Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Effects of chronic AICAR administration on the metabolic and contractile phenotypes of rat slow- and fast-twitch skeletal muscles

The present study examined the effects of chronic activation of 5'-AMP-activated protein kinase (AMPK) on the oxidative capacity and myosin heavy chain (MHC) based fibre phenotype of rodent fast- and slow-twitch muscles. Sprague-Dawley rats received daily injections for 4 weeks of the known AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) or vehicle (control). The AICAR group displayed increases in hexokinase-II (HXK-II) activity, expression, and phosphorylation in fast-twitch muscles (P<0.001) but not in the slow-twitch soleus (SOL). In the AICAR group, citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35) were elevated 1.6- and 2.1-fold (P<0.05), respectively, in fast-twitch medial gastrocnemius (MG), and by 1.2- and 1.4-fold (P<0.05) in the slower-twitch plantaris (PLANT). No changes were observed in the slow-twitch SOL. In contrast, the activity of glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12) remained unchanged in all muscles. AICAR treatment did not alter the MHC-based fibre type composition in fast- or slow-twitch muscles, as determined by immunohistochemical and electrophoretic analytical methods or by RT-PCR. We conclude that chronic activation of AMPK mimics the metabolic changes associated with chronic exercise training (increased oxidative capacity) in the fast-twitch MG and PLANT, but does not coordinately alter MHC isoform content or mRNA expression.     

Effects of aging and denervation on the expression of uncoupling proteins in slow- and fast-twitch muscles of rats

We investigated the effects of aging and denervation on the gene expression of uncoupling proteins (UCPs) in slow-twitch soleus and fast-twitch gastrocnemius muscles. In a comparison between the control limbs of 6- and 24-month-old rats, the mRNA levels of UCP3, heart-type fatty acid binding protein (HFABP), and glucose transporter-4 (GLUT4) were considerably lower in the gastrocnemius muscles of the older rats, whereas no significant differences in the mRNA levels of those genes as well as UCP2 and cytochrome oxidase subunit IV (COX-IV) were observed in the soleus muscles of young and old rats. The UCP3 and COX-IV protein levels were also reduced considerably in the aged gastrocnemius muscles with atrophy. Denervation of the sciatic nerve caused an increase in UCP3 mRNA levels in both muscles, but the regulation of other genes contrasted between the two types of skeletal muscles. In spite of the increased mRNA level, a remarkable reduction in UCP3 protein was found in the denervated gastrocnemius muscles. These results indicate that the effects of aging and denervation on the gene expression of UCPs, HFABP, GLUT4, and COX-IV are different between the muscle types. The reduction in the mitochondrial UCP3 and COX proteins in aged fast-twitch muscles may have a negative effect on energy metabolism and thermogenesis in old animals.     

Fasting-related autophagic response in slow- and fast-twitch skeletal muscle

This study investigated regulation of autophagy in slow-twitch soleus and fast-twitch plantaris muscles in fasting-related atrophy. Male Fischer-344 rats were subjected to fasting for 1, 2, or 3 days. Greater weight loss was observed in plantaris muscle than in soleus muscle in response to fasting. Western blot analysis demonstrated that LC3-II, a marker protein for macroautophagy, was expressed at a notably higher level in plantaris than in soleus muscle, and that the expression level was fasting duration-dependent. To identify factors related to LC3-II enhancement, autophagy-related signals were examined in both types of muscle. Phosphorylated mTOR was reduced in plantaris but not in soleus muscle. FOXO3a and ER stress signals were unchanged in both muscle types during fasting. These findings suggest that preferential atrophy of fast-twitch muscle is associated with induction of autophagy during fasting and that differences in autophagy regulation are attributable to differential signal regulation in soleus and plantaris muscle.     

Pre- and post-natal growth and protein turnover in smooth muscle, heart and slow- and fast-twitch skeletal muscles of the rat.

The growth of one smooth and three individual striated muscles was studied from birth to old age (105 weeks), and where possible during the later stages of foetal life also. Developmental changes in protein turnover (measured in vivo) were related to the changing patterns of growth within each muscle, and the body as a whole. Developmental growth (i.e. protein accumulation) in all muscles involved an increasing proportion of protein per unit wet weight, as well as cellular hypertrophy. The contribution of the heart towards whole-body protein and nucleic acid contents progressively decreased from 18 days of gestation to senility. In contrast, post-natal changes in both slow-twitch (soleus) and fast-twitch (tibialis anterior) skeletal muscles remained reasonably constant with respect to whole-body values. Such age-related growth in all four muscle types was accompanied by a progressive decline in both the fractional rates of protein synthesis and breakdown, the changes in synthesis being more pronounced. Age for age, the fractional rates of synthesis were highest in the oesophageal smooth muscle, similar in both cardiac and the slow-twitch muscles, and lowest in the fast-twitch tibialis muscle. Despite these differences, the developmental fall in synthetic rates was remarkably similar in all four muscles, e.g. the rates at 105 weeks were 30-35% of their values at weaning. Such developmental changes in synthesis were largely related to diminishing ribosomal capacities within each muscle. When measured under near-steady-state conditions (i.e. 105 weeks of age), the half-lives of mixed muscle proteins were 5.1, 10.4, 12.1 and 18.3 days for the smooth, cardiac, soleus and tibialis muscles respectively. Old-age atrophy was evident in the senile animals, this being more marked in each of the four muscle types than in the animal as a whole. In each muscle of the senile rats the protein content and composition per unit wet weight, and both the fractional and total rates of synthesis, were significantly lower than in the muscles of younger, mature, animals (i.e. 44 weeks). In the soleus the decreased synthesis rate appeared to be related to a further fall in the ribosomal capacity. In contrast, the changes in synthesis in the three remaining muscles correlated with significant decreases in the synthetic rate per ribosome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential modulation of carbonic anhydrase (CA III) in slow- and fast-twitch skeletal muscles of rat following denervation and reinnervation.

Carbonic anhydrase III (CA III) is influenced by neuronal factors in skeletal muscles of the rat. CA III protein and its mRNA levels were assessed in slow- and fast-twitch muscles after short-term denervation by ligature of the sciatic nerve and reinnervation following removal of the sheath tightly fixed around the nerve. Significant elevations in the CA III mRNA content of fast-twitch muscles were recorded after denervation, but they were cancelled following spontaneous muscle reinnervation. No such variations were observed in the slow-twitch soleus muscle. CA III specific activity or cytosolic CA III protein content increased in both types of muscles after denervation, while a decrease was solely observed in the soleus after reinnervation. These results suggest that neuronal mediators may be responsible for up and down variations in CA III gene expression and (or) mRNA stability in slow- and fast-twitch muscles exposed to identical stimuli. Variations of the mRNA and the protein probably reflect, in a time-related manner, the well-programmed changes in fiber type of the muscles in the context of the denervation-reinnervation model.     

Properties of slow- and fast-twitch skeletal muscle from mice with an inherited capacity for hypoxic exercise

Muscle fiber type, myosin heavy chain (MHC) isoform composition, capillary density (CD) and citrate synthase (CS) activity were investigated in predominantly slow-twitch (soleus or SOL) and fast-twitch (extensor digitorum longus or EDL) skeletal muscle from mice with inherited differences in hypoxic exercise tolerance. Striking differences in hypoxic exercise tolerance previously have been found in two inbred strains of mice, Balb/cByJ (C) and C57BL/6J (B6), and their F1 hybrid following exposure to hypobaric hypoxia. Mice from the three strains were exposed for 8 weeks to either normobaric normoxia or hypobaric hypoxia (1/2 atm). Hypoxia exposure led to a slightly higher 2b fiber composition and a lower fiber area of types 1 and 2a in SOL of all mice. In the EDL, muscle fiber and MHC isoform composition remained unaffected by chronic hypoxia. Chronic hypoxia did not significantly affect CD in either muscle from any of the three strains. There were relatively larger differences in CS activity among strains and treatment, and in SOL the highest CS activity was found in the F1 mice that had been acclimated to hypoxia. In general, however, neither differences among strains nor treatment in these properties of muscle vary in a way that clearly relates to inherited hypoxic exercise tolerance.     

Elevated growth hormone increases the Ca2+ sensitivity of slow- and fast-twitch skeletal muscle of female rats

To determine theeffect of plasma growth hormone (GH) on skeletal muscle function, wemeasured the free Ca²⁺concentration-tension relationship of slow-twitch (soleus) and fast-twitch (peroneus longus) muscles isolated from rats undergoing acromegaly in response to implanted, GH-secreting tumors. Muscles fromadult (9 mo) and aged rats (24 mo) were studied after the tumor-bearingrats weighed over 50% more than their age-matched controls.Ca²⁺-activated isometric tensionwas recorded from skinned muscle fibers. For soleus muscles, the freeCa²⁺ concentration producing 50%of maximal tension([Ca²⁺]₅₀)was 2.0 µM for rats with tumors and 3.4-3.6 µM for controls. For peroneus longus fibers,[Ca²⁺]₅₀shifted from 6.1-6.7 µM in controls to 3.5 µM after tumors were introduced into either adult or aged rats. Soleus muscle fibersfrom neonatal rats (14 days) were less sensitive toCa²⁺ than those isolated fromadult rats, having a[Ca²⁺]₅₀of 7.3 µM. The Ca²⁺ sensitivityof peroneus longus fibers did not change with age. We conclude thatsignificant increases in myofibrillarCa²⁺ sensitivity occur in skeletalmuscles undergoing rapid growth induced by GH-secreting tumors.

     

Regulation of carbonic anhydrase III by thyroid hormone: opposite modulation in slow- and fast-twitch skeletal muscle

This laboratory previously reported that a major 30 kilodalton (kDa) protein of the soluble cytoplasmic fraction of the rat slow-twitch soleus muscle is modulated by thyroid hormone. This protein has been purified and a portion of the primary structure has been determined. The sequence analysis suggested that the 30-kDa protein is carbonic anhydrase III (CA III; EC 4.2.1.1). The reaction of the protein with a CA III specific antibody and the similar modulation of CA III by thyroid hormone also support this conclusion. Immunochemical quantification of CA III and measurement of CA activity were performed in skeletal muscles of defined fiber-type composition from rats that were rendered hyperthyroid by treatment with 3,3',5-triiodo-L-thyronine. These experiments revealed that CA activity and CA III content are deinduced in the soleus muscle (primarily type I fibers) and induced in the superficial vastus lateralis muscle (primarily type IIb), whereas no changes were detected in the tibialis anterior muscle (primary type IIa). These results show that the modulation of CA III by thyroid hormone in rat skeletal muscle is not limited to the slow-twitch soleus muscle and that the amplitude and direction of this modulation are directly related to the initial fiber-type composition of the skeletal muscle.     

Quantitative morphology of stimulation-induced damage in rabbit fast-twitch skeletal muscles

Summary The purpose of this study was to examine the contention that stimulation-induced damage, resulting in degeneration with subsequent regeneration, plays a major role in the transformation of fibre type brought about by chronic electrical stimulation. Data from histological and histochemical sections of 9-day-stimulated rabbit fast-twitch muscles were analysed with multivariate statistical techniques. Fibre degeneration and regeneration varied non-systematically between sample areas at any given cross-sectional level. In the extensor digitorum longus muscle, but not in the tibialis anterior, there was more degeneration in proximal than in distal portions of the muscle. The extensor digitorum longus muscle consistently showed more degeneration than the tibialis anterior muscle. Degeneration was less extensive for an intermittent pattern of stimulation that delivered half the aggregate number of impulses of continuous stimulation. Degeneration and regeneration varied markedly between individual rabbits in each of the groups. Sections that revealed the most degeneration and regeneration also had more fibres that reacted positively with an anti-neonatal antibody. Rigorous analysis of different sources of variation has helped to explain apparent conflicts in the literature. The incidence of muscle fibre damage in the stimulated tibialis anterior muscle is low, showing that the contribution of degenerative-regenerative phenomena to fibre type conversion in this muscle is insignificant.     

Prolonged contraction-relaxation cycle of fast-twitch muscles in parvalbumin knockout mice

Thecalcium-binding protein parvalbumin (PV) occurs at high concentrationsin fast-contracting vertebrate muscle fibers. Its putative role infacilitating the rapid relaxation of mammalian fast-twitch musclefibers by acting as a temporary buffer for Ca²⁺ is still controversial. Wegenerated knockout mice for PV (PV /) and compared theCa²⁺ transients and the dynamicsof contraction of their muscles with those from heterozygous (PV+/) and wild-type (WT) mice. In the muscles of PV-deficientmice, the decay of intracellularCa²⁺ concentration([Ca²⁺]_i)after 20-ms stimulation was slower compared with WT mice and led to aprolongation of the time required to attain peak twitch tension and toan extension of the half-relaxation time. The integral [Ca²⁺]_iin muscle fibers of PV / mice was higher and consequently the force generated during a single twitch was ~40% greater than inPV +/ and WT animals. Acceleration of the contraction-relaxation cycle of fast-twitch muscle fibers by PV may confer an advantage in theperformance of rapid, phasic movements.     

Loss of fast-twitch isomyosins in skeletal muscles of the diabetic rat.

By means of pyrophosphate electrophoresis the myosin isoenzyme pattern of two fast-twitch skeletal muscles (extensor digitorum longus, gastrocnemius) and one slow-twitch muscle (soleus) was investigated in control rats and was compared with that of rats 4 weeks after induction of diabetes mellitus by streptozotocin injection. In the fast-twitch muscles the isomyosin pattern consisting of FM1 (fast isomyosin 1), FM2 and FM3 was strongly affected by diabetes, resulting in an extensive loss of FM1 and a substantial decrease of FM2. These changes were also apparent when the light chains of the fast isomyosins were analysed by two-dimensional electrophoresis: LC3f (myosin light chain 3f) largely disappeared and LC2f was significantly diminished. In contrast, the isomyosin pattern in soleus muscle, consisting of SM1 (slow isomyosin 1) and SM2, was not affected by the diabetic state, and two-dimensional electrophoresis revealed a normal light-chain pattern of LC1sa, LC1sb and LC2s. These results indicate that the isomyosins of slow-twitch oxidative myofibres are more resistant to the hormonal and metabolic disorders during diabetes mellitus than are the isomyosins of fast-twitch fibres.     

Differential effects of thyroid status on regional H2O2 production in slow- and fast-twitch muscle of ducklings

Birds seem to employ powerful physiological strategies to curb the harmful effects of reactive oxygen species (ROS) because they generally live longer than predicted by the free radical theory of aging. However, little is known about the physiological mechanisms that confer protection to birds against excessive ROS generation. Hence, we investigated the ability of birds to control mitochondrial ROS generation during physiologically stressful periods. In our study, we analyzed the relationship between the thyroid status and the function of intermyofibrillar and subsarcolemmal mitochondria located in glycolytic and oxidative muscles of ducklings. We found that the intermyofibrillar mitochondria of both glycolytic and oxidative muscles down regulate ROS production when plasma T₃ levels rise. The intermyofibrillar mitochondria of the gastrocnemius muscle (an oxidative muscle) produced less ROS and were more sensitive than the pectoralis muscle (a glycolytic muscle) to changes in plasma T₃. Such differences in the ROS production by glycolytic and oxidative muscles were associated with differences in the membrane proton permeability and in the rate of free radical leakage within the respiratory chain. This is the first evidence which shows that in birds, the amount of ROS that the mitochondria release is dependent on: (1) their location within the muscle; (2) the type of muscle (glycolytic or oxidative) and (3) on the thyroid status. Reducing muscle mitochondrial ROS generation might be an important mechanism in birds to limit oxidative damage during periods of physiological stress.     

Myosin heavy-chain-based isomyosins in developing, adult fast-twitch and slow-twitch muscles.

A modified method of electrophoresis under nondenaturing conditions made it possible to separate rat muscle extracts of defined myosin heavy chain (HC) and light chain (LC) composition into subsets of developmental, fast and slow myosin heavy-chain-based isomyosins. The fastest migrating isomyosins were the neonatal isomyosins (nM1, nM2, nM3), followed by the slightly slower migrating embryonic isomyosins (eM1, eM2, eM3, eM4). Of the nine adult fast isomyosins, the HCIIb-based isomyosins (FM1b, FM2b, FM3b) were the fastest migrating. These were followed by the HCIId-based isomyosins (FM1d, FM2d, FM3d). The HCIIa-based isomyosins (FM1a, FM2a, FM3a) were the slowest. Our results suggest that FM3a is identical with the so-called intermediate isomyosin (IM) described in the literature. The slow myosin heavy-chain-based isomyosins (SM1, SM2, SM3) migrated far behind the fast isomyosins. Whereas the gross electrophoretic mobilities of each of these isomyosin triplets is determined by the specific heavy chain complement, the different mobilities of the bands within each triplet result from different alkali light chain combinations. Thus, the fastest triplet bands of the neonatal (nM1) and adult fast isomyosins (FM1b, FM1d, FM1a) represent the LC3f homodimers, the slowest (nM3, FM3b, FM3d, FM3a) the LC1f homodimers, and the intermediate bands (nM2, FM2b, FM2d, FM2a) the LC1f/LC3f heterodimers. Different proportions of the adult fast isomyosin triplet bands indicate that the affinity for LC3f decreases in the order HCIIb, HCIId, HCIIa. The three slow isomyosins represent LC1sa (SM1) and LC1sb (SM3) homodimers and a LC1sa/LC1sb heterodimer (SM2). Circumstantial evidence suggests an inverse order in rabbit muscle where SM1 and SM3 most likely represent LC1sb and LC1sa homodimers, respectively.     

Size and metabolic properties of fibers in rat fast-twitch muscles after hindlimb suspension

Hindlimb suspension (HS) results in whole muscle atrophic and metabolic changes that vary in magnitude in different hindlimb muscles. The present study was designed to investigate these effects in single fibers. Fiber type and size and the activities of two metabolic marker enzymes were determined in a deep (close to the bone) and a superficial (away from the bone) region of the medial gastrocnemius (MG) and the tibialis anterior (TA) of control (CON) and 28-day HS adult female rats. Fibers were classified as dark or light adenosinetriphosphatase (ATPase) based on their qualitative staining reaction for myosin ATPase following alkaline preincubation. Fiber area and succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities were determined in tissue sections by use of an image analysis system. After 28 days of HS, the mean body weights of the CON and HS were similar. MG atrophied 28%, whereas TA weight was maintained in the HS. Both dark and light ATPase fibers in the deep region of the MG had smaller cross-sectional areas following HS, with the atrophic response being approximately twice as great in the light ATPase fibers. No significant changes in fiber type composition in either muscle or in fiber sizes in the superficial region of the MG or in either region of the TA were observed. Mean SDH activities of both fiber types were significantly lower in the MG and TA following HS. In contrast, mean GPD activities were either increased or maintained in light and dark ATPase fibers of both muscles in HS. Changes in SDH and GPD activity could not be directly linked to changes in fiber cross-sectional area. In summary, these data suggest an independence of the mechanisms determining muscle fiber size and metabolic adaptations associated with HS.     

Parvalbumin expression is downregulated in rat fast-twitch skeletal muscles during aging

In this study, the protein expression profile of extensor digitorum longous (EDL) and Soleus (SOL) muscles, representing fast- and slow-twitch skeletal muscles, respectively, was established using high resolution two-dimensional electrophoresis (2-DE). One protein spot was found uniquely expressed in EDL muscle. N-terminal sequence analysis identified the protein as parvalbumin. Parvalbumin is a high affinity calcium binding protein that regulates muscle contraction and relaxation. Our experiments revealed that parvalbumin expression in EDL muscle was down-regulated during aging. In addition, high-intensity exercise could reverse this age-related change. Soleus muscles do not normally express parvalbumin, but high-intensity exercise could ectopically induce its expression in both young and old SOL muscles. We have also confirmed our 2-DE findings by immunohistochemistry on muscle sections. Our results suggest that high-intensity training could be used to improve muscle functions during aging because parvalbumin play an important role in regulating skeletal muscle contraction and relaxation.