Characteristics of cat skeletal muscles grafted with intact nerves or with anastomosed nerves

Grafting of 3-g extensor digitorum longus (EDL) muscles of cats may be made with (i) severence of the nerve with spontaneous reinnervation, termed standard grafts (ii) severence of the nerve with reinnervation facilitated by anastomosis of the nerve, termed nerve-anastomosed grafts; and (iii) preservation of the nerve, termed nerve-intact grafts. In previous studies, standard grafts developed a maximum isometric tetanic tension (P0) that was 22% of the value for control EDL muscles. We hypothesized that the low values of P0 resulted from incomplete reinnervation of muscle fibers. To test this hypothesis, EDL muscles were grafted in cats with nerves intact and with nerves anastomosed. In standard grafts differences were observed in both structure and function at 120 compared with 240 days after grafting. Characteristics of the nerve-intact and nerve-anastomosed grafts did not change significantly between 120 and 240 days and the data were pooled for comparisons with control EDL muscles. Nerve-anastomosed and nerve-intact grafts developed P0 values that were 34 and 64% of the control values, respectively. Nerve-intact grafts had a mass and fiber cross-sectional area not different from control EDL muscles. Compared with control values, all grafts had fewer fibers, more connective tissue, lower absolute and normalized P0, reduced capillary density, and increased fatigability. The greater P0 of nerve-intact compared with standard and nerve-anastomosed grafts supported our hypothesis that the degree of reinnervation is a factor that limits graft development. The presence of a necrotic core and the low tension development of even the nerve-intact grafts suggested that revascularization is a significant limitation as well.     

Potentiation of the isometric twitch and mechanism of tension recruitment in mammalian skeletal muscle.

Changes produced by bromide, nitrate, and iodide in the isometric myogram of the fast-twitch extensor digitorum longus and the slow-twitch soleus muscles of the rat were studied in vitro at 35°C. Replacement of chloride with bromide, nitrate, or iodide produced only small changes in the isometric tetanic tension, but it produced a reversible potentiation of the isometric twitch tension. The order of the potentiating influence was iodide > nitrate > bromide. The tension potentiation was associated with increases in rate of tension rise and the time to peak of the isometric twitch. The results from the two muscles were essentially similar and they were qualitatively similar to those reported from frog twitch muscle. The form of tension enhancement is qualitatively similar to that obtained during summation of mechanical responses, and the results predict with sufficient accuracy the characteristics of the fully summated two-stimuli mechanical response. We suggest that changes in excitation-contraction coupling, similar to those produced by these anions, occur in mammalian skeletal muscle fiber at 35 to 38°C during summation of isometric mechanical responses.     

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.     

Influence of temperature on isometric tension development in mouse fast- and slow-twitch skeletal muscles

The influence of temperature (range 35 to 20°C) on the isometric contractile properties of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of the male C57 mice was studied in vitro, with direct stimulation. Cooling from 35 to 25°C resulted in an average twitch tension potentiation of 42% in EDL and 13% in SOL muscles. Further cooling to 20°C did not produce further potentiation in EDL muscles. The time-to-peak tension and the time to half-relaxation increased 2.4 to 3.2 times for a 10°C cooling in both muscles. The maximum tetanic tension was little changed in cooling from 35 to 30°C, but was depressed 16 to 19% in cooling to 20°C in both muscles. These results from mouse fast and slow muscles were compared with previously published data from muscles of the rat and the cat. The behaviors of the slow SOL muscles in the three species were found to be significantly different, but they could be explained on the basis of their muscle fiber-type composition.     

Abnormal distribution of skeletal muscle acetylcholinesterase molecular forms in dystrophic mice

Acetylcholinesterase (AChE) activities and molecular forms, as separated by density gradient centrifugation, were studied in dystrophic and clinically normal mouse muscle. Dystrophic hemidiaphragms exhibited normal AChE activity, but there was little or no 10 S enzyme, a form that constitutes 27% of control tissue AChE. The 10 S-AChE abnormality was similarly present in dystrophic extensor digitorum longus (EDL) muscle, but this muscle exhibited significantly reduced AChE activity. The EDL muscles also had reduced 16 S-AChE but normal 4 S enzyme activity. Chronic denervation of EDL muscles resulted in proportionally similar reductions of weight, total AChE, and 16 S enzyme in dystrophic and control muscles. We conclude that murine dystrophy involves some alterations that resemble denervation, but that there are major qualitative and quantitative differences in AChE that cannot be explained by a denervation-like effect.     

Effect of pentobarbital on contractility of mouse skeletal muscle

Pentobarbital is a hypnotic drug commonly used as anesthesia for in vivo studies in various animals. A direct effect of pentobarbital on the central nervous system and skeletal neuromuscular junction has been known for at least 30 years. A recent study using single fiber preparations from amphibian muscles indicated a significant acute and direct effect on muscle contractility at drug concentrations within the anesthetic range. The present study using whole muscles from mice demonstrated a similar augmentation of twitch tension and rate of tension development whereas tetanic tension was reduced by this drug at similar concentrations. In addition, most time parameters of contraction were prolonged. It is of interest that the slow (oxidative) muscles were considerably more sensitive to pentobarbital than the fast (primarily anaerobic) muscles. We suggest that pentobarbital should not be used as the anesthetic agent for in vivo studies of other interventions when conclusions are based on changes in muscle contractility.     

Isometric contractile properties of fast- and slow-twitch muscles in normal and spastic mice

Isometric contractile properties of the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of 24 male C57 mice were studied in vitro at 35°C. Ten animals exhibited the hereditary movement disorder known as spasticity. EDL muscles of spastic and normal mice developed similar specific tensions and they had similar twitch tension:tetanic tension ratios. The time-to-peak tension and the time to half relaxation in the twitch of spastic EDL muscles were, on average, slightly longer. SOL muscles of spastic and normal mice also developed similar specific tensions and their twitch contractions had similar times to half-relaxation. The time-to-peak tension in the twitch and the twitch tension: tetanic tension ratio of spastic SOL muscles were, on average, higher than in normal SOL. The significance of our findings remains somewhat uncertain.     

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.     

Slowing of fast-twitch muscle in the dystrophic mouse

Isometric twitch tension was measured in fast-twitch and slow-twitch muscles of normal and dystrophic ( ) mice in vivo. In dystrophic mice more than 6 months old the fast-twitch extensor digitorum longus (EDL) showed a prolongation of the time to peak tension as well as the time to relax to one-half peak tension ( ) compared with age-matched controls. In younger dystrophic mice (4 to 6 weeks) the time to peak tension was prolonged but not significantly so. This apparent “slowing” of dystrophic fast-twitch muscle was accompanied by a reduction in both cooling potentiation and post-tetanic potentiation toward values typical of slow-twitch muscle. Slow-twitch soleus muscle (SOL) of old mice was almost unaffected by the dystrophic process with regared to its contractile characteristics. However, there appeared to be a slight, but significant “speeding” of young dystrophic SOL compared with age-matched control muscles. This was apparent in reduced times to peak tension and half-relaxation as well as an enhanced cooling potentiation. We suggest that the altered contractile characteristics result from a change in some intrinsic property of the muscle fibers rather than from extrinsic factors such as the additional perimysial connective tissue seen in these muscles.     

Calcium uptake by sarcoplasmic reticulum of muscle grafts in rats

Cross transplantations were carried out in which the soleus (SOL) and extensor digitorum longus (EDL) muscles were switched to each other's muscle bed. Sixty days later, oxalate-supported calcium uptake was measured in homogenates of these grafts and compared with calcium uptake by homogenates of the contralateral control EDL and SOL muscles. With the incubation conditions used, calcium uptake was essentially limited to sarcoplasmic reticulum (SR) vesicles. The velocities of the initial rapid calcium uptake were compared in the grafts and control muscles. Subsequently calcium uptake slowed and the 30-min accumulation of calcium indicated the loading capacity of the SR. In control muscles, the EDL had a faster velocity (0.234 +/- 0.011 mumol/mg/min) of calcium uptake and higher capacity (0.527 +/- 0.017 mumol/mg) for calcium loading than the SOL (0.089 +/- 0.008 mumol/mg/min and 0.26 +/- 0.014 mumol/mg, respectively). The EDL grafts (originally SOL muscles) had faster calcium uptakes than the control SOL muscles or SOL grafts (0.196 +/- 0.013 versus 0.089 +/- 0.008 or 0.126 +/- 0.024 mumol/mg/min). Also, the calcium uptake capacities were higher in EDL grafts than in control SOL muscles (0.400 +/- 0.017 versus 0.261 +/- 0.014 mumol/mg), but not statistically higher than in SOL grafts (0.360 +/- 0.033 mumol/mg). In contrast, SOL grafts (originally EDL muscles) had slower calcium uptakes (0.126 +/- 0.024 mumol/mg/min) than did the control EDL muscles or EDL grafts and the calcium uptake capacities (0.360 +/- 0.033 mumol/mg) were lower in SOL grafts than in control EDL muscles, but not statistically lower than in EDL grafts.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of ATP on the contractions of rat and mouse fast skeletal muscle

Introduction: The aim of this study was to compare the effects of adenosine-5'-triphosphate (ATP) and adenosine on the contractility of rodent extensor digitorum longus (EDL) muscle at normal and low temperatures. Methods: Contractions of rat and mouse isolated EDL were induced by either electrical stimulation (ES) or exogenous carbachol and recorded in the presence of ATP or adenosine (both at 100 μM). Results: ATP at all temperatures caused a decrease of the contractions induced by carbachol in rat and mouse EDL and ES-induced contractions in rat EDL, while it potentiated the ES-induced contractions of mouse EDL. Adenosine reduced the contractility of rat and mouse EDL evoked by ES and did not affect the carbachol-induced contractions of rat and mouse EDL at any temperature. Discussion: Under various temperature conditions, ATP inhibits pre- but potentiates postsynaptic processes in the mouse EDL; in the rat EDL ATP causes only inhibition of neuromuscular conduction. Muscle Nerve 59:509–516, 2019     

Trophic action of nerve extract on denervated skeletal muscle in vivo: dose dependency, species specificity, and timing of treatment

Atrophy in a denervated muscle results from the disuse caused by paralysis of the muscle, and from the loss of special neurotrophic substances. Proteins extracted from rats' sciatic nerves have been shown to prevent the nondisuse atrophy of denervated rats' extensor digitorum longus (EDL) muscles when administered by intramuscular injection. The present investigation elicited more information about this neurotrophic phenomenon. Extract of liver failed to affect the rate of atrophy of denervated EDL muscles. Injection of various doses of extract of rats' or sheep's sciatic nerves indicated that the action of the extract was dose-dependent but not species-specific. Muscles denervated for 14 days and injected with extract on all 14 days or only on the first or last 7 days all exhibited significantly less atrophy than those that were not injected.     

Survival, reinnervation, and histochemical maturation of skeletal muscle xenografts in the nude mouse.

Sensory neurons of rats survive and induce the formation of taste buds in vallate papillae when grafts of rat ganglion and mouse tongue are combined in the anterior chamber of the eyes of the immunologically deficient nude mouse. The present study was undertaken to determine if cross-species neuromuscular reinnervations also occur in grafts to nude mice. The intact extensor digitorum longus muscle was taken from 20-day-old normal rats and transplanted into the site formerly occupied by the excised extensor digitorum longus muscle of nude mice. Rat muscles were placed into an innervated or denervated graft site of nude mice, into an innervated graft site of normal mice, or into an innervated graft site of nude mice immunologically reconstituted by a thymus gland graft. Autotransplantation of the extensor digitorum longus muscle to an innervated graft site was also performed in some nude mice. Many rat muscle fibers survived 60 days in nude mice, became reinnervated, and developed into histochemically defined muscle fiber types. Some rat muscle fibers survived in nude mice in a denervated graft site but these fibers were atrophic and did not form fiber types. All rat muscle fibers were rejected by normal mice or by immunologically reconstituted nude mice. Muscle fibers in autografts of nude mouse muscle also survived, became reinnervated, and formed fiber types. The present demonstration of cross-species neuromuscular reinnervation means that different animals have similar cell recognition mechanisms involved in the formation of a neuromuscular junction and in the neurotrophic induction and maintenance of muscle fiber types. Because it is quite likely that human muscle would survive in the nude mouse, this animal might be useful for elucidating the origin or reversibility of diseased human muscle.     

Influence of castration on the electrical excitability and contraction properties of the rat levator ani muscle

Castration of male rats at 2 (C₂), 20 (C₂₀), or 75 (C₇₅) days of age resulted in marked atrophy of the levator ani (LA) muscle by 105 days of age. Although the single twitch was prolonged, the twitch and tetanus tension were decreased in LA muscles from C₂ and C₂₀ rats but not from C₇₅ rats. However, the maximum force of contraction expressed per gram of muscle tissue was significantly increased after castration in all groups. Testosterone administration to rats castrated at adult age (C₇₅) reversed the changes induced by castration. The alterations induced by castration on properties of the extensor digitorum longus (EDL) muscle were quantitatively less evident than those detected for the LA muscle in the same group. However, the younger the animal was at castration, the more affected were the contraction properties of the EDL. The directly elicited action potentials of the muscle fiber in both muscles were not significantly affected by castration. These findings suggest that the effect of castration on the LA and to a lesser extent on the EDL are primarily related to the trophic influence of testosterone on the development and maintenance of the contractile system.     

Physiological and histochemical changes of the extensor digitorum longus and soleus muscles after lateral cordotomy in the albino rat

The physiological and histochemical effects of unilateral and bilateral cordotomy on the extensor digitorum longus and soleus muscles were studied in the albino rat. After unilateral cordotomy, the ipsilateral extensor digitorum longus and soleus muscles became slower and relatively faster, respectively, compared to the contralateral and normal muscles. No gross histological abnormalities were found, but changes in fiber diameter and typology were statistically significant in both muscle types compared to their contralateral muscles, and were in substantial agreement with the changes observed on the contractile force and kinetics of the isometric twitch. Bilateral cordotomy in adult animals, like unilateral cordotomy in immature animals, affected only the soleus muscle fiber types. The results have implications for the role of the upper motoneuron physiological and histochemical trophic influences in controlling slow and fast motor units.     

Post-tetanic depression of twitch tension in the cat soleus muscle

Changes produced by a preceding tetanus in the isometric twitch myogram were examined in cat soleus (slow twitch) muscle in situ, at 37°C, and with supramaximal nerve stimulation. A tetanus produced a depression in the twitch tension (P) accompanied by a shortening of time to peak (TP) and time to half-relaxation (HR), and a slight depression in the maximum rate of tension rise (MR). Recovery of P, TP, and HR, after a standard tetanus of 100 Hz for 300 ms, showed an early-fast-phase (time constant of 0.8 to 0.9 s) and a late-slow-phase (time constant of 70 to 80 s), whereas that of MR showed only the late phase of recovery. Analysis of the data indicated the occurrence of two post-tetanic influences which produce different maxima of tension depression at zero test interval and have different rates of recovery. The twitch tension depression, when examined at the same test interval (0.7 s), increased with increase in the number of stimuli in the preceding tetanus. The relationship between the post-tetanic twitch tension and the number of stimuli in the tetanus was approximately logarithmic and was similar for tetanic frequencies of 50, 100, and 200 Hz. Unlike in the fast-twitch muscles, there was no clear evidence of a post-tetanic influence which may potentiate the twitch tension: the most prominent post-tetanic effect, in this muscle, was an acceleration of the tension relaxation in the twitch.     

Effects of pH on membrane resistance in normal and dystrophic mouse skeletal muscle fibers

Membrane cable properties of skeletal muscle fibers of dystrophic mice (Rej-129) and their littermate controls were examined using a conventional two-microelectrode recording technique. Fibers from dystrophic mice had a decreased membrane resistivity (R_m) compared with those from normal mice (517 ± 27 vs 642 ± 34 Ω ? cm²), while the internal resistivities (R_i) did not differ significantly. The increase in membrane specific conductance was due to an increased Cl^? conductance (g_Cl) (2304 vs 1346 μS/cm² for normal fibers), although the K⁺ conductance (g_K) was actually decreased (234 vs 369 μS/cm² for normal fibers). With changes in pH, membrane conductances of normal and dystrophic skeletal muscle fibers varied differently, mainly due to differences in effects on the Cl^? conductance. This contrast may be due to altered regulation of internal pH in dystrophic muscle.