Muscle Plasma Membrane Changes in Dystrophin Gene Exon 52 Knockout Mouse

Changes in muscle plasma membranes in mice lacking exon 52 of the dystrophin gene (mdx52 mouse) were studied using the freeze-fracture technique. The extensor digitorum longus (EDL) muscle plasma membrane of the mdx52 mouse at 8 weeks of age showed significantly increased caveola density (p < 0.05 by two-tailed t-test) and significantly decreased densities of intramembranous particles (IMPs), orthogonal arrays (OAs) and orthogonal array subunit particles (OASPs) (p < 0.05 by two-tailed t-test, p < 0.01 by Wilcoxon rank-sum test, p < 0.05 by two-tailed t-test, respectively) on the protoplasmic face when compared with those of control EDL muscles. These changes are more similar to those seen in DMD than those in the mdx mouse at the same age as reported previously. Thus, the gene abnormality in the different exon of the mouse dystrophin gene seems to induce somewhat different changes in the muscle plasma membrane.     

Merosin (laminin-2) localization in basal lamina of normal skeletal muscle fibers and changes in plasma membrane of merosin-deficient skeletal muscle fibers

Primary deficiency of merosin causes a severe congenital muscular dystrophy (CMD) and a mouse dystrophy (dy/dy mouse). Also, its secondary deficiency is seen in some CMD with abnormal glycosylation of -dystroglycan, an extracellular membrane protein, which is the receptor of merosin and binds to dystrophin underlying the sarcolenma via -dystroglycan, a transmembrane protein. In immunogold and freeze-etch electron microscopic studies, merosin in basal lamina of normal skeletal muscles has a zonation in the distribution and is localized at the lamina lucida of muscle basal lamina, and dystrophin molecules are often closed to merosin molecules at the inside and outside surface of muscle plasma membrane. Moreover, merosin molecules exist as the short fine cross-bridge fibrils connecting the basal lamina to the neighboring outer leaflet of the muscle plasma membrane. In freeze-fracture studies, the changes in muscle plasma membranes of dy/dy mice reveal a markedly decreased density of orthogonal arrays (OAs) but normal density of intramembranous particles (IMPs), whereas depletions of IMPs with decreased OAs have been found in Fukyama-type congenital muscular dystrophy, Duchenne muscular dystrophy, and mdx mice. Thus, further studies including the functional role of OAs would be required to understand the pathomechanism of merosin-deficient CMD.     

Alterations of molecular architectures in skeletal muscle plasma membranes of dehydrated and water-loaded mice

It is likely that orthogonal arrays (OAs) and caveolae seen on the replicas of freeze-fractured muscle plasma membranes are involved in maintaining osmotic homeostasis. Therefore, using the freeze-fracture technique, we examined the ultrastructural changes in OAs and caveolae of the skeletal muscle plasma membrane of dehydrated and water-loaded mice. In the muscle plasma membranes of 6 dehydrated and 6 water-loaded mice, caveolar distribution was not altered, and the densities of caveolae and OAs did not show statistically significant differences when compared with those in 6 control mice, although the skeletal muscles of water-loaded mice sometimes had muscle plasma membranes with extremely numerous OAs. In contrast, the muscle plasma membranes of dehydrated mice often revealed changes in the distribution of OAs, which existed in a group at the confined area of the muscle plasma membranes and were frequently accompanied by the aggregations of intramembranous particles (IMPs) around OAs. Thus, on the basis of the present study, we suggest that OAs in skeletal muscles as well as those in brain may play an important role in maintaining osmotic homeostasis of these organs under abnormal water balance.     

A freeze-fracture study of sarcoplasmic reticulum from fast and slow muscle of the mouse.

Sarcoplasmic reticulum (SR) of fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles of the mouse was examined by freeze-fracture techniques. A distinctive feature of sarcoplasmic reticulum from the EDL is the presence of hillocks on the A-face within the terminal cisterns. These hillocks are usually arranged in a single row which is deployed parallel to the long axis of the adjacent T-tubule. Center-to-center spacing of hillocks within a row is about 70-75 nm. Hillocks are also found scattered within the collar region. The EDL of ten week mice was characterized by sheet-like terminal and intermediate cisterns, the latter being replaced in 37 week animals by thin tubular longitudinal elements of the SR which contain no hillocks or dimples. Hillocks occur only occasionally in SR from 10 or 20 week SOL muscle. In such cases the hillocks occur singly rather than in rows as in the terminal cisterns of EDL. The predominant form of SR in the SOL contains no hillocks. Total particles in the A-face of EDL-SR (2996 particles/mu2; S.D. = +/- 287) slightly exceeded that of SOL-SR (2558 particles/mu2; S.D. = 274 8 NM). Packing density of 8 nm particles was slightly higher for EDL (750/mu2) VS. SOL (700/mu2). The possible significance of these features of SR in fast and slow muscle is discussed.     

A freeze-fracture study of sarcoplasmic reticulum from fast and slow muscle of the mouse

Sarcoplasmic reticulum (SR) of fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles of the mouse was examined by freeze-fracture techniques. A distinctive feature of sarcoplasmic reticulum from the EDL is the presence of hillocks on the A-face within the terminal cisterns. These hillocks are usually arranged in a single row which is deployed parallel to the long axis of the adjacent T-tubule. Center-to-center spacing of hillocks within a row is about 70–75 nm. Hillocks are also found scattered within the collar region. The EDL of ten week mice was characterized by sheet-like terminal and intermediate cisterns, the latter being replaced in 37 week animals by thin tubular longitudinal elements of the SR which contain no hillocks or dimples. Hillocks occur only occasionally in SR from 10 or 20 week SOL muscle. In such cases the hillocks occur singly rather than in rows as in the terminal cisterns of EDL. The predominant form of SR in the SOL contains no hillocks. Total particles in the A-face of EDL-SR (2996 particles/μ²; S.D. = ± 287) slightly exceeded that of SOL-SR (2558 particles/μ²; S.D. = ± 274 8 nm). Packing density of 8 nm particles was slightly higher for EDL (750/μ²) vs. SOL (700/μ²). The possible significance of these features of SR in fast and slow muscle is discussed.     

Changes in contractile and metabolic parameters of skeletal muscle as rats age from 3 to 12 months

Laboratory rats are considered mature at 3 months despite that musculoskeletal growth is still occurring. Changes in muscle physiological and biochemical characteristics during development from 3 months, however, are not well understood. Whole muscles and single skinned fibres from fast-twitch extensor digitorum longus (EDL) and predominantly slow-twitch soleus (SOL) muscles were examined from male Sprague–Dawley rats (3, 6, 9, 12 months). Ca²⁺ sensitivity of contractile apparatus decreased with age in both fast- (~?0.04 pCa units) and slow-twitch (~?0.07 pCa units) muscle fibres, and specific force increased (by ~?50% and ~?25%, respectively). Myosin heavy chain composition of EDL and SOL muscles altered to a small extent with age (decrease in MHCIIa proportion after 3 months). Glycogen content increased with age (~?80% in EDL and 25% in SOL) and GLUT4 protein density decreased (~?35 and 20%, respectively), whereas the glycogen-related enzymes were little changed. GAPDH protein content was relatively constant in both muscle types, but COXIV protein decreased?~?40% in SOL muscle. Calsequestrin (CSQ) and SERCA densities remained relatively constant with age, whereas there was a progressive?~?2–3 fold increase in CSQ-like proteins, though their role and importance remain unclear. There was also ~?40% decrease in the density of the Na⁺, K⁺-ATPase (NKA) α1 subunit in EDL and the α2 subunit in SOL. These findings emphasise there are substantial changes in skeletal muscle function and the density of key proteins during early to mid-adulthood in rats, which need to be considered in the design and interpretation of experiments.     

Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch

Summary Absence of dystrophin in mdx muscles may render the muscle more susceptible to damage when submitted to high stress levels. To test this, typically slow (soleus) and fast (EDL) limb muscles of dystrophic (mdx) and normal (C57BL/10) mice were submitted (in vitro) to a series of isometric contractions, followed by a series of contractions with stretches. Muscle injury was assessed by monitoring the force signal. Membrane damage was evaluated by bathing the muscle in Procion Red, a dye that does not penetrate intact fibres, and subsequent analysis by light microscopy. After isometric contractions, only a very small force drop (<3% of maximal isometric force) was observed which indicated that no injury had occurred in soleus and EDL muscles in either mdx or C57 strains. After contractions with a stretch, a force drop of 10% was observed in soleus muscles from both strains and in EDL muscles from C57 mice. However, in mdx mice EDL muscles displayed an irreversible force drop of 40–60%. Histological analysis of the muscles indicates that force drop is associated with membrane damage. These results show that EDL muscles from mdx mice are more vulnerable than their controls, supporting the structural role hypothesis for dystrophin. Furthermore, they suggest that contractions with stretches may contribute to the muscle damage and degeneration observed in DMD-patients.     

Overexpression of inducible 70-kDa heat shock protein in mouse improves structural and functional recovery of skeletal muscles from atrophy

Heat shock proteins play a key regulatory role in cellular defense. To investigate the role of the inducible 70-kDa heat shock protein (HSP70) in skeletal muscle atrophy and subsequent recovery, soleus (SOL) and extensor digitorum longus (EDL) muscles from overexpressing HSP70 transgenic mice were immobilized for 7 days and subsequently released from immobilization and evaluated after 7 days. Histological analysis showed that there was a decrease in cross-sectional area of type II myofiber from EDL and types I and II myofiber from SOL muscles at 7-day immobilization in both wild-type and HSP70 mice. At 7-day recovery, EDL and SOL myofibers from HSP70 mice, but not from wild-type mice, recovered their size. Muscle tetanic contraction decreased only in SOL muscles from wild-type mice at both 7-day immobilization and 7-day recovery; however, it was unaltered in the respective groups from HSP70 mice. Although no effect in a fatigue protocol was observed among groups, we noticed a better contractile performance of EDL muscles from overexpressing HSP70 groups as compared to their matched wild-type groups. The number of NCAM positive-satellite cells reduced after immobilization and recovery in both EDL and SOL muscles from wild-type mice, but it was unchanged in the muscles from HSP70 mice. These results suggest that HSP70 improves structural and functional recovery of skeletal muscle after disuse atrophy, and this effect might be associated with preservation of satellite cell amount.     

A freeze-fracture study of synaptic junction development in the superficial layers of the chick optic tectum

Summary Synaptogenesis in the superficial layers of the rostral pole of the chick optic tectum has been studied using freeze-fracture techniques. The developmental sequence of intramembrane organization at synaptic junctions involves the accumulation and assembly of intramembrane particles into aggregates characteristic of the mature junctions.By embryonic day seven, areas of loosely-arranged clusters of medium-sized particles are observed on the cytoplasmic membrane leaflets (P-faces) of developing neurites. These clusters are characteristic of the intramembrane organization at presynaptic active zones. At later stages, small pits, characteristic of vesicle fusion sites, are observed interspersed among such P-face particle clusters. Complementary intramembrane specializations are also present on the external leaflets (E-faces) of presynaptic membranes at the active zones.Small solitary aggregates of large-sized particles on the E-faces of neurite plasma membranes are also seen at early embryonic stages. As development progresses, these aggregates increase in size and packing density and occupy large oval domains in postsynaptic membranes. These intramembrane specializations may represent the postsynaptic active zones of asymmetric synapses. Another type of intramembrane specialization, observed during the third week of incubation, is characterized by aggregates of small- and medium-sized particles on the P-face of postsynaptic membranes and is often seen directly apposed to the E-face of a presynaptic terminal. This type of intramembrane specialization may represent the postsynaptic active zone region at symmetrical synaptic contacts.     

Mechanical and energetic properties of dystrophic (mdx) mouse muscle

The mechanical and energetic properties of extensor digitorum longus (EDL) and soleus muscles of X chromosome-linked muscular dystrophic mutant (mdx) mice aged 4-6 weeks were studied and compared with those of the muscles of normal mice. Maximum tetanic tension, the speed of contraction of relaxation, and the heat production of mdx soleus muscles were not significantly different from those of the normal muscles. However, in mdx EDL muscles, the tension and heat production were significantly reduced, and relaxation was prolonged. To study the cause of these changes in mdx EDL muscles, tension and heat production were measured at various muscle lengths greater than optimum for tension. Both the amount of twitch heat and the heat rate for a tetanus were linearly related to the tension and had non-zero intercepts at zero tension, the activation heat. The twitch activation heat and the tension-related heat in tetani of mdx EDL muscles were not different from those in normal muscles. On the other hand, the tetanus activation heat of mdx EDL muscles was significantly smaller than that of normal muscles. Assuming that the degenerated fibers do not contribute to the active force produced, these results suggest that the amount of Ca2+ released in a contraction is not significantly different between normal and mdx muscles, but the Ca-ATPase activity of the salcoplasmic reticulum is reduced in mdx EDL, which could cause the slowing of relaxation.     

Neuromuscular junctions (NMJs): ultrastructural analysis and nicotinic acetylcholine receptor (nAChR) subunit mRNA expression in offspring subjected to protein restriction throughout pregnancy

Protein restriction during gestation can alter the skeletal muscle phenotype of offspring; however, little is known with regard to whether this also affects the neuromuscular junction (NMJ), as muscle phenotype maintenance depends upon NMJ functional integrity. This study aimed to evaluate the effects of a low protein (6%) intake by dams throughout gestation on male offspring NMJ morphology and nicotinic acetylcholine receptor (nAChR) α1, γ and ε subunit expression in the soleus (SOL) and extensor digitorum longus (EDL) muscles. Four groups of male Wistar offspring rats were studied. The offspring of dams fed low‐protein (6% protein, LP) and normal protein (17% protein, NP) diets were evaluated at 30 and 120 days of age, and the SOL and EDL muscles were collected for analysis. Morphological studies using transmission electron microscopy revealed that only SOL NMJs were affected in 30‐day‐old offspring in the LP group compared with the NP group. SOL NMJs exhibited fewer synaptic folds, the postsynaptic membranes were smooth and myelin figures were also frequently observed in the terminal axons. With regard to the expression of mRNAs encoding nAChR subunits, only 30‐day‐old LP offspring EDL muscles exhibited reduced α, γ and ε subunit expression compared with the NP group. In conclusion, our results demonstrate that a low‐protein diet (6%) imposed throughout pregnancy impairs the expression of mRNAs encoding the nAChR α, γ and ε subunits in EDL NMJs and promotes morphological changes in SOL NMJs of 30‐day‐old offspring, indicating specific differences among muscle types following long‐term maternal protein restriction.     

Power output of fast and slow skeletal muscles of mdx (dystrophic) and control mice after clenbuterol treatment

The mdx mouse is the most commonly used animal model for Duchenne muscular dystrophy. We tested the null hypothesis that 20 weeks of clenbuterol treatment ( approximately 2 mg kg-1 day-1) of mdx and control mice would have no effect on the absolute and specific force (Po, kN m-2) and absolute and normalised power output (W kg-1) of extensor digitorum longus (EDL) and soleus muscles. For mdx and control mice, clenbuterol treatment produced modest increases in the mass of the two muscles but did not increase absolute or specific force or normalised power output. For absolute power output, only the EDL muscles of mdx mice showed a difference following treatment, with the power output of treated mice being 118 % that of the untreated mice. The modest effects of clenbuterol treatment on the dynamic properties of skeletal muscle provide little support for any improvement in muscle function for the dystrophic condition.     

Sarcoplasmic reticulum function in slow- and fast-twitch skeletal muscles from mdx mice

The aim of the present study was to establish whether alterations in sarcoplasmic reticulum function are involved in the abnormal Ca(2+) homeostasis of skeletal muscle in mice with muscular dystrophy ( mdx). The properties of the sarcoplasmic reticulum and contractile proteins of fast- and slow-twitch muscles were therefore investigated in chemically skinned fibres isolated from the extensor digitorum longus (EDL) and soleus muscles of normal (C57BL/10) and mdx mice at 4 and 11 weeks of development. Sarcoplasmic reticulum Ca(2+) uptake, estimated by the Ca(2+) release following exposure to caffeine, was significantly slower in mdx mice, while the maximal Ca(2+) quantity did not differ in either type of skeletal muscle at either stage of development. In 4-week-old mice spontaneous sarcoplasmic reticulum Ca(2+) leakage was observed in EDL and soleus fibres and this was more pronounced in mdx mice. In addition, the maximal Ca(2+)-activated tension was smaller in mdx than in normal fibres, while the Ca(2+) sensitivity of the contractile apparatus was not significantly different. These results indicate that mdx hindlimb muscles are affected differently by the disease process and suggest that a reduced ability of the Ca(2+)-ATPase to load Ca(2+) and a leaky sarcoplasmic reticulum membrane may be involved in the altered intracellular Ca(2+) homeostasis.     

Developmental alterations in membrane organization of rat subpial astrocytes

Summary Subpial astrocytic processes were examined in developing rats, mainly with complementary replicas, to see how orthogonal arrays of particles (OAs) are formed and become numerous in membranes covered by basal lamina. Only a few (4.2%) endfeet in the membranes contacting the basal lamina (subpial membranes) had acquired OAs by the 19-day foetal stage. The number of endfeet provided with OAs increased drastically in the prenatal period, continued to increase at birth (P0), and somewhat more slowly in the early postnatal period (P0–P3), reaching 100% at P10. There were neuronal processes as well abutting on the basal lamina at the pial surface but they were easy to distinguish from astrocytic endfeet because of their larger intramembrane particles (IMPs), which are sparsely distributed and in patch-like aggregations. The distribution density of OAs in differentiated astrocytic endfeet also increased very gradually with age until P0, a little faster in the early postnatal period, and drastically from P10 to adult. Ordinary globular IMPs increased in number with age and continued to increase in the lateral membrane where OAs were still very few, though less rapidly in the subpial membrane as OAs became numerous. With maturation, larger IMPs became conspicuous in the lateral membrane but not in the subpial, suggesting that larger IMPs were predominantly used to form OAS. We have proposed the idea that relatively large IMPs line up to form single linear arrays (SLs), appearing as grooves on the E face, and that occasionally some SLs line up in multiple rows multiple linear arrays (MLs)] and that SLs or MLs fuse with one another to become rod-like strands, then divide into squares to become OAS. SLs and MLs appeared ontogenetically earlier than OAs, and continued to appear in membranes provided with OAS. In areas where membranes were bent, transition of these three structures was observable and the proportion of OAs increased with age. Further, in such areas, alignment of OAs was different according to membrane curvature: concentric in and around protrusions, perpendicular to the edge of invaginations. This unique association of OA alignment with membrane curvature suggests that OAs contribute to some membrane stability in the area covered by the basal lamina and provide the membrane with special resistance to bending.     

Effect of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca-ATPase, on skeletal muscles from normal and mdx mice

AIM: In this study, we investigated Ca2+ loading by the sarcoplasmic reticulum in skeletal muscle from mdx mice, an animal model of human Duchenne's muscular dystrophy, at two stages of development: 4 and 11 weeks. METHOD: Experiments were conducted on fast- (extensor digitorum longus, EDL) and slow- (soleus) twitch muscles expressing different isoforms of Ca2+-ATPase, which is responsible for the uptake of Ca2+ by the sarcoplasmic reticulum. RESULTS: In sarcoplasmic reticulum vesicles, the ATP-dependent activity and sensitivity to cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, were similar in mdx and normal EDL muscle. Furthermore, in chemically-skinned fibres from both normal and mdx muscles, the presence of CPA induced a decrease in Ca2+ uptake by the sarcoplasmic reticulum. However, the sensitivity to CPA was lower in mdx EDL muscle than in normal muscle. In addition, in EDL muscle from 4-week-old mdx mice, the expression of the slow Ca2+-pump isoform (SERCA2a) was significantly increased, without any accompanying change in slow myosin expression. In contrast, the expression and function of the Ca2+-ATPase in mdx soleus muscles at 4- and 11-weeks of development did not differ from those in age-matched controls. CONCLUSION: These findings show that in dystrophic muscle, where the Ca2+ homeostasis was perturbed, the Ca2+ handling by the sarcoplasmic reticulum was altered in fast-twitch muscle, and this was associated with the expression of the slow isoform of SERCA. In these muscles, reduced Ca2+ uptake could then contribute to an elevated concentration of Ca2+ in the cytosol, and also to Ca2+ depletion of the sarcoplasmic reticulum.     

Functional regeneration in the hindlimb skeletal muscle of the mdx mouse

Summary The pattern of spontaneous skeletal muscle degeneration and clinical recovery in hindlimb muscles of the mdx mutant mouse was examined for functional and metabolic confirmation of apparent structural regeneration. The contractile properties, histochemical staining and myosin light chain and parvalbumin contents of extensor digitorum longus (EDL) and soleus (Sol) muscles of mdx and age-matched control mice were studied at 3–4 and 32 weeks. Histochemical staining (myofibrillar ATPase and NADH-tetrazolium reductase) revealed no significant change in slow-twitch-oxidative (SO) or fast-twitch-oxidative-glycolytic (FOG) fibre type proportions in mdx Sol apart from the normal age-related increase in SO fibres. At 32 weeks mdx EDL, however, showed significantly smaller fast-twitch-glycolytic (FG) and larger FOG proportions than those in control EDL. These fibre type distributions were confirmed by differential staining with antibodies to myosin slow-twitch and fast-twitch heavy chain isozymes. Frequency distribution of cross-sectional area for each fibre type showed a wider than normal range of areas especially in FOG fibres of mdx Sol, and FG fibres of mdx EDL, supporting previous observations using autoradiography of myofibre regeneration. Isometric twitch and tetanic tensions in Sol were significantly less than in controls at 4 weeks, but by 32 weeks, values were not different from age-matched controls. In mdx EDL at 3 weeks, twitch and tetanus tensions were significantly less, and time-to-peak twitch tensions were significantly faster than in control EDL. By 32 weeks, mdx EDL twitch and tetanus tensions expressed relative to muscle weight continued to be significantly lower than in age-matched controls, despite normal absolute tensions. The maximum velocity of shortening in 32-week mdx EDL was significantly lower than in control EDL. Myosin light chain distribution in mdx Sol exhibited significantly less light chain 2-slow (LC2s) and more light chain 1b-slow(LC1bs) at 32 weeks than age-matched control Sol. Gels of EDL from 32-week-old mdx mice showed significantly less light chain 2-fast-phosphorylated (LC2f-P) and light chain 3-fast (LC3f) and significantly more light chain 1-fast (LC1f) and light chain 2-fast (LC2f), but normal parvalbumin content compared to age-matched controls. These observations suggest that mdx hindlimb muscles are differentially affected by the disease process as it occurs in murine models of dystrophy. However, the uniqueness of mdx Sol and to a lesser extent EDL is that they also undergo an important degree of functional regeneration which is able to compensate spontaneously for degenerative influences of genetic origin. The mdx mutant may therefore be an important model for the study of regeneration by skeletal muscle, and of the nerve-muscle interactions which enable or restrict that regeneration.     

Electron microscopic and autoradiographic characterization of hindlimb muscle regeneration in the mdx mouse

The pattern of postnatal growth and development of skeletal muscle in mdx mice was studied by light and transmission electron microscopy and by autoradiography and was compared with that in their normal age-matched controls at 4 and 32 weeks of age. The muscle weights of both the extensor digitorum longus (EDL) and soleus muscles of mdx mice were significantly greater than those in control mice at both ages. Body weights of male and female mdx mice were also increased over controls up to 12 weeks of age. At 4 weeks, both the EDL and soleus muscles exhibited focal areas of degeneration, necrosis, and regeneration of centrally nucleated extrafusal fibers resulting in a wide range of fiber sizes. By 32 weeks, the majority of fibers in both muscles were centrally nucleated, and focal areas of recent regeneration were observed. By electron microscopy, the course of macrophage infiltration into areas of degenerating fibers and the ongoing regeneration of myofibers within redundant cylinders of external lamina were noted. This pattern was frequent in 4-week-old mdx muscles and was present to a lesser degree at 32 weeks. A notable lack of both adipose tissue infiltration and fibrotic change in the endomysium were observed in muscles at both ages. Autoradiograms of muscles from 4-week-old mdx mice injected with tritiated thymidine showed an increased proportion of labeled sublaminal nuclei at 24 and 48 hours after injection compared to controls. At 32 weeks of age, labeling of nuclei in muscles of mdx mice was also greater than in controls, but was reduced compared to muscle labeling in 4-week-old mdx mice. The observed features of mdx muscle tissue suggest that this animal model is more applicable to the study of regeneration dynamics than to Duchenne-type human muscular dystrophy.     

Ultrastructural and metabolic characteristics of single muscle fibres belonging to the same type in various muscles in rats

Summary Single muscle fibres from soleus (SOL) as well as extensor digitorum longus (EDL) muscles from Wistar male rats in relaxing solution were divided into three types by their histochemical features — slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), or fast-twitch glycolytic (FG) fibres. The relationship between ultrastructural profiles (mitochondrial volume, number, and Z-line width) and metabolic profiles (glycolytic and oxidative enzymes' activity) were analysed using the same types of fibres dissected from different SOL and EDL muscles using stereological and biochemical techniques. The Z-line width is specialized in different fibre types. Fast-twitch (FG and FOG) fibres have narrow Z-line width compared to slow-twitch (SO) fibre in SOL and EDL muscles. A significant difference was observed between SOL muscle SO and FOG fibres and EDL muscle SO and FOG fibres. All glycolytic (lactate dehydrogenase (LDH), phosphofructokinase (PFK), pyruvate kinase (PK) and creatine kinase (CK) activities in FOG fibres from EDL muscles were significantly higher (p<0.01) than those dissected from SOL muscles. The oxidative enzyme (succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) activity in SO and FOG fibres dissected from SOL muscle were significantly higher (p<0.01) than those dissected from EDL muscles. Mitochondrial volume and number in SO fibres dissected from SOL muscle were significantly higher (volume,p<0.01, number,p<0.01) than those dissected from EDL muscles. A significant difference was not observed in mitochondrial volume of FOG fibres between SOL and EDL muscles. Significant positive correlation was observed in FOG (p<0.05) and FG (p<0.01) fibres between mitochondrial volume and number dissected from EDL muscle.The results suggest that the same type of single muscle fibres in different muscles have different ultrastructural and metabolic profiles, and these profiles resembled those of the fibre types primarily constituting those muscles.     

Late reinnervation of the rat soleus muscle is differentially suppressed by chronic stimulation and by ectopic innervation

Soleus (SOL) and extensor digitorum longus (EDL) muscles in adult rats were kept denervated for 2 months by four repeated freezes at 2-week intervals of the sciatic nerve. Reinnervation was studied in the absence or presence of chronic muscle stimulation, starting 1 month before reinnervation began. In addition, reinnervation was studied in SOL muscles where a previously transplanted fibular (FIB) nerve had formed ectopic neuromuscular junctions outside the original endplate area. After repeated freezes only, reinnervation was complete judged by tension measurements and histochemical examinations in SOL (n = 7) and EDL (n = 8) muscles. In directly stimulated muscles reinnervation was incomplete, and the force tensions evoked from indirect stimulation was on average 87 (n = 5) and 82% (n = 5) of direct muscle stimulation in SOL and EDL muscles, respectively. Of ectopically innervated SOL muscle fibres, only 26% became reinnervated in 12 muscles. Denervation and reinnervation increased the number of muscle fibres in stimulated (n = 4) and unstimulated (n = 5) EDL muscles by 18 and 15%, respectively. In stimulated (n = 4) and unstimulated (n = 7) SOL muscles, on the other hand, the number of muscle fibres remained normal. The stronger suppression of reinnervation in ectopically reinnervated compared to chronically stimulated SOL fibres indicates that reinnervation can also be suppressed by activity independent influences from the foreign nerve.     

Physiological and biochemical characteristics of skeletal muscles in sedentary and active rats

Laboratory rats are sedentary if housed in conditions where activity is limited. Changes in muscle characteristics with chronic inactivity were investigated by comparing sedentary rats with rats undertaking voluntary wheel running for either 6 or 12 weeks. EDL (type II fibers) and soleus (SOL) muscles (predominantly type I fibers) were examined. When measured within 1–2 h post-running, calcium sensitivity of the contractile apparatus was increased, but only in type II fibers. This increase disappeared when fibers were treated with DTT, indicative of oxidative regulation of the contractile apparatus, and was absent in fibers from rats that had ceased running 24 h prior to experiments. Specific force production was ~?10 to 25% lower in muscle fibers of sedentary compared to active rats, and excitability of skinned fibers was decreased. Muscle glycogen content was ~?30% lower and glycogen synthase content?~?50% higher in SOL of sedentary rats, and in EDL glycogenin was 30% lower. Na⁺, K⁺-ATPase α1 subunit density was ~?20% lower in both EDL and SOL in sedentary rats, and GAPDH content in SOL?~?35% higher. There were no changes in content of the calcium handling proteins calsequestrin and SERCA, but the content of CSQ-like protein was increased in active rats (by ~?20% in EDL and 60% in SOL). These findings show that voluntary exercise elicits an acute oxidation-induced increase in Ca²⁺ sensitivity in type II fibers, and also that there are substantial changes in skeletal muscle characteristics and biochemical processes in sedentary rats.