Normal Soleus Research Articles

Measurement of excitation-contraction coupling time in lower extremities.

The aim of this study was to apply a novel method to measure excitation-contraction coupling time (ECCT) in normal soleus muscles. We performed simultaneous recordings of soleus compound muscle action potential (CMAP) and foot movement-related potential (MRP), and measured ankle plantar flexion torque in 36 healthy subjects. We calculated ECCT and examined the relations between CMAP, MRP, ECCT and ankle plantar flexion torque. Statistical analyses established reference ranges (mean ± SE) for CMAP (13.4 ± 0.9 mV), MRP (5.3 ± 0.4 m/s2), ECCT (5.2 ± 0.1 ms), torque (85.9 ± 6.4 Nm) and torque/body weight (1.4 ± 0.1 Nm/kg). The torque showed a positive linear correlation with CMAP (p = 0.041) and a negative linear correlation with ECCT (p = 0.045). Soleus ECCT can be recorded easily, and is useful to assess the impairment of E-C coupling in muscles of the lower extremities.

Muscular hypertrophy and atrophy in normal rats provoked by the administration of normal and denervated muscle extracts.

This study was conducted to determine the effects of extracts obtained from both normal and denervated muscles on different muscle types. Wistar rats were used and were divided into a control group and four experimental groups. Each experimental group was treated intraperitoneally during 10 consecutive days with a different extract. These extracts were obtained from normal soleus muscle, denervated soleus, normal extensor digitorum longus, and denervated extensor digitorum longus. Following treatment, the soleus and extensor digitorum longus muscles were obtained for study under optic and transmission electron microscope; morphometric parameters and myogenic responses were also analyzed. The results demonstrated that the treatment with normal soleus muscle and denervated soleus muscle extracts provoked hypertrophy and increased myogenic activity. In contrast, treatment with extracts from the normal and denervated EDL had a different effect depending on the muscle analyzed. In the soleus muscle it provoked hypertrophy of type I fibers and increased myogenic activity, while in the extensor digitorum longus atrophy of the type II fibers was observed without changes in myogenic activity. This suggests that the muscular responses of atrophy and hypertrophy may depend on different factors related to the muscle type which could be related to innervation.

Sagittal gait patterns in cerebral palsy: The plantarflexor–knee extension couple index

The identification of gait patterns in cerebral palsy offers a common language for clinicians and contributes to management algorithms. We describe a quantitative classification of sagittal gait patterns based on the plantarflexor–knee extension couple index. This consists of a scatter plot based on ankle and knee scores, and allows objective identification of the sagittal gait pattern.Sagittal kinematic data from 200 limbs of 100 patients with bilateral spastic cerebral palsy were utilized to validate the algorithm against the assessment of a clinician with expertise in gait pattern identification. A dataset of 776 cerebral palsy patients, 1552 limbs, was used to compare the sagittal gait patterns against k-means statistical clustering. The classification was further explored with respect to the knee kinetics during the middle of stance and physical examination measurements of the gastrocnemius–soleus complex. Two supplementary materials (Appendices 2 and 3) provide in-depth discussion about statistical properties of the plantarflexor–knee extension couple index as well as its relationship with statistical clustering.The plantarflexor–knee extension index achieved 98% accuracy and may be suitable for the computational classification of large patient cohorts and multicentre studies. The sagittal gait patterns were strongly related to k-means statistical clustering and physical examination of the gastrocnemius–soleus complex. Patients in crouch gait had normal soleus and gastrocnemius lengths but spasticity in the gastrocnemius. Patients in jump gait exhibited a short gastrocnemius and soleus and gastrocnemius spasticity. Patients in true equinus presented with a moderately contracted soleus and gastrocnemius and gastrocnemius spasticity. Patients in apparent equinus did not show abnormal physical examination measurements for the gastrocnemius–soleus complex.

Does patellar tendon shortening improve the outcome of multilevel surgery in CP children with flexed knee gait?—A randomized clinical trial

The identification of gait patterns in cerebral palsy offers a common language for clinicians and contributes to management algorithms. We describe a quantitative classification of sagittal gait patterns based on the plantarflexor–knee extension couple index. This consists of a scatter plot based on ankle and knee scores, and allows objective identification of the sagittal gait pattern.Sagittal kinematic data from 200 limbs of 100 patients with bilateral spastic cerebral palsy were utilized to validate the algorithm against the assessment of a clinician with expertise in gait pattern identification. A dataset of 776 cerebral palsy patients, 1552 limbs, was used to compare the sagittal gait patterns against k-means statistical clustering. The classification was further explored with respect to the knee kinetics during the middle of stance and physical examination measurements of the gastrocnemius–soleus complex. Two supplementary materials (Appendices 2 and 3) provide in-depth discussion about statistical properties of the plantarflexor–knee extension couple index as well as its relationship with statistical clustering.The plantarflexor–knee extension index achieved 98% accuracy and may be suitable for the computational classification of large patient cohorts and multicentre studies. The sagittal gait patterns were strongly related to k-means statistical clustering and physical examination of the gastrocnemius–soleus complex. Patients in crouch gait had normal soleus and gastrocnemius lengths but spasticity in the gastrocnemius. Patients in jump gait exhibited a short gastrocnemius and soleus and gastrocnemius spasticity. Patients in true equinus presented with a moderately contracted soleus and gastrocnemius and gastrocnemius spasticity. Patients in apparent equinus did not show abnormal physical examination measurements for the gastrocnemius–soleus complex.

Effects of Mechanical Over-Loading on the Properties of Soleus Muscle Fibers, with or without Damage, in Wild Type and Mdx Mice

Effects of mechanical over-loading on the characteristics of regenerating or normal soleus muscle fibers were studied in dystrophin-deficient (mdx) and wild type (WT) mice. Damage was also induced in WT mice by injection of cardiotoxin (CTX) into soleus muscle. Over-loading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mouse muscles by ablation of the distal tendons of plantaris and gastrocnemius muscles. All of the myonuclei in normal muscle of WT mice were distributed at the peripheral region. But, central myonuclei were noted in all fibers of WT mice regenerating from CTX-injection-related injury. Further, many fibers of mdx mice possessed central myonuclei and the distribution of such fibers was increased in response to over-loading, suggesting a shift of myonuclei from peripheral to central region. Approximately 1.4% branched fibers were seen in the intact muscle of mdx mice, although these fibers were not detected in WT mice. The percentage of these fibers in mdx, not in WT, mice was increased by over-loading (∼51.2%). The fiber CSA in normal WT mice was increased by over-loading (p<0.05), but not in mdx and CTX-injected WT mice. It was suggested that compensatory hypertrophy is induced in normal muscle fibers of WT mice following functional over-loading. But, it was also indicated that muscle fibers in mdx mice are susceptible to mechanical over-loading and fiber splitting and shift of myonuclei from peripheral to central region are induced.

Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt

Although it is well known that administration of the selective β(2)-adrenergic agonist clenbuterol (CB) protects muscle following denervation (DEN), the underlying molecular mechanism remains unclear. We report that in vivo treatment with CB (3 mg/kg sc) for 3 days induces antiproteolytic effects in normal and denervated rat soleus muscle via distinct mechanisms. In normal soleus muscle, CB treatment stimulates protein synthesis, inhibits Ca(2+)-dependent proteolysis, and increases the levels of calpastatin protein. On the other hand, the administration of CB to DEN rats ameliorates the loss of muscle mass, enhances the rate of protein synthesis, attenuates hyperactivation of proteasomal and lysosomal proteolysis, and suppresses the transcription of the lysosomal protease cathepsin L and of atrogin-1/MAFbx and MuRF1, two ubiquitin (Ub) ligases involved in muscle atrophy. These effects were not associated with alterations in either IGF-I content or Akt phosphorylation levels. In isolated muscles, CB (10(-6) M) treatment significantly attenuated DEN-induced overall proteolysis and upregulation in the mRNA levels of the Ub ligases. Similar responses were observed in denervated muscles exposed to 6-BNZ-cAMP (500 μM), a PKA activator. The in vitro addition of triciribine (10 μM), a selective Akt inhibitor, did not block the inhibitory effects of CB on proteolysis and Ub ligase mRNA levels. These data indicate that short-term treatment with CB mitigates DEN-induced atrophy of the soleus muscle through the stimulation of protein synthesis, downregulation of cathepsin L and Ub ligases, and consequent inhibition of lysosomal and proteasomal activities and that these effects are independent of Akt and possibly mediated by the cAMP/PKA signaling pathway.

Differential expression of sarcoplasmic and myofibrillar proteins of rat soleus muscle during denervation atrophy.

Denervation is known to induce skeletal muscle atrophy and fiber-type transitions, the molecular mechanisms of which are poorly understood. To investigate the effect of denervation on skeletal muscle, proteomic analysis was performed to compare denervated soleus muscle with normal soleus muscle. The muscles were fractionated to myofibrillar and sarcoplasmic fractions, which were analysed using two-dimensional gel electrophoresis (2-DE), followed by MALDI-TOF-MS. At least 30 differentially regulated proteins were identified in the sarcoplasmic fractions of normal and denervated soleus muscles. This group included metabolic enzymes, signaling molecules, chaperones, and contractile proteins. We also found two proteins, APOBEC-2 (RNA-editing enzyme) and Gamma-synuclein (breast cancer related protein), which have not been recognized as denervation-induced proteins to date. Our results might prove to be beneficial in elucidating the molecular mechanisms of denervation-induced muscle atrophy.

Mechano-sensitivity of normal and long term denervated soleus muscle of the rat

Introduction and objectives: Evidence showed that physical forces, as passive stretching or active contraction, may counteract various kinds of skeletal muscle atrophy due, for instance, to muscle immobilization, pathophysiology or denervation. Accordingly, active muscle contraction induced by functional electric stimulation is helpful to reduce the muscle atrophic state in denervated man. Moreover, there is evidence that also passive mechanical stimulation of the sarcolemnic membrane may reduce the atrophic muscle state. As to the mechanisms by which mechanical stimulation modulates muscle physiology and pathophysiology, there is a growing list of facts that signaling pathway to the nucleus involves stretch activated channels (SACs) of the sarcolemma and the cytoskeleton. SACs activation allowed a Ca2+ inflow that activates Ca2+-dependent molecular signals. Cytoskeleton may be activated by Ca2+-dependent and -independent paths and its contraction and elongation represent not only a mechanical signal to the nucleus but also a stimulus for many molecular signals. The aim of this work was to evaluate in soleus muscle of the rat, the mechano-sensitivity of SACs before and after medium and long term denervation.Methods: Electrophysiologic experiments were made in normal and denervated Soleus muscle of Wistar rats. Currents were recorded in voltage clamp by intracellular microelectrodes inserted in a single fiber.Results: Our findings demonstrated that SACs were expressed in normal soleus muscle and that SAC currents were potentiated by muscle stretching. Another important result was that the sensitivity to stretching increased after denervation and was particularly evident in long term denervated muscles.Discussion: The reported effects are in agreement with the effects of exercise on inducing muscle hypertrophy or with the positive effects on repairing the atrophic state of skeletal muscles by mechanical stimulation or, in denervated humans, by the functional electrical stimulation (FES).

Effect of precocious locomotor activity on the development of motoneurones and motor units of slow and fast muscles in rat

We have investigated the effect of precociously increasing locomotor activity during early postnatal development by daily treatment with the monoaminergic precursor L-DOPA on the survival of motoneurones supplying the slow soleus (SOL) muscle and the fast, tibialis anterior (TA) and extensor digitorum longus (EDL) muscles as well as the contractile and histochemical properties of these muscles. L-DOPA treatment resulted in a significant loss of motoneurones to the slow SOL muscle, but not to the fast TA and EDL muscles. Moreover, motoneurones to fast muscles also die as when exposed to increased activity in early life, if their axons are repeatedly injured. The loss of normal soleus motoneurones was accompanied by an increase in force of the remaining motor units and sprouting of the surviving axons suggesting a remodelling of motor unit organisation. The time to peak contraction of both SOL and EDL muscles from L-DOPA treated rats was prolonged at 8 weeks of age. At 4 weeks the soleus muscles of the L-DOPA treated animal developed more tension than the saline treated one. This difference between the two groups did not persist and by 8 weeks of age the muscle weight and tetanic tension from either group were not significantly different from control animals. The present study shows that early transient, precocious locomotor activity induced by L-DOPA is damaging to normal soleus but not to normal EDL/TA motoneurones.

NFATc1 nucleocytoplasmic shuttling is controlled by nerve activity in skeletal muscle

Calcineurin-NFAT signaling has been shown to control activity-dependent muscle gene regulation and induce a program of gene expression typical of slow oxidative muscle fibers. Following Ca2+-calmodulin stimulation, calcineurin dephosphorylates NFAT proteins and induces their translocation into the nucleus. However, NFAT nuclear translocation has never been investigated in skeletal muscle in vivo. To determine whether NFATc1 nucleocytoplasmic shuttling depends on muscle activity, we transfected fast and slow mouse muscles with plasmids coding for an NFATc1-GFP fusion protein. We found that NFATc1-GFP has a predominantly cytoplasmic localization in the fast tibialis anterior muscle but a predominantly nuclear localization in the slow soleus muscle, with a characteristic focal intranuclear distribution. Two hours of complete inactivity, induced by denervation or anaesthesia, cause NFATc1 export out of the nucleus in soleus muscle fibers, whereas electrostimulation of tibialis anterior with a low-frequency tonic impulse pattern, mimicking the firing pattern of slow motor neurons, causes NFATc1 nuclear translocation. The activity-dependent nuclear import and export of NFATc1 is a rapid event, as visualized directly in vivo by two-photon microscopy. The calcineurin inhibitor cain/cabin1 causes nuclear export of NFATc1 both in normal soleus and stimulated tibialis anterior muscle. These findings support the notion that in skeletal muscle NFATc1 is a calcineurin-dependent nerve activity sensor.

Effects of Ovarian Hormones on Rat Soleus Skeletal Muscle Regrowth during Reloading after Disuse

1034 PURPOSE: Ovarian hormones can mediate skeletal muscle damage and may play a role in the mediation of regrowth processes regulated by reloading after disuse. The purpose of this study was to determine what role ovarian hormones play in skeletal muscle regrowth after disuse atrophy. METHODS: Female Sprague Dawley rats (N = 65) were randomly assigned to control (Normal) and ovariectomized (OVX) treatments. Animals were then assigned to cage control (Con), 10-days of hindlimb suspension (Sus), 10-days of hindlimb suspension with 7-days of reloading (Sus+7), or 10-days of hindlimb suspension with 14-days of reloading (Sus+14) treatment groups. RESULTS: Muscle wt/tibia length (mg/mm) returned to Con values with Sus+7 in Normal soleus muscle, but remained decreased 32% with Sus+7 and 11% with Sus+14 in OVX soleus muscle. Normal soleus muscle mean cross sectional area (CSA) decreased 46% with Sus (1280 ± 103 μm2) but returned to Con (2363 ± 118 μm2) values with Sus+14 (2245 ± 226 μm2). OVX soleus CSA decreased 52% with Sus (1158 ± 196 μm2) and remained decreased 37% from Con (2423 ± 116 μm2) values with Sus+14 (1518 ± 233 μm2). Sus (9.4 ± 2.3%), Sus+7 (22.4 ± 3.7%), and Sus+14 (17.8 ± 1.7%) increased the percentage of small (< 500 μm2) fibers 1243%, 762%, and 128% respectively, in Normal soleus muscle. Only Sus+14 (30.7 ± 6.9%), however, increased small fibers 333% in the OVX soleus. CONCLUSIONS: The results of this study suggest that ovariectomy impairs the normal regenerative processes associated with reloading after disuse atrophy. Supported by a Grant from the South Carolina Space Grant Consortium and an American College of Sports Medicine/NASA Student Grant.

Effects of nerve growth factor on the neurotization of denervated muscles.

Studies on surgical repair techniques of the peripheral nerve are still trying to improve the outcome. There are many studies on the effects of various neurotrophic factors on the transected peripheral nerve. Muscular neurotization, which is the direct implantation of the nerve to the target denervated skeletal muscle, is one of the techniques used when the primary repair of the peripheral nerves is not possible. The effects of nerve growth factor (NGF), which is one of the primary neurotrophic factors, on the reinnervation of denervated muscles by neurotization is investigated in this experimental study. The denervated soleus muscle was neurotized via peroneal nerve implantation (group 1), and NGF was administered to the neurotized muscle (group 2). All animals were evaluated at weeks 8, 10, and 12 using electromyography. Muscle contractility, muscle weight, and histological morphometric tests were performed at week 12. The experimental groups were compared with each other and normal control values. Electromyographically, group 2 (direct nerve implantation + NGF) demonstrated better reinnervation in all evaluations. The study of muscle weight showed that the muscle mass was 75% of the normal soleus muscle in group 1 and was 85% of the normal side in group 2 at the end of week 12. In group 1, the twitch force was 56% of the normal soleus muscle and was 71% in group 2. Tetanic force was 53% of the normal soleus muscle in group 1 and 68% in group 2. Histological morphometric studies revealed that there was a decrease in the density of the motor end plates in group 1, but there was no statistically significant difference between the normal soleus muscles and the NGF applied to group 2. The positive effects of NGF on the neurotization of denervated muscles seen in this study suggest that it may be useful for treating some difficult reconstructions caused by denervation.

Transection of the sciatic nerve and reinnervation in adult rats: muscle and endplate morphology.

The functional recovery after peripheral nerve lesions is generally poor. We studied whether changes in muscles after reinnervation might explain such disappointing results. The functional recovery after peripheral nerve lesions is generally poor. Changes in muscle morphology and neuromuscular innervation might partly explain this lack of compensation. In order to test this hypothesis, we studied muscular differentiation in the soleus, gastrocnemius and tibialis anterior muscles at 7, 15 and 21 weeks after a sciatic nerve lesion in adult rats. In the gastrocnemius and tibialis muscles the percentages of type II muscles fibres were decreased at 7 and 15 weeks but at 21 weeks they again approached normal values. The soleus muscle, however, was permanently decreased in size and this muscle, in contrast to the normal soleus muscle, contained mainly type II fibres. The morphology of the endplates showed distinct stages of degeneration and reinnervation. Two weeks after denervation, in rats in which reinnervation was prevented, all 3 muscles contained considerable numbers of morphologically abnormal endplates and, after 7 weeks, no endplates were detected. During reinnervation, endplates showing signs of acetylcholinesterase activity were observed in all 3 muscles from 7 weeks. At later ages a shift towards morphologically normal endplates occurred, but complete recovery was not observed. Endplates in all 3 muscles were polyneurally innervated at 7 weeks. Although these percentages decreased over age, polyneural innervation was still present at 21 weeks. We conclude that the changes in the distribution of fibre types, abnormal endplate morphology and polyneural innervation may in part explain the poor functional recovery after peripheral nerve lesions.

Treatment of the neuromuscular junction with 4-aminopyridine results in improved reinnervation following nerve injury in neonatal rats.

During early postnatal development, nerve injury results in the death of a large proportion of motoneurones and poor recovery of muscle function. Our previous results have shown that premature enhancement of transmitter release from nerve terminals prevents the death of motoneurones following neonatal nerve injury. Whether this increase in motoneurone survival is reflected in an improvement in the reinnervation of muscle was studied here. The muscles in one hindlimb of newborn rats were treated with 4-aminopyridine. Three days later, the sciatic nerve was crushed in the treated leg. When the animals were seven, 14 and 21days of age, the soleus and extensor digitorum longus muscles were removed and processed for GAP-43 (a 43-kDa growth-associated protein) and synaptophysin immunocytochemistry. Both GAP-43 and synaptophysin were expressed in normal soleus and extensor digitorum longus muscles at seven days. Synaptophysin was still expressed at 14 days, but GAP-43 expression had declined. Following nerve injury at three days of age, there was no GAP-43 or synaptophysin immunoreactivity in nerve terminals at seven days. By 21 days, there were 17.3+/-2.1 GAP-43-positive terminals per section in the soleus and 17.7+/-1.4 in the extensor digitorum longus, with mean terminal areas of 47.5+/-3.3 and 49.8+/-2.6 microm(2), respectively. In animals in which nerve crush was preceded by 4-aminopyridine treatment, at 21 days there were 32.9+/-2.6 GAP-43-immunoreactive terminals in the soleus and 44.9+/-2.3 in the extensor digitorum longus, with a mean area of 122.7+/-6.6 microm(2) in the soleus and 136.2+/-9.7 microm(2) in the extensor digitorum longus. These results indicate that in muscles pretreated with 4-aminopyridine, prior to nerve crush at three days, there are significantly more terminals, which occupy a larger area than in untreated muscles. Thus, increasing transmitter release prior to nerve injury significantly improved the ability of axons to reinnervate muscle.

Ciliary neurotrophic factor prevents unweighting-induced functional changes in rat soleus muscle.

The purpose of the present work was to see whether changes in rat soleus characteristics due to 3 wk of hindlimb suspension could be modified by ciliary neurotrophic factor (CNTF) treatment. Throughout the tail suspension period, the cytokine was delivered by means of an osmotic pump (flow rate 16 microg. kg(-1). h(-1)) implanted under the hindlimb skin. In contrast to extensor digitorum longus, CNTF treatment was able to reduce unweighting-induced atrophy in the soleus. Twitch and 146 mM potassium (K) tensions, measured in small bundles of unloaded soleus, decreased by 48 and 40%, respectively. Moreover, the time to peak tension and the time constant of relaxation of the twitch were 48 and 54% faster, respectively, in unloaded soleus than in normal muscle. On the contrary, twitch and 146 mM K contracture generated in CNTF-treated unloaded and normal soleus were not different. CNTF receptor-alpha mRNA expression increased in extensor digitorum longus and soleus unloaded nontreated muscles but was similar in CNTF-treated unloaded muscles. The present results demonstrate that exogenously provided CNTF could prevent functional changes occurring in soleus innervated muscle subject to unweighting.

Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles.

The mitotic activity in muscles of growing rats and the effect of denervation were studied by means of continuous infusion of 5-bromo-2-deoxyuridine (BRDU). Denervated muscles after 10 weeks contained 20 to 60% fewer muscle nuclei than normal; BRDU labeled about 25% of the nuclei of normal soleus and extensor digitorum longus (EDL) and of denervated EDL muscles but only 5% in the denervated soleus muscle. Labeled nuclei persisted in denervated but not in normal muscles. After the main growth period, the turnover of myonuclei was at most 1 to 2% per week. The behavior of connective tissue nuclei was similar to that in muscle fibers. Infusion of BRDU had no effect on contractile properties. It is suggested that the exceptionally rapid atrophy of the denervated rat soleus associated with loss of satellite cells was due to loss of myonuclei and differentiation and fusion of satellite cells. The cause may possibly be that the phase of postdenervation fibrillation is shorter than in other muscles.

Parathyroid hormone-related protein is rapidly up-regulated in blood vessels of rat skeletal muscle by low-frequency stimulation.

To identify early changes in gene expression of fast-twitch rat muscle exposed to chronic low-frequency stimulation, differential display was applied comparing mRNA patterns between control and 12-h stimulated tibialis anterior (TA) muscles. Among the signals of differentially expressed mRNAs, a cDNA of approximately 300 bp was identified as specific to the parathyroid-hormone-related protein (PTHrP). As verified by semi-quantitative reverse transcriptase polymerase chain reaction, this mRNA was present at low levels in normal slow-twitch soleus and fast-twitch TA muscles, but was approximately 80-fold elevated in TA muscles after 12 h of low-frequency stimulation. With ongoing stimulation for 2 or 4 days PTHrP mRNA returned to basal levels. PTHrP was located exclusively in blood vessels of both control and stimulated muscles. PTHrP immunohistochemistry revealed enhanced capillarization in 8-day stimulated muscles. In view of its vasodilative effect the up-regulation of PTHrP could be an important initial step related to enhanced capillarization in response to increased contractile activity.

Age effect on expression of myosin heavy and light chain isoforms in suspended rat soleus muscle.

This study was designed to test the hypothesis that myosin heavy (MHC) and light chain (MLC) plasticity resulting from hindlimb suspension (HS) is an age-dependent process. By using an electrophoretic technique, the distribution of MHC and MLC isoforms was quantitatively evaluated in the soleus muscles from 3- or 12-wk-old rats after 1-3 wk of HS treatment was maintained. In normal 12- and 15-wk-old rats, the soleus muscles contained a predominance of MHCI ( approximately 94%) with small amounts of MHCIIa, but not MHCIId or MHCIIb. The suspended muscles of adult rats were characterized by the appearance of MHCIIb and MHCIId, the latter reaching approximately 6% after 3 wk of HS treatment. In contrast to changes in MHC, HS did not induce a transition in the MLC pattern in the soleus muscles from adult rats. Compared with adult rats, in juveniles HS had a much more pronounced effect on the shift toward faster MHC and MLC isoform expression. The soleus muscles of 6-wk-old rats after 3 wk of HS were composed of 37.0% MHCI, 19.1% MHCIIa, 23.7% MHCIId, and 20.2% MHCIIb. Changes in MLC isoforms consisted of an increase in MLC1f and MLC2f concomitant with a decrease in MLC2s. These results indicate the existence of a differential effect of HS on MHC and MLC transitions that appears to be age dependent. They also suggest that the suspended soleus muscles from young rats may acquire the intrinsic contractile properties that are intermediate between those in the normal soleus and typical fast-twitch skeletal muscles.

Muscle LIM protein: expressed in slow muscle and induced in fast muscle by enhanced contractile activity.

To identify early changes in gene expression during the fast-to-slow transition induced by chronic low-frequency stimulation, total RNA was extracted from 12-h-stimulated tibialis anterior (TA) muscles of rats and amplified by differential display RT-PCR. Among the signals of differentially expressed mRNAs, a cDNA approximately 300 bp in length, which was almost undetectable in control TA muscles but prominent in stimulated TA and normal soleus muscles, was identified. This cDNA was cloned and identified as corresponding to the mRNA of the muscle LIM protein (MLP). Its differential expression in control, stimulated TA, and soleus muscles was verified by Northern blotting. Antibodies against MLP were used to identify by immunoblot analysis a protein of 22 kDa, the predicted molecular mass of MLP. Immunohistochemistry revealed strong reactivity for MLP in all fibers of normal soleus muscle and faint staining of some type IIA and type I fibers in control TA muscle. These fibers increased in number and staining intensity in 4-day-stimulated TA muscle. MLP thus seems to play an essential role during the rearrangement of cytoskeletal and/or myofibrillar structures in transforming adult muscle fibers.

PGAM-M expression is regulated pretranslationally in hindlimb muscles and under altered loading conditions.

Enzymatic activity from the muscle-specific isoform of phosphoglycerate mutase (PGAM-M) is higher within glycolytic skeletal muscles than in oxidative muscles. The hypothesis that PGAM-M is regulated pretranslationally among muscles of the hindlimb was tested using enzymatic assays, Western blots, and Northern blots. We further investigated the regulatory level(s) at which PGAM-M gene expression is controlled during hindlimb unweighting. PGAM-M mRNA and immunoreactive protein levels were fourfold lower in the rat soleus muscle than in the tibialis anterior (TA), plantaris, and extensor digitorum longus muscles. Four weeks of unweighting induced a 2.5-fold increase in PGAM enzymatic activity within the soleus muscle, a 1.8-fold increase in PGAM-M immunoreactivity, and a 3. 5-fold increase in PGAM-M mRNA. To examine potential transcriptional regulatory mechanisms, the proximal 400 bp of the rat PGAM-M promoter were linked to a firefly luciferase and injected into normal and unweighted TA and soleus muscles. Firefly luciferase activity was elevated two- to threefold in the TA and the unweighted soleus over the normal soleus muscle. These data suggest that PGAM-M expression is pretranslationally regulated among muscle types and within unweighted slow-twitch muscle. Furthermore, the proximal 400 bp of the PGAM-M promoter contains cis-acting sequences to allow muscle-type-specific expression of a reporter gene and responsiveness to soleus muscle unweighting.