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.

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.

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.

Further aspects of muscular dystrophy in mdx mice.

The histopathological aspects of leg muscles in young, adult and old mdx mice were compared. Before 1 yr of age, the mutant showed a vigorous regeneration, following the fibre necrosis, which led to hypertrophic muscles. Hypertrophy resulted from a mixture of hypertrophic and small fibres but not from fibrosis. From the age of 15 months, the muscles progressively atrophied and the fibres were atrophic or split. An important degree of fibrosis occurred particularly in soleus and plantaris muscles. These aspects in old mdx mice are similar to those described in other dystrophinopathies in animals and humans.

The basic fibroblast growth factor-saporin mitotoxin impairs in vivo skeletal regeneration following injury.

Basic fibroblast growth factor (bFGF) is known to promote the proliferation of myogenic cells in vitro and has been detected in regenerating muscle in vivo. We attempted to modify in vivo post-injury muscle regeneration by acting through the potential involvement of bFGF and the presence of bFGF receptors on regenerating myogenic cells. For this purpose, bFGF conjugated to saporin, a potent mitotoxin, was injected in denervated-devascularized extensor digitorum longus of mice. Cytotoxicity occurred in the regeneration process, as shown by the significantly reduced number and the smaller diameter of the regenerating myotubes, probably determined by the binding of the toxic conjugate to bFGF receptors of myogenic cells. This study describes a suitable in vivo model to develop this specifically-mediated cytotoxicity, which offers new perspectives for acting on muscle regeneration disorders.

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.

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.

In vivo 2D 1H NMR of mdx mouse muscle and myoblast cells during fusion: evidence for a characteristic signal of long chain fatty acids.

We have previously demonstrated that 2D 1H NMR is suitable for studying cerebral metabolism. The same technique was used to study the hind leg muscle of normal (C57BL10) and dystrophic (mdx) mice. The results were compared to preliminary results for cultured muscle cells to determine the origin of fatty acid signals.

Muscle regeneration following injury can be modified in vivo by immune neutralization of basic fibroblast growth factor, transforming growth factor beta 1 or insulin-like growth factor I.

Previous in vitro studies pointed out the role played by several growth factors (basic fibroblast growth factor or bFGF, transforming growth factor beta-1 or TGF beta 1, insulin-like growth factor-I or IGF-I) on the proliferation, the differentiation and the fusion of myogenic precursor cells. We attempted to modify the muscle regeneration which follows the denervation-devascularization of extensor digitorum longus in mice, by acting on the growth factors which are possibly involved in this process. The injection of neutralizing antibodies against either bFGF or IGF-I into the muscle at the time of lesion reduced the number and diameter of regenerating myofibres, suggesting a delay in proliferation and/or fusion of activated satellite cells. The neutralization of TGF beta 1 led to an increased number of small regenerating myofibres, which would be due to the promoting effects of the remaining growth factors (i.e. bFGF and IGF-I) on myoblast proliferation. These contrasted results strongly suggest that the growth factors regulate in vivo muscle regeneration and would be accessible tools for future therapy of muscular disorders.

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.

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.

Functional recovery induced by satellite cell grafts in irreversibly injured muscles.

Grafting autologous cultured satellite cells in irreversibly injured rat extensor digitorum longus EDL muscle leads to myofiber regeneration at the grafting site. In this study, we investigated whether cell grafts induced functional improvement and correlated mechanophysiological findings with histological observations. In cell grafted muscles, the number of myofibers did not differ significantly between 2 wk and 3 mo, whereas no regenerating myofibers were observed in ungrafted controls. During this period, the total number of myofibers in the cell grafted muscles represented 48.2-51.9% of that in normal muscles. The mean diameter of regenerated myofibers increased with time, reaching a maximum (32 microns) at the second mo and remained smaller than that of normal myofibers (47 microns). Muscle function was measured by mechanophysiological recordings of muscle response to supramaximal electrical stimulation of the nerve in situ. Cell grafted muscles exhibited a progressive improvement of all contractile parameters. After 3 mo, a 4-fold increase in absolute values of twitch and tetanic tension outputs was measured in cell grafted muscles when compared to ungrafted controls. However, these parameters remained much lower than in normal muscles (23.4% and 22.3% of control, respectively). This study showed that myogenic cell grafts replace degenerated myofibers and form functional myofibers. Functional improvement observed, between 2 wk and 3 mo after cell grafting, correlated with the development, differentiation, and maturation of the regenerated myofibers rather than with an increase in the number of regenerated myofibers.

Electrophysiological study of conditioning lesion effects on rat sciatic nerve regeneration following either prior section or freeze. II. Blocking by prior tenotomy.

The conditioning lesion effects refer to the earlier formation and the accelerated regeneration of axonal sprouts following two successive axotomies. In a previous study, we observed that a prior freeze or a prior cut of rat sciatic nerve resulted in differences in the enhancement of the regeneration rate and the reduction of the initial delay. These differences were interpreted as a possible non-neuronal cells influence on the intrinsic regulation of the conditioning lesion phenomenon. In the present study, we attempted to modify the status of the muscles using tenotomy before the prior nerve injury to determine the respective influence of the muscular cells on conditioning lesion effects. Thus, the conditioning lesion, which was either a cut or a freeze of the tibial nerve at the ankle, was performed 14 days after foot sole muscles were tenotomized, close to their insertion into the calcaneus bone. The test lesion was always a freeze of the sciatic nerve at midthigh performed 7 days following the prior lesion. The elongation of the regenerating sprouts was electrophysiologically evaluated and the regeneration rate as well as the initial delay were calculated by means of regression analysis. Tenotomy did not influence the regeneration as was demonstrated in a group with a single sciatic nerve lesion. In contrast, when prior lesion was performed, the tenotomy prevented both the enhancement of the rate of regeneration and the reduction of the initial delay, whatever was the type of the conditioning lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological study of conditioning lesion effect on rat sciatic nerve regeneration following either prior section or freeze. I. Intensity and time course.

A peripheral nerve lesion performed distally prior to a proximal axotomy is known to result in an increase in the rate of regeneration of both sensory and motor fibres. This phenomenon is called the 'conditioning lesion effect'. The aim of this study was to determine whether or not the kind of the conditioning lesion influences the intensity and the time course of the conditioning lesion effect. The prior lesion was performed on the tibial nerve of rats at the ankle either by cutting the nerve or freezing it by means of a 1 mm diameter liquid nitrogen cryod. At several points in time up to 28 days a second (or test) lesion consisting of a freeze was performed on the sciatic nerve at the middle part of the thigh. The regeneration of the fastest growing fibres of the sciatic nerve was measured electrophysiologically 5, 7 and 9 days after the test lesion. The nerve was surgically removed, immediately mounted in a recording nerve chamber and stimulated proximally to the test lesion. The distance between the test lesion and the most distal point where an evoked nerve potential was detectable was taken as the regenerated nerve length. Then the rate of regeneration was calculated and the initial delay was estimated by means of a linear regression plotting the regenerated nerve lengths against the days of recording. All the results were compared to those of a control group where the test lesion alone was performed. The increase in the maximal rate of regeneration was greater following a prior section (+25%) than following a prior freeze (+12%). Following a prior section, the rate of regeneration began to be significantly increased for a conditioning interval of 4 days, and went on until a conditioning interval of 28 days. By contrast, after a prior freeze the rate of regeneration was significantly increased solely for an interval of 14 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Conditioning lesion effects on rat sciatic nerve regeneration are influenced by electrical stimulation delivered to denervated muscles.

The rate of regeneration and the initial delay of the fastest growing fibers of the rat sciatic nerve were electrophysiologically evaluated after a freeze at mid-thigh. A prior section or a prior freeze at the ankle level increased the rate of regeneration and decreased the initial delay with different magnitudes. These phenomena are named 'conditioning lesion effects'. A daily electrical stimulation transcutaneously delivered to the foot sole muscles from the day following their denervation by the prior lesion did not modify the increased rate of regeneration but prevented the decrease of the initial delay whatever the type of the prior lesion. Therefore, the initiation of earlier sprouting of the parent axons seems to be specifically controlled by a signal associated with muscle denervation properties.

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.

Respective importance of different nerve conduction velocities in leprosy.

Motor and sensory nerve conduction studies were performed in the distal part of the ulnar, median and radial nerves of 12 tuberculoid and 12 lepromatous leprosy patients, compared with 15 normal subjects. Slowing of sensory conduction velocity (SCV) was shown in all nerves with no difference between tuberculoid and lepromatous patients. The radial SCV slowing is correlated (P less than 0.001) with the clinical findings. Impairment of motor distal latencies was observed only in tuberculpoid patients. It is concluded that the radial SCV is the most reliable conduction test and is proposed as an early diagnostic test for leprosy.

The Hoffmann reflex of the soleus muscle. A study in leprosy.

The H reflex of the soleus muscle was used to investigate the monosynaptic reflex arc of two groups of leprosy patients compared with a similar group of normal subjects. The H reflex recordings show two abnormalities: (1) An increase in the latency of the reflex without difference between lepromatous and borderline patients. (2) A decrease of the Hmax:Mmax amplitude ratio more pronounced in the lepromatous group. The discrepancy between these results and the commonly described preservation of the deep tendon reflexes in leprosy is discussed and the hypothesis that leprosy neuropathy would affect all nerve trunks related to blood vessel changes in suggested.

mdx mice show progressive weakness and muscle deterioration with age.

The time-course of degeneration/regeneration was investigated in leg muscles throughout the life of the mdx mutant mouse, which is a biochemical homologue of Duchenne muscular dystrophy (DMD). In young and adult mice (up to 52 weeks old), muscle fibre necrosis was compensated by a vigorous regeneration, but in old mdx mice (65-104 weeks) this regeneration slightly declined, while the necrotic process persisted. Body and muscles weights declined strikingly after 52 weeks. Life span of mdx mutants was reduced in comparison with the control C57BL/10 animals. Immunostaining of old mdx muscles showed clusters of dystrophin-positive fibres. Muscle fibres in old mdx mice showed great variation in size, many being atrophied or split. Endomysial fibrosis became increasingly conspicuous, and there was some accumulation of adipose tissue. These progressive degenerative changes of old mdx mice resemble those found in DMD and imply that basic pathological similarities between the murine and human diseases previously observed in diaphragm of mdx mice may be extended to other skeletal muscles.