Effects of hypobaric hypoxia on the oxidative capacity of the extensor digitorum longus motor units in the rat

The fiber number, fiber type distribution, and succinate dehydrogenase activity were investigated from the fast-twitch extensor digitorum longus muscle of male rats exposed to 7 weeks of hypobaric hypoxia. The oxidative metabolic capacity of the motoneurons in the extensor digitorum longus neuron pool was also determined from quantitative histochemical analyses. The fiber number and oxidative enzyme activity of the muscle were not changed by hypoxia. An increase in the percentage of fast-twitch oxidative (FO) fibers and a concemitant decrease in the percentage of fast-twitch (F) fibers were observed in the hypoxic muscle. On the other hand, the oxidative capacity of small-to medium-sized alpha motoneurons (25–45 m average soma diameter) was increased. The increase in the oxidative capacity of small- to medium-sized motoneurons and the type shift of muscle fibers from F (low-oxidative) to FO (high-oxidative) indicate that hypoxia enhances the oxidative capacity of particular motor units in the neuron pool.     

Fiber type-specific expression of major proteolytic systems in fast- to slow-transforming rabbit muscle

The present study investigatesthe role of two major proteolytic systems in transforming rabbit andrat muscles. The fast-to-slow transformation of rabbit muscle bychronic low-frequency stimulation (CLFS) induces fast-to-slowtransitions of intact, mature fibers and replacement of degeneratingfibers by newly formed slow fibers. Ubiquitination, an indicator of theATP-dependent proteasome system, and calpain activity were measured inhomogenates of control and stimulated extensor digitorum longusmuscles. Calpain activity increased similarly (~2-fold) in stimulatedrat and rabbit muscles. CLFS had no effect on protein ubiquitination inrat muscle but led to elevations in ubiquitin protein conjugates inrabbit muscle. Immunohistochemistry was used to study the distributionof µ-calpain and m-calpain and of ubiquitinated proteins in myosinheavy chain-based fiber types. The findings suggest that bothproteolytic systems are involved in fiber transformation andreplacement. Transforming mature fibers displayed increases inµ-calpain and accumulation of ubiquitin protein conjugates. Themajority of these fibers were identified as type IIA. Enhancedubiquitination was also observed in degenerating and necrotic fibers.Such fibers additionally displayed elevated m-calpain levels.Conversely, p94, the skeletal muscle-specific calpain, decayed rapidlyafter stimulation onset and was hardly detectable after 4 days of CLFS.

     

Histochemical fiber type distribution and epinephrine sensitivity in normal and cross-transplanted rat muscles

The metabolic integrity of fully regenerated transplants was investigated by measuring induced changes in glycogen concentration. The extensor digitorum longus and the soleus muscles were cross transplanted: the extensor digitorum longus into the soleus muscle bed (SOLT) and the soleus muscle into the extensor digitorum longus bed (EDLT). The histochemical fiber type distribution of the regenerated muscles was determined and was found to transform in cross-transplanted EDLT and SOLT. After transplantation and regeneration, both muscles had initially low glycogen concentrations. However, the EDLT glycogen concentration was not significantly different from that of the contralateral extensor digitorum longus control muscle after 60 days. In the SOLT, glycogen gradually increased but remained less than in the contralateral soleus control muscle. SOLT and control soleus muscles responded with a significant glycogen depletion to an epinephrine dose two orders of magnitude less than the lowest dose affecting glycogen levels in EDLT and extensor digitorum longus muscles. These results indicate that transplanted muscles are capable of regenerating normal glycogenolytic responses and that the sensitivity of the response observed depends on the site of transplantation and is related to the type of innervation and histochemical fiber type.     

Postnatal growth and differentiation in three hindlimb muscles of the rat

Summary The postnatal development, between 0 and 90 days, of three hindlimb muscles and diaphragm of the rat was investigated with respect to fiber types and diameter (histochemistry) and substrate oxidation rates and enzyme activities (biochemistry). The process of muscle fiber differentiation into mature patterns was evaluated by visual classification into 3 or 4 groups having different staining intensities for 3 enzyme-histochemical reactions, enabling 26 fiber types to be distinguished. These exhibited specific sizes and growth rates that varied among the muscles. One of the hindleg muscles (flexor digitorum brevis) remained much more immature than soleus and extensor digitorum longus.The histochemical and biochemical findings correlated well. The capacity for pyruvate and palmitate oxidation, and the activities of cytochrome c oxidase and citrate synthase, increased markedly between 9 and 37 days in soleus and extensor digitorum longus (except citrate synthase in the latter) but not in flexor digitorum brevis. Creatine kinase activity increased in all hindlimb muscles. Both the capacity and the activity of pyruvate oxidation (determined in homogenates and intact isolated muscles, respectively), were in accordance with the fiber type composition. In contrast to oxidation capacity, the activity of pyruvate oxidation decreased after birth until the mature stage, when a value of 18–42% of that of early postnatal muscles was recorded.     

Identical responses of fast muscle to sustained activity by low-frequency stimulation in young and aging rats

Toinvestigate effects of sustained activity on major phenotypicproperties, the left extensor digitorum longus muscle of young (15 wk)and aging (101 wk) male Brown Norway rats was subjected to 50 days ofchronic low-frequency stimulation (CLFS; 10 Hz, 10 h/day).The contralateral muscle served as control. Changes in metabolicenzymes were analyzed by using glyceraldehyde-3-phosphate dehydrogenaseand lactate dehydrogenase as reference enzymes of glycolysis and byusing citrate synthase and 3-hydroxyacyl-CoA dehydrogenase asmitochondrial enzymes representative of aerobic-oxidative metabolism. Myosin heavy chain (MHC) isoforms wereanalyzed by SDS-PAGE. No differences existed between the enzymeactivity profiles of control muscles from young and aging rats. CLFSinduced similar increases in mitochondrial enzymes, as well as similardecreases in glycolytic enzymes. Although the MHC composition of thecontrol muscles in the aging rats displayed a shift toward slowerisoforms, the ultimate changes induced by CLFS led to nearly identicalMHC phenotypes in both young and aging rats. These results demonstrate an unaltered adaptability of skeletal muscle to increased neuromuscular activity in the aging rat.

     

Skeletal muscle is protected from disuse in hibernating dauria ground squirrels

The purpose of this study is to test the hypothesis that muscle fibers are protected from undue atrophy in hibernating dauria ground squirrels (Spermophilus dauricus, Brandt). Muscle mass, fiber cross sectional area (CSA, video analysis) and fiber type distribution (m-ATPase staining) were determined in extensor digitorum longus (EDL) muscle from non-hibernating control animals (Pre-H), from animals who hibernated for one (H1) or two (H2) months, and from animals 2-4 days after arousal (Post-H; N = 8 each). Muscle wet weight decreased less than body weight in hibernating animals, resulting in a steady increase in muscle-to-body mass ratio (+ 37% in Post-H compared to Pre-H, p < 0.001). In the Pre-H group, Type I (6.3 ± 2.0%) and II (93.7 ± 2.0%) fiber CSAs were 1719 ± 201 and 2261 ± 287 μm², respectively. There was a tendency (n.s.) of larger CSA of type I in hibernators compared to pre-H. In the Post-H group, fiber CSA and type distribution were not different from Pre-H. We are the first to report data on EDL fiber type distribution and confirm a protective effect that prevents muscle atrophy in spite of prolonged disuse during hibernation in dauria ground squirrels.     

Effect of thoracoabdominal breathing patterns on inspiratory effort sensation

The present study examined the effects of elastase-induced emphysema on the structure of the external oblique and transverse abdominis muscles and a non-respiratory muscle, the extensor digitorum longus. Muscle structure was assessed from the cross-sectional area (CSA) and percent of individual fiber types in histochemically stained sections and from the number of sarcomeres arranged in series along the length of individual fibers. Data were obtained in eight hamsters with emphysema and nine saline-injected controls. In the normal (control) animals the external oblique was thicker but contained fewer sarcomeres than the transverse abdominis. Fiber size was similar in the two muscles. In the transverse abdominis the percents of fast-glycolytic and fast-oxidative fibers were greater and smaller, respectively, than in the external oblique. Lung volume of emphysematous hamsters was 168% of control values (P less than 0.001). In emphysematous compared with control animals, the CSA of fast-twitch fibers in the external oblique and transverse abdominis was significantly reduced. Fiber length and sarcomere number were significantly decreased in the transverse abdominis but not in the external oblique in emphysematous hamsters. In contrast, fiber size and composition of the extensor digitorum longus was similar in emphysematous and control animals. These data indicate that cellular responses of the ventilatory muscles to chronic hyperinflation and altered thoracic geometry induced by emphysema are not present in limb skeletal muscle. We speculate that changes in fiber length and CSA of fast fibers in the abdominal expiratory muscles reflect responses to chronic alterations in the mechanics of breathing that may affect muscle load, length, or the pattern of activity.     

Denervated muscle fibers explain the deficit in specific force following reinnervation of the rat extensor digitorum longus muscle

The authors tested the hypothesis that, after denervation and reinnervation of skeletal muscle, observed deficits in specific force can be completely attributed to the presence of denervated muscle fibers. The peroneal nerve innervating the extensor digitorum longus muscle in rats was sectioned and the distal stump was coapted to the proximal stump, allowing either a large number of motor axons (nonreduced, n = 12) or a drastically reduced number of axons access to the distal nerve stump (drastically reduced, n = 18). A control group of rats underwent exposure of the peroneal nerve, without transection, followed by wound closure (control, n = 9). Four months after the operation, the maximum tetanic isometric force (Fo) of the extensor digitorum longus muscle was measured in situ and the specific force (sFo) was calculated. Cross-sections of the muscles were labeled for neural cell adhesion molecule (NCAM) protein to distinguish between innervated and denervated muscle fibers. Compared with extensor digitorum longus muscles from rats in the control (295 +/- 11 kN/m2) and nonreduced (276 +/- 12 kN/m2) groups, sFo of the extensor digitorum longus muscles from animals in the drastically reduced group was decreased (227 +/- 15 kN/m2, p < 0.05). The percentage of denervated muscle fibers in the extensor digitorum longus muscles from animals in the drastically reduced group (18 +/- 3 percent) was significantly higher than in the control (3 +/- 1 percent) group, but not compared with the nonreduced (9 +/- 2 percent) group. After exclusion of the denervated fibers, sFo did not differ between extensor digitorum longus muscles from animals in the drastically reduced (270 +/- 20 kN/m2), nonreduced (301 +/- 13 kN/m2), or control (303 +/- 10 kN/m2) groups. The authors conclude that, under circumstances of denervation and rapid reinnervation, the decrease in sFo of muscle can be attributed to the presence of denervated muscle fibers.     

Chronic electrical stimulation of nongrafted and grafted skeletal muscles in rats

Our purpose was to determine the effects of chronic electrical stimulation on the structure and function of neve-intact grafts in rats. Fourteen days after grafting, extensor digitorum longus (EDL) grafts (n = 6) and nongrafted EDL muscles (n = 4) were stimulated 8 h/day at 10 Hz for 26 days. Measurements were made subsequently of cytochrome c concentration, capillary density, contraction and relaxation times, developed tension, and the resistance to fatigue. Compared with contralateral nonstimulated grafts, chronically stimulated grafts demonstrated a 65% greater cytochrome c concentration, 45% greater number of capillaries per millimeter squared, 30% greater resistance to fatigue, 35% longer contraction time, 30% longer relaxation time, and 30% lower maximum tetanic tension. The differences that resulted from the stimulation of nongrafted EDL muscles were significant but of less magnitude. Chronic stimulation of 8 h/day provided a mixed stimulus for adaptation that enhanced the metabolic and endurance characteristics of fibers in muscles and grafts, but decreased the total fiber cross-sectional area and development of force.     

Calpain activity in fast, slow, transforming, and regenerating skeletal muscles of rat

Fiber-type transitions in adult skeletal muscleinduced by chronic low-frequency stimulation (CLFS) encompasscoordinated exchanges of myofibrillar protein isoforms. CLFS-inducedelevations in cytosolic Ca²⁺ could activate proteases,especially calpains, the major Ca²⁺-regulated cytosolicproteases. Calpain activity determined by a fluorogenic substrate inthe presence of unaltered endogenous calpastatin activities increasedtwofold in low-frequency-stimulated extensor digitorum longus (EDL)muscle, reaching a level intermediate between normal fast- andslow-twitch muscles. µ- and m-calpains were delineated by acalpain-specific zymographical assay that assessed total activitiesindependent of calpastatin and distinguished between native andprocessed calpains. Contrary to normal EDL, structure-bound, namelymyofibrillar and microsomal calpains, were abundant in soleus muscle.However, the fast-to-slow conversion of EDL was accompanied by an earlytranslocation of cytosolic µ-calpain, suggesting that myofibrillarand microsomal µ-calpain was responsible for the twofold increase inactivity and thus involved in controlled proteolysis during fibertransformation. This is in contrast to muscle regeneration wherem-calpain translocation predominated. Taken together, we suggest thattranslocation is an important step in the control of calpain activityin skeletal muscle in vivo.

     

Coexistence of fast and slow types of myosin light chains in a single fiber of rat soleus muscle

Single muscle fibers were isolated from soleus and extensor digitorum longus muscle of adult rats. The muscle fiber type of single fibers was determined physiologically by the skinned fiber method according to the sensitivity to strontium (Sr) ions. The fiber type of single fibers was contrasted to the pattern of myosin light chains analyzed by one and two dimensional gel-electrophoreses. All the type 2 fibers isolated from soleus muscle contained both fast and slow types of myosin light chains.     

Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo

Reiser, Peter J., William O. Kline, and Pal L. Vaghy.Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo. J. Appl. Physiol. 82(4): 1250-1255, 1997.Fast-twitch skeletal muscles contain more neuronal-type nitricoxide synthase (nNOS) than slow-twitch muscles because nNOS is presentonly in fast (type II) muscle fibers. Chronic in vivo electricalstimulation of tibialis anterior and extensor digitorum longus musclesof rabbits was used as a method of inducing fast-to-slow fiber typetransformation. We have studied whether an increase in musclecontractile activity induced by electrical stimulation alters nNOSexpression, and if so, whether the nNOS expression decreases to thelevels present in slow muscles. Changes in the expression of myosinheavy chain isoforms and maximum velocity of shortening of skinnedfibers indicated characteristic fast-to-slow fiber type transformationafter 3 wk of stimulation. At the same time, activity of NOS doubled inthe stimulated muscles, and this correlated with an increase in theexpression of nNOS shown by immunoblot analysis. These data suggestthat nNOS expression in skeletal muscle is regulated by muscle activityand that this regulation does not necessarily follow the fast-twitchand slow-twitch pattern during the dynamic phase of phenotypetransformation.

     

Slow/cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban mRNAs are expressed in chronically stimulated rabbit fast-twitch muscle

Fast-twitch extensor digitorum longus muscles of the rabbit were subjected to chronic low-frequency stimulation during different time periods. Changes in the relative amounts of mRNAs encoding fast and slow/cardiac Ca2+-ATPase isoforms were assessed through the use of an RNase-protection assay. Stimulation-induced increases in slow cardiac Ca2+-ATPase and phospholamban mRNAs were quantified by mRNA hybridization. Prolonged stimulation resulted in an exchange of the fast with the slow/cardiac Ca2+-ATPase isoform mRNAs. The exchange was complete after 72 d of stimulation as compared with normal slow-twitch soleus muscle. The tissue content of phospholamban mRNA reached levels similar to that found in normal slow-twitch soleus muscle by the same time. The conversion of the sarcoplasmic reticulum coincided with the fast-to-slow troponin C isoform transition, previously investigated in the same muscles.     

Structure of tendon organs of the rat after neonatal de-efferentation

Summary The number, size and structure of tendon organs were examined in leg muscles of the rat 3–19 weeks after de-efferentation performed in newborn animals by removal of the lumbosacral spinal cord. After this operation, tendon organs differentiated and grew in disused muscles and were innervated by primary sensory neurons, the dorsal roots of which had been disrupted.Three weeks after de-efferentation extensor digitorum longus muscles contained 14.1±1.0 (mean±standard error) and soleus muscles had 14.2±1.6 tendon organs, which corresponds to the mean number of tendon organs in the respective control muscles. The mean size of tendon organs was, however, changed. Tendon organs became on the average by 53% longer and by 35% thinner in de-efferented extensor digitorum longus muscles that were prolonged due to immobilization, as compared with shorter and wider tendon organs in de-efferented soleus muscle that remained in the shortened position.The ultrastructural differentiation of tendon organs was completed after the operation as under normal conditions. Thus it can be concluded that elimination of muscle function during the period of postnatal development indirectly affects the mean size of these receptors, but does not otherwise interfere with their morphogenesis.     

Stimulation-induced damage in rabbit fast-twitch skeletal muscles: a quantitative morphological study of the influence of pattern and frequency

The aim of this study was to determine whether muscle fibre degeneration brought about by chronic lowfrequency electrical stimulation was related to the pattern and frequency of stimulation. Rabbit fast-twitch muscles, tibialis anterior and extensor digitorum longus, were stimulated for 9 days with pulse trains ranging in frequency from 1.25 Hz to 10 Hz. Histological data from these muscles were analysed with multivariate statistical techniques. At the lower stimulation frequencies there was a significantly lower incidence of degenerating muscle fibres. Fibres that reacted positively with an antineonatal antibody were most numerous in the sections that revealed the most degeneration. The dependence on frequency was generally similar for the two muscles, but the extensor digitorum longus muscles showed more degeneration than the tibialis anterior at every frequency. Muscles subjected to 10 Hz intermittent stimulation showed significantly less degeneration than muscles stimulated with 5 Hz continuously, although the aggregate number of impulses delivered was the same. The incidence of degeneration in the extensor digitorum longus muscles stimulated at 1.25 Hz was indistinguishable from that in control, unstimulated muscles; for the tibialis anterior muscles, this was also true for stimulation at 2.5 Hz. We conclude that damage is not an inevitable consequence of electrical stimulation. The influence of pattern and frequency on damage should be taken into account when devising neuromuscular stimulation régimes for clinical use.     

The effect of a growth promoting drug,clenbuterol, on fibre frequency and area in hind limb muscles from young male rats

The effect of dietary administration of clenbuterol on soleus and extensor digitorum longus muscles was studied after 4 and 21 days. Both muscles showed an increase in wet weight with no significant change in total fibre number. After 4 days fibre cross-sectional areas were increased in soleus, but not in extensor digitorum longus, and after 21 days there was a change in fibre frequencies in extensor digitorum longus but not soleus muscles.     

Changes in satellite cell content and myosin isoforms in low-frequency-stimulated fast muscle of hypothyroid rat

Chroniclow-frequency stimulation was used to study the effects of enhancedcontractile activity on satellite cell content and myosin isoformexpression in extensor digitorum longus muscles from hypothyroid rats.As verified by immunohistochemical staining for desmin, vimentin, andmyosin heavy chain (MHC) isoforms and by histological analysis,stimulation induced a transformation of existing fast fibers towardslower fibers without signs of fiber deterioration or regeneration.Immunohistochemically detected increases in MHC I and MHC IIa isoforms,as well as reduced numbers of fibers expressing the faster MHCisoforms, mirrored the rearrangement of the thick-filament composition.These changes, especially the upregulation of MHC IIa, were accompaniedby an induction of developmental MHC isoforms in the transforming adultfibers. Satellite cell content rose 2.6-, 3.0-, and 3.7-fold over thatof corresponding controls (P < 0.05 in all cases) in 5-, 10-, and 20-day-stimulated muscles, respectively.Hypothyroidism alone had no effect on satellite cell content butresulted in a significant reduction in fiber size. The relativesatellite cell contents increased (P < 0.05) from 3.8% in euthyroid control muscles to 7.9, 11.5, and13.8% in the 5-, 10-, and 20-day-stimulated hypothyroid muscles,respectively. In 20-day-stimulated muscles, the relative satellite cellcontent reached an almost twofold higher level than that of normalslow-twitch soleus muscle. This increase occurred concomitantly with arise in myonuclear density, most probably because of the fusion of satellite cells with existing fibers.

     

Intrafusal muscle fibers of duck muscle spindles

Serial transverse paraffin sections of intrafusal muscle fibers of spindles from the extensor pollicis and the extensor digitorum communis of ducks show that only one type of intrafusal muscle fiber exists, based on the mid-equatorial nucleation pattern, diameter, and length. Although the overall range in fiber diameter at the mid-equatorial region is between 4.2-20.0 microns, the average caliber is 10.4 +/- 3.18 microns (S.D.) for spindles of the extensor pollicis and 9.3 +/- 2.11 microns (S.D.) for spindles of the extensor digitorum communis muscles. The range in spindle length for the extensor pollicis is 290-2,090 microns, average 1,120 +/- 569 microns (S.D.), and for the extensor digitorum communis 1,160-2,500 microns, average 1,745 +/- 367 microns (S.D.). The range in number of fibers per spindle for the extensor pollicis muscle is 5-12, average 8.2, and for the extensor digitorum muscle it is 1-11. In the extensor digitorum communis, there appear to be two groups, based on fiber number. Spindles of one group have a range of 5-11 fibers per spindle with an average of 7.2, whereas the second group has a range of 1-4 with an average of 2.7 fibers per spindle. The second group of spindles constitutes 52.5% of the 40 spindles studied, and of these 7.5% were monofibril spindles, 15.0% difibril, 17.5% trifibril, 12.5% quadrifibril spindles.     

Presence in chicken tibialis anterior and extensor digitorum longus muscle spindles of reactive and unreactive intrafusal fibers after incubation with monoclonal antibodies against myosin heavy chains

Cross and longitudinal sections from the encapsulated portions of chicken tibialis anterior and extensor digitorum longus muscle spindles were examined to determine whether their intrafusal fibers were a structurally homogeneous or heterogeneous population. The techniques used were the histochemical actomyosin (mATPase) reaction, and fluorescence immunohistochemistry employing two monoclonal antibodies, CA-83 and CCM-52, that are specific for myosin heavy chains. After incubation with antibody CCM-52, intrafusal fibers fluoresced either strongly or weakly to moderately. Antibody CA-83 was even more selective. In addition to identifying the strongly reactive category, it clearly separated the remaining fibers into unreactive and moderately reactive groups. As a whole, after incubation for mATPase, pH 9.6 preincubation, unreactive fibers stained darker than strongly reactive fibers. Moreover, the cross-sectional area of the unreactive fibers was significantly larger than that of the strongly reactive fibers. In the average-size muscle spindle with six intrafusal fibers, there were four unreactive fibers and two strongly reactive fibers. In about one-third of the receptors examined, one moderately reactive fiber was present. Taken together, the data indicate that intrafusal fibers of chicken tibialis anterior and extensor digitorum longus muscles are not structurally homogeneous. The observed variations can be better explained in terms of different fiber types than of continuous gradients within one type of fiber.