Regulation of HSP25 expression and phosphorylation in functionally overloaded rat plantaris and soleus muscles.

Functional overload (FO) is a powerful inducer of muscle hypertrophy and both oxidative and mechanical stress in muscle fibers. Heat shock protein 25 (HSP25) may protect against both of these stressors, and its expression can be regulated by changes in muscle loading and activation. The primary purpose of the present study was to test the hypothesis that chronic FO increases HSP25 expression and phosphorylation (pHSP25) in hypertrophying rat hindlimb muscle. HSP25 and pHSP25 levels were quantified in soluble and insoluble fractions of the soleus and plantaris to determine whether 3 or 7 days of FO increase translocation of HSP25 and/or pHSP25 to the insoluble fraction. p38 protein and phosphorylation (p-p38) was measured to determine its association with changes in pHSP25. HSP25 mRNA showed time-dependent increases in both the soleus and plantaris with FO. Three or seven days of FO increased HSP25 and pHSP25 in the soluble fraction in both muscles, with a greater response in the plantaris. In the insoluble fraction, HSP25 was increased after 3 or 7 days in both muscles, whereas pHSP25 was only increased in the 7-day plantaris. p38 and p-p38 increased in the plantaris at both time points. In the soleus, p-p38 only increased after 7 days. These results show that FO is associated with changes in HSP25 expression and phosphorylation and suggest its role in the remodeling that occurs during muscle hypertrophy. Increases in HSP25 in the insoluble fraction suggest that it may help to stabilize actin and/or other cytoskeletal proteins during the stress of muscle remodeling.     

Effect of hindlimb immobilization and recovery on compensatory hypertrophied rat plantaris muscle

Compensatory hypertrophy of the rat plantaris muscle (PLT) was induced by ipsilateral gastrocnemius muscle ablation. Following 8 weeks (wks) of hypertrophy, hindlimbs were cast immobilized (HI) for 4 weeks after which weight bearing was unrestricted for 8 wks (recovery). Compensatory hypertrophy increased PLT wet weight/body weight ratio (83%), muscle fiber cross-sectional areas (1.5 to 2 fold), and the percent of slow oxidative (% SO) fibers (2 fold) in the experimental compared to the contralateral sham control muscle. PLT protein content and maximal activities of phosphofructokinase (PFK), mitochondrial glycerol phosphate dehydrogenase, and succinate dehydrogenase were unaltered with muscle hypertrophy. HI produced significant decreases in PFK activity (50%) and muscle fiber cross-sectional areas (50%) but did not significantly change the histochemical myofibrillar ATPase profile. Following remobilization, muscle weight/body weight ratio and maximal enzyme activities recovered to that of aged matched controls. Muscle fiber areas returned to pre-immobilization sizes but were approximately 25% smaller than aged matched control hypertrophy muscles. The % SO fibers in the hypertrophied muscle remained higher than controls but did not return to pre-immobilization values. These results indicate that biochemical and histochemical characteristics of hypertrophied rat PLT recover from HI during 8 wks of normal weight bearing similar to that of normal control muscle. However, the recovery time period was insufficient to allow complete compensation of fiber size to that of the age-matched control animals.     

Effects of gravitational loading on rat soleus muscle fibers following hindlimb suspension.

Effects of 16 days of hindlimb suspension and 16 days of ambulation recovery at 1-G or 2-G environment on the characteristics of soleus muscle fibers were studied in male Wistar Hannover rats. The mean cross-sectional area and myonuclear number in isolated single fibers at the termination of suspension were approximately 30% and 25% of the controls, respectively. Satellite cells were distributed evenly throughout the fiber length in the control. However, the number of satellite cells distributed at the middle of the fiber was less in the unloaded rats immediately after the termination of suspension. Both the numbers of quiescent and mitotic active satellite cell per fiber were approximately 57% less immediately after the termination of suspension than controls. The number of satellite cells at the end of fibers was increased first during the early phase of reloading. Subsequently, the number at the middle was gradually increased. The myonuclear number per fiber was also less (approximately 25%) in the unloaded than the age-matched control at the termination of suspension, but was increased following the recovery. Although the mean in vivo sarcomere length of the soleus muscle was shortened in response to plantarflexion of ankle joint, the length at the certain ankle joint angle was increased after 16 days of suspension due to sarcomere remodeling. The length at the proximal and distal, rather than the middle, portion of the fiber was stretched in both reloaded and control rats in response to dorsiflexion of the ankle joint. But it was noted that the magnitude of stretch was greater in the unloaded rats. It is suggested that the fiber end is more stimulated rapidly than the middle portion by the load applied to the muscle during the ambulation recovery.     

Enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles of hypocaloric rats.

1. The effect of hypocaloric feeding (25% of normal food intake for 21 days) of rats on the enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles was studied. 2. In control and hypocaloric rats the muscle relaxation rates at 100 Hz were 35.76 and 11.38% force loss/10 ms respectively. Control rats exhibited enhanced force of muscle contraction as the frequency of stimulation increased from 10 to 100 Hz, with maximum force being at 100 Hz. Hypocaloric rats exhibited a decrease in the increment of force being exerted at high frequencies, with maintenance of force at lower stimulatory frequencies. 3. In muscles of hypocaloric rats, there were significant decreases in the maximal activities of hexokinase (17.6-37.0%), 6-phosphofructokinase (22.7-34.2%), pyruvate kinase (21.2-36.0%), citrate synthase (34.1-41.5%), oxoglutarate dehydrogenase (29.4-52.4%) and 3-hydroxyacyl-CoA dehydrogenase (26.7-32.1%), whereas the activities of glycogen phosphorylase increased (23.8-43.4%) compared with control values. 4. In soleus-muscle strip preparations of hypocaloric rats, there were significant decreases in the rates of lactate production (28.1%) and glucose oxidation (32.6%) compared with control preparations. 5. Mitochondrial preparations from muscles of hypocaloric rats incubated with various substrates exhibited decreased rates of oxygen uptake compared with control preparations. 6. In muscles of hypocaloric rats (gastrocnemius and soleus), there were significant decreases in the concentrations of glycogen (P less than 0.001) and phosphocreatine (P less than 0.001) and increases in those of pyruvate (P less than 0.001), lactate (P less than 0.001) and ADP (P less than 0.001), whereas those of ATP and AMP remained unchanged. 7. Calculated [lactate]/[pyruvate] and [ATP]/[ADP] ratios exhibited significant increases (P less than 0.05) and decreases (P less than 0.05) in muscles of hypocaloric rats respectively. 8. The results are discussed in relation to the genesis of muscle dysfunction caused by malnutrition.     

Force-length properties and functional demands of cat gastrocnemius, soleus and plantaris muscles.

The purpose of this study was to measure isometric force-length properties of cat soleus, gastrocnemius and plantaris muscle-tendon units, and to relate these properties to the functional demands of these muscles during everyday locomotor activities. Isometric force-length properties were determined using an in situ preparation, where forces were measured using buckle-type tendon transducers, and muscle-tendon unit lengths were quantified through ankle and knee joint configurations. Functional demands of the muscles were assessed using direct muscle force measurements in freely moving animals. Force-length properties and functional demands were determined for soleus, gastrocnemius and plantaris muscles simultaneously in each animal. The results suggest that isometric force-length properties of cat soleus, gastrocnemius and plantaris muscles, as well as the region of the force-length relation that is used during everyday locomotor tasks, match the functional demands.     

Proteomic analysis of rat soleus muscle undergoing hindlimb suspension-induced atrophy and reweighting hypertrophy

A proteomic analysis was performed comparing normal rat soleus muscle to soleus muscle that had undergone either 0.5, 1, 2, 4, 7, 10 and 14 days of hindlimb suspension-induced atrophy or hindlimb suspension-induced atrophied soleus muscle that had undergone 1 hour, 8 hour, 1 day, 2 day, 4 day and 7 days of reweighting-induced hypertrophy. Muscle mass measurements demonstrated continual loss of soleus mass occurred throughout the 21 days of hindlimb suspension; following reweighting, atrophied soleus muscle mass increased dramatically between 8 hours and 1 day post reweighting. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 29 soleus proteins. Reweighting following atrophy demonstrated statistically significant changes in the relative levels of 15 soleus proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both atrophied and hypertrophied soleus muscle. Five differentially regulated proteins from the hindlimb suspended atrophied soleus muscle were identified while five proteins were identified in the reweighting-induced hypertrophied soleus muscles. The identified proteins could be generally grouped together as metabolic proteins, chaperone proteins and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the skeletal muscle proteome occur during disuse-induced soleus muscle atrophy and reweighting hypertrophy.     

Preventive effect of nucleoprotein on hindlimb unloading-induced capillary regression in rat soleus muscle

We investigated the preventive effects of nucleoprotein on capillary regression and mitochondrial dysfunction induced by unloading in the soleus muscle of rats. Nucleoprotein is a supplement made from soft roe of salmon, and its major components are nucleotides and protamine. Adult male Sprague-Dawley rats were divided randomly into control, hindlimb unloading (HU), and hindlimb unloading plus nucleoprotein administration (HU+ NP) groups. Hindlimb unloading was carried out for 2 weeks in the rats belonging to the HU and the HU+ NP groups. The rats of the HU+ NP group were administered nucleoprotein (500 mg/kg) using a feeding needle twice a day for 2 weeks. Hindlimb unloading resulted in capillary regression, decreased succinate dehydrogenase activity of the muscle fiber, and decreased PGC-1α expression in the soleus muscle. These effects were prevented by administration of nucleoprotein. Nucleoprotein appears to prevent capillary regression and mitochondrial dysfunction caused by unloading of the skeletal muscle. Therefore, nucleoprotein supplementation may be an effective therapy for maintaining capillary network and mitochondrial metabolism of the muscle fiber during an unloading period.     

The relationship of voluntary running to fibre type composition, fibre area and capillary supply in rat soleus and plantaris muscles

Twenty 4-week-old Wistar rats exercised voluntarily in running wheels each day for 45 days. Fibre type composition, fibre cross-sectional area and the number of capillaries around a fibre of the slow-twitch soleus and fast-twitch plantaris muscles were examined and compared with animals which had no access to running wheels. The exercise group had a higher percentage of fast-twitch oxidative glycolytic (FOG) fibres and a lower percentage of fast-twitch glycolytic (FG) fibres in the deep portion of the plantaris muscle. The area of FOG fibres in the surface portion of the plantaris muscle was also greater in the exercise group. In the exercised animals, there was a positive relationship between the running distance and the area of FOG fibres in both the deep and surface portions of the plantaris muscle. In addition, the running distance correlated positively with the percentage of FOG fibres and negatively with that of FG fibres in the deep portion of the plantaris muscle. There were no relationships between the running distance and fibre type composition, or fibre area and capillary supply in the soleus muscle. These results suggested that the increase in the percentage and area of FOG fibres in the fast-twitch muscle was closely related to voluntary running.     

Adaptation of rat lateral gastrocnemius muscle motor units during hindlimb unloading

Contractile and fatigue-resistance properties of 71 lateral gastrocnemius muscle (LG) motor units (MU) following 14 days of hindlimb unloading (HU) were compared to those of 60 LG MU from control rats. The MU properties were assessed from isolated and stimulated individual motor axons. The MU were classified using standard criteria (shape of unfused tetani and fatigue resistance). The HU did not affect LG MU composition, but diminished the maximal tetanic tension (Po) of all MU types: P0 was significantly reduced by about 40% for the slow and fast-resistant MU, and by 18% for the fast-fatigable ones. The speed-related properties of fast-resistant MU became more similar to those of slower MU. The fatigue properties of MU were evaluated during a 5-min exercise test, using two fatigue indexes, FI2 and FI5, which expressed the relative capacity of MU to generate tension after 2 and 5 min, respectively. Results showed that 14 days of HU did not change the fatigue sensitivity of the LG MU. However, when F15 was compared to FI2, a greater decrease was observed after HU than in control conditions for the fast-resistant and fast-intermediate MU. It was concluded that a prolonged fatigue test may show changes in metabolic properties of muscle fibres during 14 days of HU. Specific adaptations of LG MU as well as comparisons with those of the soleus muscle under the same conditions are discussed.     

Enhancement of hybrid-fiber types in rat soleus muscle after clenbuterol administration during hindlimb unloading

Our objective was to determine the effects of a clenbuterol (CB) treatment orally administered (2 mg per kg) to rats submitted to 14 days of hindlimb unloading (HU). The morphological and the contractile properties as well as the myosin heavy chain isoforms contained in each fiber type were determined in whole soleus muscles. As classically described after HU, a decrease in muscle wet weight and in body mass associated with a loss of muscular force, an evolution of the contractile parameters towards those of a fast muscle type, and the emergence of fast myosin heavy chain isoforms were observed. The CB treatment in the HU rats helped reduce the decrease in 1) muscle and body weights, 2) force and 3) the proportion of slow fibers, without preventing the emergence of fast myosin isoforms. Clenbuterol induced a complex remodelling of the muscle typing promoting the combination of both slow and fast myosin isoforms within one fiber. To conclude, our data demonstrate that CB administration partially counteracts the effects produced by HU, and they allow us to anticipate advances in the treatment of muscular atrophy.     

Endothelium-dependent vasodilation in different rat hindlimb skeletal muscles.

Few studies have examined potential for endothelium-dependent vasodilation in skeletal muscles of different fiber-type composition. We hypothesized that muscles composed of slow oxidative (SO)- and/or fast oxidative glycolytic (FOG)-type fibers have greater potential for endothelium-dependent vasodilation than muscles composed of fast glycolytic (FG)-type fibers. To test this hypothesis, the isolated perfused rat hindlimb preparation was used with a constant-flow, variable-pressure approach. Perfusion pressure was monitored continuously, and muscle-specific flows were determined by using radiolabeled microspheres at four time points: control, at peak effect of acetylcholine (ACh I; 1-2 x 10(-4) M), at peak effect of ACh after infusion of an endothelial inhibitor (ACh II), and at peak effect of sodium nitroprusside (SNP; 4-5 x 10(-4) M). Conductance was calculated by using pressure and flow data. In the SO-type soleus muscle, conductance increased with ACh and SNP, but the increase in conductance with ACh was partially abolished by the endothelial inhibitor N(G)-nitro-l-arginine methyl ester (control, 0.87 +/- 0.19; ACh I, 2.07 +/- 0.29; ACh II, 1.32 +/- 0.15; SNP, 1.76 +/- 0.19 ml. min(-1). 100 g(-1). mmHg(-1); P < 0.05, ACh I and SNP vs. control). In the FOG-type red gastrocnemius muscle, similar findings were obtained (control, 0.64 +/- 0.11; ACh I, 1.36 +/- 0.21; ACh II, 0.73 +/- 0.16; SNP, 1.30 +/- 0.21 ml. min(-1). 100 g(-1). mmHg; P < 0.05, ACh I and SNP vs. control). In the FG-type white gastrocnemius muscle, neither ACh nor SNP increased conductance. Similar findings were obtained when muscles were combined into high- and low-oxidative muscle groups. Indomethacin had no effect on responses to ACh. These data indicate that endothelium-dependent vasodilation is exhibited by high-oxidative, but not low-oxidative, rat skeletal muscle. Furthermore, endothelium-dependent vasodilation in high-oxidative muscle appears to be primarily mediated by nitric oxide.     

Parallel and divergent adaptations of rat soleus and plantaris to chronic exercise and hypergravity

It has been demonstrated that endurance exercise and chronic acceleration, i.e., hypergravity, produce comparable adaptations in a variety of physiological systems, including decreased adiposity, increased energy metabolism, and altered intermediary metabolism. Similar adaptations have not been demonstrated for skeletal muscle per se. To further differentiate between these general responses with respect to gravity and exercise, this study tested the hypothesis that chronic exercise (voluntary wheel running) and chronic acceleration (2 G via centrifugation) will induce similar changes in muscle myosin heavy chain (MHC) isoform expression in rat plantaris, a fast extensor, and in rat soleus, a slow "antigravity" extensor. The experimental design involved four groups of mature male rats (n = 8/group): 1 G and 2 G with running wheels, and 1 G and 2 G controls without running wheels. The primary observations from the study were as follows: 1) 8 wk of 2 G are an adequate stimulus for MHC compositional changes in rat plantaris and soleus muscle; 2) both exercise and +G caused an increase in the slow MHC1 isoform in soleus muscle, suggesting that loading is a primary stimulus for this shift; and 3) 2 G and exercise appeared to have differential effects on the plantaris muscle MHC isoforms, with 2 G causing an increase in MHC2b, and exercise causing a decrease in MHC2b with a concomitant increase in MHC1, suggesting that factors other than enhanced loading, possibly locomotor activity levels, are the primary stimulus for this shift.     

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.     

Regression of capillary network in atrophied soleus muscle induced by hindlimb unweighting.

Little is known about the mechanisms responsible for the adaptation and changes in the capillary network of hindlimb unweighting (HU)-induced atrophied skeletal muscle, especially the coupling between functional and structural alterations of intercapillary anastomoses and tortuosity of capillaries. We hypothesized that muscle atrophy by HU leads to the apoptotic regression of the capillaries and intercapillary anastomoses with their functional alteration in hemodynamics. To clarify the three-dimensional architecture of the capillary network, contrast medium-injected rat soleus muscles were visualized clearly using a confocal laser scanning microscope, and sections were stained by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) and with anti-von Willebrand factor. In vivo, the red blood cell velocity of soleus muscle capillaries were determined with a pencil-lens intravital microscope brought into direct contact with the soleus surface. After HU, the total muscle mass, myofibril protein mass, and slow-type myosin heavy chain content were significantly lower. The number of capillaries paralleling muscle fiber and red blood cells velocity were higher in atrophied soleus. However, the mean capillary volume and capillary luminal diameter were significantly smaller after HU than in the age-matched control group. In addition, we found that the number of anastomoses and the tortuosity were significantly lower and TUNEL-positive endothelial cells were observed in atrophied soleus muscles, especially the anastomoses and/or tortuous capillaries. These results indicate that muscle atrophy by HU generates structural alterations in the capillary network, and apoptosis appears to occur in the endothelial cell of the muscle capillaries.     

Acid phosphatase activity in soleus and plantaris muscle fibres of normal and dystrophic hamsters

Summary The activity of acid phosphatase in skeletal muscle fibres of the plantaris and soleus of normal and dystrophic male hamsters was quantified using a histochemical post-coupling semipermeable membrane technique. Althoug the absolute levels of activity were found to vary widely from one animal to another, the ratio of the mean activities in the two muscles in each animal was virtually constant. In normal muscles, the ratio was about 0.73 and in dystrophic muscles, about 0.77. The activity in plantaris muscle fibres was always significantly lower than that in the corresponding soleus fibres, and in normal fibres compared to dystrophic ones. Another difference was that in normal fibres the mean activity declined to a constant level in mature animals older than about 3 months. In contrast, the activity in dystrophic muscles appeared to fall exponentially throughout life. The functional significance of these findings is discussed.In honour of Prof. P. van Duijn     

Contractile responses of the rat gastrocnemius and soleus muscles to isotonic resistance exercise.

Male rats were divided into control and weight-trained (WT) groups. WT rats performed squat-type exercises twice daily, 5 days/wk, for 14 wk. They averaged 36 lifts/day, with an average weight of 555 g. Muscle-to-body weight ratio (mg/g) of the soleus (Sol) was not different from control, but it increased 11 and 6% in the gastrocnemius (Gast) and plantaris, respectively (P < 0.05). The normalized twitch tension of the in situ Sol was elevated by 21%, whereas single-skinned type I fibers from the Sol showed an increased rate constant of tension redevelopment (K(tr)) but no other contractile adaptations to WT. In contrast, the Gast type I fibers showed an increase (P < 0.05) in maximal velocity of shortening (25%), peak power (15%), K(tr) (18%), and normalized tension (7%). The K(tr) and normalized tension of the Gast type IIa fibers increased by 24% (P < 0.05) and 12% (P < 0.05), respectively, whereas velocity and power showed a tendency to increase. Fiber size, determined by myosin ATPase histochemistry, was not different for any fiber type from the Gast or Sol. These results indicate that isotonic resistance exercise of the calf targets the Gast (type I and type IIa fibers) and has little effect on the Sol.