 EDL muscles. Following crush injury, both 12- and 20-week mdx and C57BL/10 EDL exhibited a slowed time to peak (TTP) and half-relaxation time (H1/2R) of twitch. There was no difference in posttetanic potentiation between the different groups. Crushed EDL of both strains showed an increased resistance to fatigue, compared to the non-crushed controls. The present study provides morphological and functional evidence for the greater recovery of mdx muscle compared to C57BL/10 muscle following iterative crush injury; however, the recovery does not completely prevent the appearance of necrosis/regeneration features.

The cellular events of injured muscle regeneration depend on the nature of the injury.

The cellular events of muscle degeneration and regeneration and their time course were studied in two experimental models of muscle injury mice; (i) the denervation-devascularization (DD) of the extensor digitorum longus (EDL) muscle, which is an ischaemic lesion; (ii) the injection of notexin (NOT), a snake venom, in the tibialis anterior (TA) muscle, resulting in a toxic lesion. Compared to the ischaemic lesion, the toxic lesion was characterized by a more extensive inflammatory infiltrate and a shortened phase of phagocytosis of the damaged myofibres. This allowed the proliferation and differentiation of muscle precursor cells (mpc) to take place earlier and may be further promoted by growth factors released by inflammatory cells. Compared to DD-EDL, NOT-TA showed also a greater conservation of the basement membranes of the necrotic myofibres, that can support the fusion of mpc into myotubes, and a better microvascularization. The onset of muscle regeneration is tightly related to the events which occur during the phase of degeneration.

Effects of treadmill exercise and high-fat feeding on muscle degeneration in mdx mice at the time of weaning.

1. Dystrophin-deficient hindlimb muscles of mdx mice undergo necrosis at the time of weaning when the motor activity of the mice greatly increases and muscle energy metabolism becomes more dependent on insulin and carbohydrates. 2. We have attempted to determine if the onset of myofibre necrosis in mdx mice at the time of weaning is related to the development of motor activity and/or the change in diet. 3. Fourteen-day-old mdx mice were divided into two groups after weaning. One group was trained to run on a treadmill and the other group was kept on a high-fat diet. Muscle necrosis was assessed histologically in the soleus and extensor digitorum longus muscles of mice in both experiments. 4. Keeping mice on a high-fat milk diet from the time of weaning up to 42 days of age did not influence the occurrence of necrosis in the soleus and extensor digitorum longus muscles of the mdx pups. In contrast, treadmill exercise greatly increased necrosis in both muscles. 5. We conclude that an increase in motor activity exacerbates the degeneration of hindlimb muscles of mdx mice at the time of weaning.

Age-related differences in regeneration of dystrophic (mdx) and normal muscle in the mouse.

The mdx mouse, a genetic homologue of human Duchenne muscular dystrophy (DMD), has been attributed with a greater regenerative capacity of its skeletal muscles. Here, we have tested the hypothesis that muscles of mdx mice regenerate better than those of nondystrophic animals. We studied muscle regeneration resulting from a denervation-devascularization injury (DD) of extensor digitorum longus muscle (EDL) at 3 weeks and 2 months in mdx and wild-type (C57BL/10) mice. Histological and morphometrical studies of muscle regeneration were made from 3 to 180 days later. When DD was performed in 3-week-old C57BL/10 mice, the percentages of nonperipheral nuclei in regenerated fibers decreased progressively over 3 months. This decrease did not occur in animals where DDs were performed at 2 months, suggesting that two different populations of muscle precursor cells are mobilized in muscle regeneration in mice at these two ages. Moreover, mdx EDL muscle regenerated similarly to the controls for up to 60 postoperative days, as shown by distribution of mean diameters and percentage of nonperipheral nuclei of muscle fibers. After 60 postoperative days, necrosis/regeneration characteristics of mdx muscles recurred, suggesting that mdx-regenerated muscle fibers remain susceptible to degeneration.

Phenotype of dystrophinopathy in old mdx mice.

BACKGROUND: Mdx mutant mice, like patients with Duchenne Muscular Dystrophy (DMD), lack dystrophin, a subsarcolemmal protein, that results in myofiber necrosis. However young mdx mice, in contrast to DMD children, exhibit a successful muscle regeneration and not an extensive fibrosis. METHODS: Old mdx mice were monitored clinically up to their spontaneous death, and most of their organs were studied histologically to look for differences with those of the wild C57BL/10 mice strain. RESULTS: In old mdx mice (at least 20 months of age), we report clinical and pathological features of muscular dystrophy, i.e., progressive motor weakness and loss of myofibers replaced by extensive connective tissue, similar to the phenotype of dystrophinopathy observed in DMD patients. Various degrees of dystrophic involvement were observed in cardiac, respiratory, postural, and hindlimb skeletal mdx muscles and also in smooth muscles of the digestive and urinary tracts. No gross histological abnormalities were found in other tissue than muscular tissue. CONCLUSIONS: Late in life, mdx mice develop a muscular dystrophy close to DMD dystrophinopathy. We suggest that the study of the effects of ageing in mdx mice would give clues to better understand the pathophysiology of DMD.