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.     

Identification of regenerated dystrophic minced muscle transplanted in normal mice

The origin of the reconstituted normal and dystrophic transplants in normal mice of the Bar Harbor 129 ReJ strain was investigated by transplanting [³H]thymidine-labelled minced tibialis anterior muscles into the legs of unlabelled hosts. After 20 days the transplants were processed for autoradiography and histology. At varying time intervals between 0 and 50 days radioactivity counts were made on the transplants and compared with those from the adjacent EDL and contralateral tibialis anterior muscles of the hosts.Both autoradiography and radioactivity counts showed that the transplanted muscles were formed from muscle cells derived from within the donor tissue. Moreover, normal and dystrophic transplants from normal hosts were histologically similar.     

Membrane elasticity of erythrocytes from normal and dystrophic mice

The membrane deformability of erythrocytes from normal and dystrophic mice was determined using a flow channel technique whereby erythrocytes attached to the floor of a parallel plate channel were deformed by fluid shear forces. A nonlinear stress-strain experimental behavior was observed for both populations of erythrocytes which was best described with a polynominal expression: τ_S = aε_x + [bε_x³/(2ε_x + 1)]. A comprehensive statistical analysis of the data indicated that a large percentage of the variance of the data was due to the experimental design. Furthermore, the 2 populations of cells were different in terms of the strain-stress relationship which best fitted the data, i.e., ε_x = ατ_s + βτ_s² + γτ_s³. Up to a shear stress of 5.5 dyn/cm², where 95% of the data points were found, the dystrophic erythrocytes were slightly but significantly more deformable than the normal erythrocytes.     

Functional properties of muscles transplanted between normal and dystrophic mice

Tibialis anterior muscles were transplanted between 12-week-old normal and dystrophic mice with intact or polydimethyl silicone-capped peroneal nerve. After 150 days the transplants were removed and their isometric twitch contraction properties were studied in vitro at 20 C. Intact normal and dystrophic muscles of equivalent age were used as controls. Dystrophic muscles developed lower twitch and tetanus tension than normal muscles and showed prolonged half relaxation time. The contraction time and twitch/tetanus ratio of both types of muscle were similar. Of all transplantations performed, only those in normal mice with intact nerve responded upon stimulation. Both normal and dystrophic transplants in normal hosts showed similar isometric properties. Although intact dystrophic muscles and viable dystrophic transplants in normal hosts were similar in weight, the transplants developed about three to four times more tension. In addition, dystrophic transplants showed relaxation times similar to normal muscles. It is suggested that the dystrophic lesion in mice may have a neural origin.     

Peripheral mechanisms of fatigue in muscles of normal and dystrophic mice.

We evaluated the contribution of different processes to fatigue of normal and dystrophic mouse muscles using an in vitro electromyography chamber. Fatigue was induced by repetitive nerve stimulation at 30 Hz for 0.5 s, every 2.5 s until tension decreased by about 50%. We monitored the compound nerve action potential (AP), compound muscle AP, and isometric tension responses to nerve stimulation, and compound muscle AP and tension responses to direct muscle stimulation. In normal mice, about 50% reduction in nerve-evoked tension occurred by 2.4 min in extensor digitorum longus (EDL), 4.8 min in diaphragm, and 9 min in soleus. Analysis of the responses revealed that the fatigue was caused by failure of more than one process in all muscles, and failure of nerve conduction did not contribute to fatigue in any muscle. Failure of neuromuscular transmission, muscle membrane excitation, and excitation-contraction (E-C) coupling and contractility accounted for 55, 45, and 0%, respectively, of the fatigue in EDL, for 21, 74, and 5% of the fatigue in diaphragm, and for 2, 54, and 44% of the fatigue in soleus. In dystrophic mice, while about 50% reduction in nerve-evoked tension occurred by 8.1 min in EDL and 5.6 min in diaphragm, only 29% reduction in tension occurred by 80 min in soleus. Failure of neuromuscular transmission, muscle membrane excitation, E-C coupling and contractility accounted for 22, 63 and 15% of the fatigue in EDL, for 21, 79, and 0% of the fatigue in diaphragm, and for 15, 59, and 26% of the fatigue in soleus. The proportion of slow-twitch oxidative fibers was more than normal in dystrophic EDL, but the same as normal in dystrophic diaphragm and soleus. The slower onset of fatigue was attributable to lesser failure of neuromuscular transmission in dystrophic EDL, and to lesser failure of E-C coupling and contractility in dystrophic soleus.     

Intracellular activity of sodium in normal and dystrophic skeletal muscle from mice

Potassium and sodium ion-selective microelectrodes were used in vitro to investigate the depolarization of skeletal muscle fibers associated with muscular dystrophy. In dystrophy there was a large increase of intracellular Na activity and an associated decrease in K activity in fibers of extensor digitorum longus muscles. Despite this, the recorded membrane potential was very close to the calculated potassium equilibrium potential (E_k) in dystrophic fibers. In contrast, in normal muscle fibers, E_m was significantly depolarized with respect to E_K. The data suggest that in dystrophic fibers there is an increase in the relative membrane permeability to potassium over sodium.     

Ouabain binding sites in skeletal muscle from normal and dystrophic mice.

The specific binding of tritiated ouabain was used to estimate the density of Na+-K+-ATPase sites ("Na+-pump" sites) in segments of skeletal muscle from normal and dystrophic mice. Ouabain binding was approximately 4 times greater in red (soleus) muscle than in white (superficial gastrocnemius) muscle from normal animals. In dystrophic soleus muscles, ouabain binding was decreased by nearly one-half. Because Na+-K+-ATPase activity is associated with plasma membranes, these observations constitute further evidence for a sarcolemmal abnormality in dystrophic mice.     

X-ray diffraction from striated muscles and nerves in normal and dystrophic mice

The structure of striated muscle (thick and thin filaments, filament lattice, and collagen), peripheral nerve myelin, and tendon collagen were studied in tissues from dystrophic and normal mice using small-angle x-ray diffraction. There were increases in the amount of disorganized tissue in the dystrophic mice, and the time course of the changes was monitored over the first 42 weeks of life. As the dystrophic mice became older, the contractile apparatus of the muscles appeared to atrophy, while the amount of collagen increased. In general, the molecular structure and packing appeared to remain unchanged as the disease progressed, although changes in the relative amounts and the organization of proteins were noted. In both normal and dystrophic mice, the collagen periodicity (65.7 nm) was 2% smaller when detected in muscle tissue compared with that detected in tendon tissue.     

Axoplasmic flow of protein in the sciatic nerve of normal and dystrophic mice

The axoplasmic flow of proteins has been studied in the sciatic nerve in both normal and dystrophic mice ofter injection of radioactive leucine into the spinal cord. In both normal and dystrophic mice three rates of flow are described corresponding to 2000, 500, and 0.7 mm per day. The flow patterns differ in that in the dystrophic mouse sciatic nerve less material flows at the rate of 2000 mm per day than in the corresponding normal nerve but two or three times more flows at 500 mm per day. Leucine metabolites in both aqueous and lipid solvent extracts follow a similar pattern. The abnormal flow pattern in the dystrophic nerve is discussed in relation to the possible passage of substances with trophic influence along the nerve. The role of substances, supplied to the muscle by axoplasmic flow, which may repress gene expression in the muscle is considered in the light of these findings and those of others which indicate an influence by the nervous system on muscle characteristics.     

Effects of insulin on protein synthesis in muscles from normal and dystrophic mice

Protein synthesis in soleus and extensor digitorum longus (EDL) muscles was measured in vitro to test the hypothesis that the lack of muscle protein accumulation in dystrophic conditions could be caused by a reduced sensitivity to insulin. We demonstrate that physiological insulin concentrations stimulate protein synthesis in soleus muscles from normal mice but not from muscles obtained from dystrophic (dy) animals. The difference is lost at very high insulin concentrations (1 microM) and could not be shown at any concentration with EDL muscles. These results, together with the reported reduced inhibitory effect of insulin on protein synthesis in dystrophic hamsters and on protein breakdown in dystrophic mice, suggest that protein metabolism in certain muscles from dystrophic animals may be less responsive to the anabolic effects of insulin.     

Long-term peripheral nerve and muscle recordings from normal and dystrophic mice

A method for long-term recording of electrical activity from small mammalian nerves and muscles is described. Electrodes for stimulating and recording activity were implanted on nerves and muscles subserving ankle flexion and extension in normal and dystrophic mice. Activity was monitored on a regular basis for up to 200 days following implantation. Neural compound action potentials, compound EMG potentials and twitch tension were recorded. Shortly after implantation, evoked EMG and twitch tension declined, but recovered progressively to values measured at the time of implantation and subsequently remained steady in normal mice. However, while dystrophic mice did recover, with EMG levels reaching 50-60% of the values recorded at implantation, tension eventually dropped to 10% in flexor muscles and 25% in extensors.     

Intracellular activity of sodium in normal and dystrophic skeletal muscle from C57BL/6J mice

Potassium and sodium ion-selective microelectrodes were used in vitro to investigate the depolarization of skeletal muscle fibers associated with muscular dystrophy. In dystrophy there was a large increase of intracellular Na activity and an associated decrease in K activity in fibers of extensor digitorum longus muscles. Despite this, the recorded membrane potential was very close to the calculated potassium equilibrium potential (Ek) in dystrophic fibers. In contrast, in normal muscle fibers, Em was significantly depolarized with respect to Ek. The data suggest that in dystrophic fibers there is an increase in the relative membrane permeability to potassium over sodium.     

Intracellular potassium activities in muscles of normal and dystrophic mice: An in vivo electrometric study

Intracellular potassium ion activity (aK_i⁺) was measured in vivo in gastrocnemius muscle fibers of normal and dystrophic ($\text{dy-2J}\text{dy-2J}$) $\text{C57B1}\text{6}\text{J}$ mice. Rapid measurements were made by means of a double-barrel ion selective microelectrode. The aK_i⁺ in muscles of dy-2J mice was about 8% lower than in muscles of normal mice. This reduction in aK_i⁺ was not sufficient to account for the low resting potential of the dy-2J fibers. The fibers measured were from the superficial region of the muscle, a region previously shown to be composed of fast-twitch fibers. This region shows only minor morphologic changes in young dy-2J mice. The reduction of aK_i⁺ in these fibers is consistent with the hypothesis that there is a general membrane defect associated with muscular dystrophy. It is also suggested, however, that the neurally derived pseudomyotonia associated with the dy-2J mutation and the concomitant increase in the oxidative capacity in the measured muscle fibers might also affect the concentration of intracellular K⁺.     

Myosin isoforms in hindlimb muscles of normal and dystrophic (ReJ129 dy/dy) mice.

Myosin isoform expression was studied in hindlimb muscles of control (Dy/Dy) and dystrophic (dy/dy) mice of the ReJ129 strain during postnatal development. Three myosin heavy chain isoforms (fast II-B MHC, neonatal MHC, and slow or I MHC) were identified using monoclonal antibodies. Only original fibers, i.e., fibers formed during fetal life, were studied. Necrotic and regenerating fibers were excluded. The disappearance of neonatal MHC was found to be delayed in all muscles of dystrophic mice, except the soleus. The fraction of fibers containing I MHC was similar in control and dystrophic animals at all ages, except during the third postnatal week. The developmental increase in the fraction of fibers expressing II-B MHC was interrupted in dystrophic mice by two marked declines. The first occurred during the second postnatal week at the beginning of the main wave of fiber necrosis, and the second occurred at between 30 and 90 postnatal days.     

Intracellular activity of potassium in normal and dystrophic skeletal muscle from C57BL/6J mice

Potassium-sensitive intracellular microelectrodes were used in vitro to investigate the cause of the depolarization of the resting membrane seen in dystrophic skeletal muscle fibers. In dystrophic fibers the membrane potential (E_m) was found to agree closely with the potassium equilibrium potential (E_K), calculated from measurements of intra- and extracellular potassium activity (a_K), whereas the E_m of normal fibers was depolarized with respect to E_K. However, when (a_K)_o was varied the value of E_m in normal and dystrophic fibers did not conform to that predicted from the Nernst equation. Calculations showed that a relatively high membrane permeability to Na could only partly explain the differences. Na loading of fibers indicated that dystrophic fibers may have a greater electrogenic capacity despite a reported decrease in membrane ATPase activity. It was concluded that measurement of the intracellular activities of Na and Cl as well as K is necessary before the depolarization of dystrophic fibers may be fully described. The changes observed were in agreement with the hypothesis that there is a general membrane disorder associated with muscular dystrophy.     

Beta-endorphin decreases fatigue and increases glucose uptake independently in normal and dystrophic mice

beta-Endorphin and a C-terminal analogue have been shown to decrease muscle fatigue and increase glucose uptake in muscles of normal mice. In order to provide evidence whether these peptides might be useful in muscle-wasting conditions and whether the two actions of the peptides are interdependent, the effect of beta-endorphin on muscle fatigue and glucose uptake was studied using isolated hemidiaphragm preparations of dystrophic mice as well as normal mice. Muscle contractions were elicited by high-frequency stimulation of the phrenic nerve. Glucose uptake was measured using (nonmetabolizable) 2-deoxy-D-[1-(3)H]glucose. beta-Endorphin and the C-terminal analogue reduced fatigue in normal muscles of males but not females. Insulin had no effect in either sex. The peptides increased 2-deoxyglucose uptake in contracting and noncontracting muscles of normal males and females. beta-Endorphin reduced fatigue and increased deoxyglucose uptake in dystrophic muscles. The effect on fatigue was not due to increased glucose uptake, as the energy substrate present was pyruvate. Nerve stimulation released beta-endorphin immunoreactivity from intramuscular nerves of dystrophic mice. It is hypothesized that beta-endorphin released from motor nerves as well as from the pituitary could be responsible for improving muscle function during exercise. beta-Endorphin or analogues could have therapeutic use in muscle-wasting disease.     

Effect of denervation on adenine nucleotides in skeletal muscle from normal and dystrophic mice

The effect of denervation on the adenine nucleotide content of fast- and slow-twitch skeletal muscle of the C57BL mouse was studied by high-performance liquid chromatography. From the adenine nucleotide content the energy charge, a measure of high-energy phosphate available to the cell, was calculated. The energy charge of the extensor digitorum longus muscle was significantly higher than that of the same muscle from dystrophic mice (C57BL/6J dy2j/dy2j) and on denervation decreased to the values found in the innervated muscle from dystrophic animals. Denervation of the muscle in dystrophic mice did not change the energy charge of that muscle. The energy charge of the soleus muscle from both normal and dystrophic mice was similar and did not change on denervation. It is proposed that in the dystrophic process a functional denervation of skeletal muscle occurs which preferentially affects fast-twitch muscle, leading to a reduction in the energy charge.     

Developmental changes in succinate dehydrogenase activity in muscle fibers from normal and dystrophic mice

The question of whether or not the development of dystrophic muscles is similar to that of normal muscles, prior to the manifestations of the symptoms of the disease, is investigated here. The developmental change in the activity of succinate dehydrogenase was therefore measured in individual fibers of prospectively dystrophic muscles from 10- to 28-day-old mice (strain C57Bl/6J dy2j) and compared with that of muscles from normal mice of the same age. It was found that up to 10 days of age, muscle fibers from normal and prospective dystrophic animals had low succinate dehydrogenase activities, and were all more or less uniform. Thereafter in the normal muscle the overall activity of the enzyme increased and the fibers became more heterogeneous with age. By 21 days the extensor digitorum longus muscle resembled that of the adult. At that time, fibers from prospectively dystrophic muscles had lower succinate dehydrogenase activities and were more homogeneous. Thus fibers from prospectively dystrophic muscles fail to achieve their adult characteristics by 21 days. On the basis of these results, it is suggested that muscle maturation is retarded in dystrophic animals.