Parabiotic reinnervation in normal and dystrophic mice. Part 1. Muscle weight and physiological studies.

A technique called parabiotic reinnervation has been developed. This technique consisted of suturing the sectioned peripheral end of the common peroneal nerve of a normal mouse, strain 129 ReJ +/+ to that of the distal stump remaining after sectioning that same nerve in a dystrophic mouse, strain 129 ReJ dy/dy. The animals were then parabiosed and allowed to recover and thus it was possible to innervate a dystrophic muscle with a normal nerve and vice-versa. This made it possible to test the hypothesis that a derangement of the trophic function normally exerted by a nerve on a muscle might be an element in the pathogenesis of muscular dystrophy in the adult mouse. Studies on the weights and isometric twitch characteristics of such parabiotically reinnervated muscles led to the conclusion that a derangement of the trophic function exerted by a nerve on a muscle is not an element in the pathogenesis of muscular dystrophy in the adult 129 ReJ dy/dy strain of mouse.     

Response of normal and dystrophic muscles to increased functional demand

Immature fast muscles appear to be unfavorably influenced by excessive activity and there is evidence suggesting that the maturation of muscles from animal models of muscular dystrophy and patients suffering from Duchenne dystrophy is impaired. We therefore examined the effects of chronic functional overload applied at different stages of postnatal life on a fast muscle in normal and dystrophic mice (C57B1/6J dy2j/dy2j). "Overload" of tibialis anterior muscle was produced by removal of its synergist extensor digitorum longus in one hind limb. Our results suggest that increased functional demand can be damaging to immature muscles and that in dystrophic animals, the inability to adjust to overload persists into adult life.     

Muscular transverse relaxation time measurement by magnetic resonance imaging at 4 Tesla in normal and dystrophic dy/dy and dy(2j)/dy(2j) mice

Muscular transverse relaxation times values were measured in vivo in normal mice (strain C57BL6/J, n=14) and in murine models of human congenital muscular dystrophy (dy/dy, n=9; dy(2j)/dy(2j), n=8). A single-slice multi-echo sequence was used. Gastrocnemius/soleus complex, thigh and buttock muscles were studied. Muscular transverse relaxation times values were compared between different muscle groups in each type of animal and between animal groups. Differences were observed between normal and dy(2j)/dy(2j) mice from 3 to 12 weeks of age, and between normal and dy/dy mice at 6 weeks. In specific age ranges, the values of muscular transverse relaxation times in two dystrophic models are different from those in normal mice, and could thus be used as an index of modifications in dystrophic muscle to evaluate therapies.     

Is there a maturation defect related to calcium in muscle mitochondria from dystrophic mice and Duchenne and Becker muscular dystrophy patients?

In our study, mitochondria were isolated from skeletal muscle in 2-, 3-, 4-, 6-, 8-, and 12-week-old normal (C57BL6j dy/+), and 4-, 8-, and 12-week-old dystrophic (C57BL6j dy/dy) mice and in normal subjects and patients with Duchenne or Becker muscular dystrophy. A deficit was observed in a calcium-specific mitochondrial protein in the very young control mouse, compared with the adult mouse. In the adult dystrophic mouse this deficit was found in clinically affected hindleg muscles as well as in apparently normal front leg muscles; it was also found in quadriceps muscles from patients with Duchenne and Becker muscular dystrophy. It is not observed in normal adult mice or in normal subjects. The body of our results suggests that in the forms of muscular dystrophy studied there would be a maturation defect in this calcium-binding mitochondrial protein (“calmitine”), a defect which might be generalized in the entire skeletal muscle system and conceivably could be the cause of muscle degeneration in certain myopathies such as Duchenne and Becker muscular dystrophy.     

Increased T3 deiodination in dystrophic and neonatally denervated mouse muscle

T₃, free iodide levels and morphological features were compared in normal, 129 ReJ dy/dy dystrophic and neonatally denervated skeletal muscles of mice, killed 6 hr after a single intraperitoneal injection of iodine-labelled hormone.Both the dystrophic and the denervated muscles were found to contain equal amounts of T₃ but more iodide than the normal specimens.Increased iodide levels are considered to reflect augmented T₃-catabolism by muscle dehalogenase and may indicate low free hormone levels in the soluble enzyme fraction of the cell, despite its normal overall T₃ content. The close light-microscopic resemblance of 129 ReJ dy/dy dystrophy and neonatal denervation is confirmed. The possibility is discussed that one or several of the muscular dystrophies are forms of target organ hypothyroidism.     

Potassium and caffeine contractures in limb muscles of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice

The strength of contractures, produced by 15 to 146 mM [K]0 (as L-glutamate), was measured in isolated small bundles of muscle fibers from the fast-twitch extensor digitorum longus and from the slow-twitch soleus of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice. The analysis of the relation between the maximal amplitude of the contracture vs the membrane potential and the time constant of relaxation of the K-contractures has shown that dystrophy induced an attenuation of the differences between fast- and slow-twitch muscles. The repriming of K-contractures was more affected by changes in [Ca]0 in normal soleus than in normal extensor digitorum longus and this difference was unaffected by dystrophy. For both types of muscles, the ability of caffeine to produce contractures was reduced in dystrophic muscle and this modification was not related to a change in the fiber typing.     

Effects of slow frequency electrical stimulation on muscles of dystrophic mice.

The hind leg muscles of dystrophic mice (C57 BL dy2J/dy2J) wer chronically stimulated at 10 Hz for 30 minutes six times a day. After 14 days of such activity a clinical improvement in the use of the stimulated leg was noticed. The twitch and tetanic tensions developed by the stimulated tibialis anterior and extensor digitorum longus muscles were higher than those developed by the control, unstimulated muscles on the contralateral side. Histochemically visualised activity of the oxidative enzyme succinic dehydrogenase was greater in fibres of the stimulated muscles. The stimulated muscles contained more muscle fibres than unstimulated controls. It is concluded that slow frequency activity has a beneficial effect on muscles of dystrophic mice.     

Study of neurotrophism in normal/dystrophic parabiotic mice

The trophic influences of nerve and muscle on one another were studied in normal and dystrophic littermates of C57BL/6J dy^2J mice parabiosed at 20 to 23 days after birth. Each parabiont had a soleus muscle cross-reinnervated by a tibial nerve of its partner. Ultrastructural abnormalities of muscle and endplate were quantified and compared 6 to 7 months postoperatively. The dystrophic nerve degenerated despite reinnervation to a normal muscle. The normal muscle did not prevent the dystrophic nerve from degenerating, and the dystrophic nerve induced degenerative changes in the reinnervated normal muscle. Normal nerve did not retard the genetically programmed degeneration of the dystrophic muscle. The dystrophic muscle, however, did not appear to cause normal nerve terminals to degenerate. We conclude that both nerve and muscle cells in dystrophic mice express characteristics of muscular dystrophy. Muscle fibers of a few motor units further suffer from abnormal neurotrophic influence because of the degeneration of the motor neurons. Myotrophic influence on nerve was not observed.     

Immunoblot analysis of sarcoplasmic calcium binding proteins in Duchenne muscular dystrophy

The Western blotting technique was used to detect parvalbumin and S-100 protein in muscles from 10 Duchenne muscular dystrophy (DD) patients, 13 patients with other muscle diseases and 5 age-matched healthy subjects. DD muscles were found to contain decreased amounts of parvalbumin and the S-100 protein. The parvalbumin level did not relate to the age of the patients and the stage of the disease. The S-100 protein decreased progressively with the age of the patients. In a very advanced DD case the S-100 protein was present in trace amounts. In other primary myopathies, including Becker dystrophy, and neurogenic muscular atrophy both parvalbumin and S-100 protein levels were similar to that observed in healthy subjects. The decrease in the amount of both calcium binding proteins may contribute to the elevation of free intracellular Ca²⁺ level in the sarcoplasm of dystrophic muscle and would result in abnormalities in processes regulated by these proteins. The mechanism(s) responsible for the decrease of parvalbumin and S-100 protein in DD muscles are discussed.     

Na(+)/Ca(2+) exchange in human myotubes: intracellular calcium rises in response to external sodium depletion are enhanced in DMD

This study aims to investigate the sodium/calcium exchanger expression in human co-cultured skeletal muscle cells and to compare the effects of Na(+)/Ca(2+) exchange activity in normal and dystrophic (Duchenne's muscular dystrophy) human co-cultured myotubes. For this purpose, variations of intracellular calcium concentration ([Ca(2+)](int)) were monitored, as the variations of the fluorescence ratio of indo-1 probe, in response to external sodium depletion. External sodium withdrawal induced [Ca(2+)](int) rises within several seconds in both normal and Duchenne's muscular dystrophy myotubes. These Na(+)-free-induced [Ca(2+)](int) elevations were attributed to the reverse mode of the Na(+)/Ca(2+) exchange mechanism since the phenomenon was dependent on extracellular calcium concentration ([Ca(2+)](ext)), and since it was sensitive to external Ni(2+) ions. Amplitudes of Na(+)-free-induced [Ca(2+)](int) rises were significantly greater in Duchenne's muscular dystrophy cells than in normal ones. Such a difference disappeared when the sarcoplasmic reticulum was pharmacologically blocked, suggesting that the reverse mode of the Na(+)/Ca(2+) exchange mechanism was able to generate enhanced calcium-induced calcium-release in Duchenne's muscular dystrophy myotubes. Immunostaining images of Na(+)/Ca(2+) exchanger (NCX) isoforms, obtained by confocal microscopy, revealed the presence of NCX1 and NCX3 at the sarcolemmal level of both normal and Duchenne's muscular dystrophy myotubes. No differences were observed in the location of NCX isoforms expression between normal and Duchenne's muscular dystrophy co-cultured myotubes.     

Distribution of ten laminin chains in dystrophic and regenerating muscles.

Using immunohistochemical methods, we assessed the distribution of all 10 known laminin chains (alpha1-5, beta1-3, gamma1 and gamma2) in skeletal muscles from patients with Duchenne, congenital, limb girdle, or Emery-Dreifuss muscular dystrophies. The alpha2, beta1 and gamma1 chains were abundant in the basal lamina surrounding muscle fibers in normal controls; alpha1, alpha3-alpha5, beta3, and gamma2 were undetectable; and beta2 was present at a low level. Compared to controls, levels of the alpha5 chain were increased in muscles from many dystrophic patients; levels of beta1 were reduced and/or levels of beta2 were increased in a minority. However, these changes were neither specific for, nor consistent within, diagnostic categories. In contrast, levels of alpha4 were increased in muscles from all patients with alpha2 laminin (merosin)-deficient congenital muscular dystrophy. Loss of alpha2 laminin in congenital dystrophy is disease-specific but some other changes in laminin isoform expression in dystrophic muscles could be secondary consequences of myopathy, denervation, regeneration or immaturity. To distinguish among these possibilities, we compared the laminins of embryonic, denervated, regenerating, and mutant mouse muscles with those in normal adult muscle. Embryonic muscle basal lamina contained alpha4 and alpha5 along with alpha2, and regenerating muscle re-expressed alpha5 but not alpha4. Levels of alpha5 but not alpha4 were increased in dystrophin (mdx) mutants and in dystrophin/utrophin double mutants (mdx:utrn -/-), models for Duchenne dystrophy. In contrast, laminin alpha4 was upregulated more than alpha5 in muscles of laminin alpha2 mutant mice (dy/dy; a model for alpha2-deficient congenital dystrophy). Based on these results, we hypothesize that the expression of alpha5 in many dystrophies reflects the regenerative process, whereas the selective expression of alpha4 in alpha2-deficient muscle is a specific compensatory response to loss of alpha2.     

A preparation for studying dystrophic avian muscle and neuromuscular junctions

The extensor of the second digit of the chicken wing (EDII) is a small, fast-twitch muscle susceptible to chicken muscular dystrophy and well-suited for correlated studies of the morphology and physiology of identified nerve and muscle fibers. In cross section, the dystrophic EDII show morphological abnormalities common to other well-studied dystrophic chicken muscles. However, in contrast to other dystrophic chicken muscles, living endplates can be viewed in the EDII, facilitating electrophysiological studies. A survey of electrophysiological properties of the EDII muscle in birds 5–16 weeks ex ovo revealed that compared to normal preparations, the dystrophic preparations had: (1) lower resting potentials, (2) lower miniature endplate potential frequency, (3) abortive nerve-evoked action potentials recorded extrajunctionally, and (4) multiple muscle fiber action potentials to a single depolarizing current pulse.     

Isotonic fatigue in laminin alpha2-deficient dy/dy dystrophic mouse diaphragm

Laminin alpha2 deficiency causes approximately 50% of human congenital muscular dystrophies. Muscle in the corresponding dy/dy mouse model has reduced force but increased fatigue resistance during isometric contractions. To determine whether a similar pattern of alterations is present during isotonic contractions, dy/dy diaphragm was studied in vitro. During 20% load, dystrophic diaphragm had significantly reduced shortening, shortening velocity, work and power deficits, which persisted during the fatigue-inducing stimulation. In contrast, during 40% load, isotonic contractile performance of diseased muscle was impaired only mildly and only for some contractile parameters. At both loads, rate of isotonic fatigue when expressed relative to initial contractile values was similar for dystrophic and normal muscle, or in some instances slightly higher for dystrophic muscle. Therefore, fatigue resistance is considerably impaired during isotonic contractions relative to that reported previously for isometric contractions. This has important implications for increased susceptibility to respiratory failure in laminin alpha2-deficient muscular dystrophy.     

Effect of neonatal denervation-reinnervation on the functional capacity of a 129ReJ dy/dy murine dystrophic muscle

The sciatic nerves of 14-day-old 129 ReJ normal (++) and dystrophic (dy/dy) mice were transected in the mid-thigh region. The cut ends of the nerves were approximated to facilitate regeneration. One hundred days after denervation, contractile properties of denervated-reinnervated, normal and dystrophic extensor digitorum longus (EDL) muscles were compared to age-matched normal and dystrophic muscles. In dystrophic muscle, in vitro twitch and tetanic tensions were reduced, compared to those of normal muscle. The denervation-reinnervation procedure resulted in an increase in these parameters as compared to unoperated dy muscle. These data correlated with increases in total myofiber cross-sectional areas. Twitch contraction time was not significantly affected by the dystrophic condition or by the denervation-reinnervation protocol. Whereas dystrophic muscle had a longer half-relaxation time than normal muscle, denervation-reinnervation of the dystrophic EDL resulted in a significantly faster half-relaxation time. While fatigue resistance was greater in dystrophic muscles than in normal muscle, there was a significant decrease in fatigue resistance in the denervated-reinnervated dystrophic muscle. Transient neonatal denervation results in modification of both the morphological and physiological characteristics of murine dystrophy.     

Muscle regeneration and mitochondrial calmitine increase in the dystrophic dy/dy mouse after intramuscular chlorpromazine injection

We studied the effect of chlorpromazine injection on the gastrocnemius muscles of C57BL/6J dy/dy dystrophic mice. Changes in mitochondrial calmitine concentrations and differences in microscopy studies, fibre typing and morphometry were compared in gastrocnemius muscles of dystrophic and control mice before and 2 and 21 days after injection. In both cases, calmitine reduction associated with muscle degeneration was observed 2 days after drug injection. Calmitine then increased, reaching a level at day 21 nearly identical to that of controls before injection. This increase was associated with muscle regeneration. These results clearly indicate that dystrophic mouse muscle can regenerate calmitine after drug-induced damage.     

Transient and chronic neonatal denervation of murine muscle: a procedure to modify the phenotypic expression of muscular dystrophy

The extensor digitorum longus muscles of 14-d-old normal (129 ReJ ++) and dystrophic (129 ReJ dy/dy) mice were denervated by cutting the sciatic nerve. One denervation protocol was designed to inhibit reinnervation of the shank muscles, the other to promote reinnervation. Chronically denervated muscles (muscles that remained denervated for 100 d after nerve section) exhibited marked atrophy, but the number of myofibers in these muscles (1066 +/- 46 and 931 +/- 62 for the denervated normal and dystrophic muscles, respectively) was similar to the number of myofibers found in age-matched, unoperated normal muscles [922 +/- 28 (Ontell et al., 1984)] and was significantly greater than the number of myofibers found in age-matched dystrophic muscles [547 +/- 45 (Ontell et al., 1984)]. Similar effects on myofiber number were obtained when denervated muscles were allowed to reinnervate. Reinnervation of both normal and dystrophic muscles mitigated the marked atrophy that characterized chronically denervated muscles. The dystrophic reinnervated muscles appeared "healthier" than age-matched, unoperated dystrophic muscles, having 70% more myofibers, less myofiber diameter variability, substantially less connective tissue infiltration, and a greater amount of contractile tissue at their widest girths. The present study demonstrated that it is possible to alter the phenotypic expression of the histopathological changes associated with murine dystrophy, in dystrophic myofibers that are formed during fetal development, by subjecting the muscle to neonatal denervation.     

Glycogen cycle enzymes and cyclic nucleotides in avian muscular dystrophy

Fast-frozen pectoralis muscle samples were taken from normal chickens (lines 200 and 412) and chickens having hereditary muscular dystrophy (line 304). The glycogen phosphorylase activity ratio (activity without AMP/activity with AMP) was significantly greater in dystrophic muscles (0.306 +/- 0.046) than it was in normal muscles (0.090 +/- 0.023). Glucagon treatment did not cause any changes in phosphorylase activity ratios. Isoproterenol treatment of both normal and dystrophic muscles raised the phosphorylase activity ratio of normal muscles to 0.446 +/- 0.054, which was not significantly different from that of the dystrophic muscles. The dystrophic muscles had significantly less glycogen than normal muscles (23.3 +/- 2.8 compared with 36.8 +/- 2.8 mumoles glucosyl units/g of muscle). There was no relationship of muscular dystrophy to total phosphorylase activity (measured in the presence of 1 mM AMP) and to glycogen synthase activities measured without and with glucose 6-phosphate. Normal muscles had 28% less cAMP and 49% less cGMP than dystrophic muscles, but these differences were eliminated by treatment of the chickens with glucagon.     

The absence of dystrophic characteristics in normal muscles successfully cross-reinnervated by nerves of dystrophic genotype: physiological and cytochemical study of crossed solei of normal and dystrophic parabiotic mice.

Dystrophic mice and normal littermates were joined in parabiotic union between 20 to 23 days of age with a cross of the “fast” tibial nerve of one partner to the slow soleus muscle of the other. This preparation allowed us (a) to monitor the “neurotrophic” influences of “fast” nerves on slow muscles and (b) to assess the influence of these nerves on the expression of hereditary muscular dystrophy. Twelve parabiotic pairs examined 3 to 6 months postoperatively reveal that the cross-reinnervated solei, either normal or dystrophic, exhibit post-tetanic potentiation, a mean 30% decrease in contraction time, and a mean 48% decrease in half-relaxation time as compared to the contralateral control muscles which were self-reinnervated. Self-reinnervated dystrophic solei generate 60% less tension than do self-reinnervated normal solei. The contractile capabilities of solei of the dystrophic mice cross-reinnervated by nerves of the normal partners are not enhanced, nor are these parameters reduced in normal solei receiving nerves of dystrophic genotype. Instead, cross-reinnervated solei exhibit twitch and tetanic tensions similar to those of their contralateral control muscles. Cytochemical and structural analyses indicate that the crossed “fast” nerves of normal or of dystrophic genotype are effective in altering the cytochemical pattern of the slow muscles to fiber types characteristic of fast muscles. However, normal nerves innervating solei of the dystrophic parabiont do not arrest the progress of the disease, and nerves of dystrophic genotype innervating muscles of the normal parabiont do not induce a pathological state. The results indicate that the peripheral motor nerves of dystrophic mice are normal in exerting “neurotrophic” influences and that muscular dystrophy progresses in spite of the presence of normal “neurotrophic” influences. This unique approach of a double nerve cross achieved through parabiosis gives strong evidence that the etiology of hereditary muscular dystrophy in this species is not nerve mediated.     

Parabiotic reinnervation in normal and dystrophic mice. Part 2. Morphological studies.

The technique of parabiotic reinnervation has been used to test directly the neurogenic theory of the aetiology of muscular dystrophy in mice. Dystrophic muscles contain significantly fewer muscle fibres than their normal controls; they also have a much broader spectrum of fibre size because of a much higher proportion of very small fibres and are poorly differentiated into histochemical fibre types. These criteria were used to assess whether there was any amelioration of the dystrophic process in response to the introduction of a normal nerve supply, or whether dystrophic changes were induced in normal muscle reinnervated with a dystrophic nerve. Self-reinnervated normal and dystrophic TA and EDL muscles contained the same numbers of fibres as unoperated controls. The process of parabiosis alone resulted in no changes in normal or dystrophic muscles. In the process of parabiotic reinnervation, the efficiency of the reinnervation process was not affected by the parabiotic state. The parabiotic reinnervation of dystrophic muscle by normal nerve resulted in no significant increase in fibre numbers and the spectrum of fibre sizes was essentially the same as in unoperated dystrophic muscle. The parabiotic reinnervation of normal muscle by dystrophic nerve resulted in a reduction of fibre numbers in only some of the muscles examined. However, the spectrum of fibre diameters remained essentially normal, and the differentiation of the fibres into histochemical fibre types was characteristic of reinnervated normal muscle. There was a marked absence of necrosis or of other histological signs of dystrophy in these muscles. Since there was no positive evidence to show that conversion of normal to dystrophic, or dystrophic to normal muscle occurred under the influence of parabiotic nerve transposition, two alternative conclusions were admissible. Firstly, the influence of dystrophic nerve upon muscle may be operative in fetal or neonatal life and may be irreversible by means of the subsequent introduction of a normal nerve supply. Secondly, the dystrophic state in muscle may be determined by genetic factors independent of nerve supply.