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.     

Effects of hindlimb suspension on soleus muscle fibers and their spinal motoneurons in Wistar Hannover rats

Cross-sectional areas and succinate dehydrogenase (SDH) activities of soleus muscle fibers and their spinal motoneurons in male Wistar Hannover rats were determined after 16 days of hindlimb suspension. A decreased percentage of type I fibers and an increased percentage of type I+II fibers were observed after hindlimb suspension. Cross-sectional areas of all types of fibers were smaller in the hindlimb suspended than control rats. SDH activities of all types of fibers did not change after hindlimb suspension. Numbers, cross-sectional areas, or SDH activities of spinal motoneurons did not change after hindlimb suspension. It is suggested that spinal motoneurons innervating the rat soleus muscle are not affected by decreased neuromuscular activity on Earth and that gravity itself is important for maintaining of spinal motoneuron metabolic properties.     

Changes in fiber composition of soleus muscle during rat hindlimb suspension

Chronic reduction of gravitational load in the rear limbs of rats to simulate the influence of near-zero gravity in skeletal muscles has been shown previously to elicit atrophy in the soleus muscle. Use of this model by the present investigation indicates that soleus atrophy was characterized by a decline in the number of fibers in groups that contained the slow isoenzyme of myosin and which were classified as type I from intensity of staining to myofibrillar actomyosin adenosinetriphosphatase (ATPase) and to NADH tetrazolium reductase. Furthermore total fiber number was not changed, whereas fibers containing the intermediate isoenzyme and those classified as type IIa increased. There results could be explained by either a change in the composition within existing fibers or a simultaneous loss of slow fibers and de novo synthesis of intermediate and fast fibers. Evidence for transformation included an absence of embryonic or neonatal myosin in muscles from suspended rats and the constant fiber number that was unchanged by 4 wk of suspension. Furthermore although fiber areas of both groups of type I and IIa fibers declined during suspension, variability of the fiber areas within each group did not increase.     

Effects of oxidation on the power of chemically skinned rat soleus fibres

Oxidation alters calcium sensitivity, and decreases maximum isometric force (Po) and shortening velocity (Vmax) of single muscle fibres. To examine the effect of oxidation on the curvature of the force-velocity relationship, which determines muscle power in addition to Po and Vmax, skinned rat type I fibres were maximally activated at 15°C in a solution with pCa 4.5 and subjected to isotonic contractions before and after 4-min incubation in 50 mM H?O? (n=10) or normal relaxing solution (n=3). In five oxidised and four control fibres the rate of force redevelopment (ktr), following a rapid release and re-stretch, was measured. This gives a measure of the sum of the rate constants for cross-bridge attachment (f) and detachment (g?): (f+g?). H?O? reduced Po, Vmax and ktr by 19%, 21% and 24% respectively (P<0.001), while the shape of the force-velocity relationship was unchanged. Fitting data to the Huxley cross-bridge model suggested that oxidation decreased both the rate constant for cross-bridge attachment (f), and detachment of negatively strained cross-bridges (g?), similar to the effect of reduced activation. This suggests that oxidative modification is a possible cause of the variation in contractile properties between muscle fibres of the same type.     

Effects of the beta-adrenoceptor agonist isoprenaline on insulin-sensitivity in soleus muscle of the rat.

The interactions between a beta-adrenoceptor agonist (isoprenaline) and insulin on rates of hexose transport, glucose phosphorylation, glycogen synthesis and glycogenolysis were investigated in the incubated stripped soleus-muscle preparation of the rat. In the presence of 1 microM-isoprenaline, insulin was less effective in stimulating glucose phosphorylation and glycogen synthesis. The stimulation of glycogenolysis by isoprenaline was only slightly decreased even at high (10000 microunits/ml) concentrations of insulin. Insulin-stimulated phosphorylation of 2-deoxyglucose was decreased by isoprenaline. It is suggested that this decrease in the rate of glucose phosphorylation is caused by the observed elevated concentration of glucose 6-phosphate, which inhibits hexokinase activity. This conclusion is supported by the fact that isoprenaline had no effect on the stimulation of 3-O-methylglucose transport by insulin.     

Effects of oxygen deprivation on incubated rat soleus muscle

Isolated soleus muscle deprived of oxygen produces more lactate and alanine than oxygen-supplied muscle. Oxygenated muscle synthesized glutamine, while anoxic muscle used this amino acid. Oxygen deprivation decreased adenine nucleotides leading to the efflux of nucleosides. Protein synthesis and degradation responded differently to anoxia. Synthesis almost completely ceased, while proteolysis increased. Therefore, protein degradation in soleus muscle is enhanced when energy supplies and oxygen tension are low.     

Effects of inactivity on fiber size and myonuclear number in rat soleus muscle.

The effects of short-term (4 days) and long-term (60 days) neuromuscular inactivity on myonuclear number, size, and myosin heavy chain (MHC) composition of isolated rat soleus fibers were determined using confocal microscopy and gel electrophoresis. Inactivity was produced via spinal cord isolation (SI), i.e., complete spinal cord transections at a midthoracic and a high sacral level and bilateral deafferentation between the transection sites. Compared with control, there was an increase in the percentage of fibers containing the faster MHC isoforms after 60, but not 4, days of SI. The mean sizes of type I and type I+IIa fibers were 41 and 27% and 66 and 56% smaller after 4 and 60 days of SI, respectively. Thus atrophy occurred earlier than the shift in myosin heavy chain (MHC) profile. The number of myonuclei was approximately 30% higher in type I than type I+IIa fibers in control soleus, but after 60 days of SI these values were similar. The number of myonuclei per millimeter in type I fibers was significantly lower than control after 60 days of SI, whereas there was no change in type I+IIa fibers. Thus myonuclei were eliminated from fibers containing only type I MHC. Because the magnitude of the loss of myonuclei was less than the level of atrophy, the myonuclear domains of both type I and type I+IIa fibers were significantly lower than control. Thus chronic (60 days) inactivity results in smaller, faster fibers that contain a higher than normal amount of DNA per unit of cytoplasm. The absence of activation of muscle fibers that are normally the most active (pure type I fibers) resulted in most, but not all, fibers expressing some fast MHC isoforms. The results also indicate that a loss of myonuclei is not a prerequisite for sustained muscle fiber atrophy.     

Size and metabolic properties of single muscle fibers in rat soleus after hindlimb suspension

The effects of 28 days of hindlimb suspension (HS) and HS plus 10 daily forceful lengthening contractions on rat soleus muscle fibers were studied. Compared with age-matched controls (CON), soleus wet weights of suspended rats were significantly decreased (approximately 49%). In HS rats, the light adenosinetriphosphatase (ATPase) fibers (staining lightly for myosin ATPase, pH = 8.8) atrophied more than the dark ATPase fibers (staining darkly for myosin ATPase, pH = 8.8). Single-fiber alpha-glycerophosphate dehydrogenase (GPD) and succinate dehydrogenase (SDH) activities and the proportion of dark ATPase fibers were higher in HS than CON rats. Daily forceful lengthening contractions did not prevent the suspension-induced changes. These results considered in conjunction with a collaborative study on the mechanical properties of HS rats (Roy et al., accompanying paper) suggest a shift in the contractile potential of the muscle following HS without a deficit in SDH, a metabolic property commonly associated with resistance to fatigue. The results support the view that soleus muscle fibers can change from a slow-twitch oxidative to a fast-twitch oxidative-glycolytic profile, but rarely to a fast-twitch glycolytic one, and that SDH and GPD activity per volume of tissue can be maintained or increased even when there are severe losses of contractile proteins.     

Contractile properties of rat soleus muscle after 15 days of hindlimb suspension

The properties of the contractile elements interacting to develop force in atrophied rat soleus muscle were studied by using single skinned fibers, which permitted direct access to the contractile apparatus. Muscle atrophy was induced by 15 days of hindlimb suspension. Suspension resulted in a decrease of maximal tension relative to an important decline in fiber diameter. Ca affinity of the contractile proteins was not changed insofar as the tension-pCa relationship was not shifted along the pCa axis. However, after hindlimb suspension 1) the value of the Hill coefficient from the tension-pCa curve was found to be higher, 2) a higher Ca threshold for activation was reported, and 3) a significant increase in contraction kinetics was described. All these results suggested that after suspension the mechanical properties of the slow-twitch soleus appeared to resemble more closely those of a fast-twitch muscle. Our results were in complete agreement with published histochemical data.     

Variation in the determinants of power of chemically skinned type I rat soleus muscle fibres

We explored to which extent maximal velocity of shortening (Vmax), force per cross-sectional area (specific tension, Po) and curvature of the force–velocity relationship (a/Po in the Hill equation) contribute to differences in peak power of single, chemically skinned rat type I fibres. Force–velocity relationships were determined from isotonic contractions of 94 maximally activated fibres. Peak power (±SD) was 3.50 ± 1.64 W L⁻¹. There was a tenfold range of peak power and five-, six- and fourfold ranges for Po, Vmax and a/Po, respectively. None of the differences between fibres was explicable by differences in myosin heavy or light chain composition. The inverse relationship between a/Po and Vmax suggests a similar underlying cause. Fitting the data to the Huxley (Progr Biophys Biophys Chem 7:255–318, 1957) cross-bridge model showed that the rate constant g ₂ and the sum of the rate constants (f + g ₁) co-varied, both being low in the slowest fibres. Approximately 16% of the variation in Po could be explained by variation in the proportion of attached cycling cross-bridges (f/(f + g ₁)), but the origin of most of the variance in Po remains unknown.     

The absence of support loading on the rat m. soleus leads to an increase in the number of intrafusal muscle fibres in muscle spindles

The ultrastructure of muscle spindles (incapsulated mechanoreceptors of stretch of extrafusal muscle fibres) of m. soleus in adult Wistar rats after repeated unloading of support on hind limbs with preservation of support loading on fore limbs has been studied by transmissing electron microscopy. It was shown that, along with muscle spindles with the ordinary number of intrafusal muscle fibres (four), m. soleus contains spindles with an increased number of intrafusal fibers (five to six). It was assumed that the increase in the number of intrafusal muscle fibers is due to the proliferation of their satellite cells.     

Effects of cobalt, magnesium, and cadmium on contraction of rat soleus muscle.

The effects on isometric tension of three divalent ions that block calcium channels, magnesium, cobalt, and cadmium, were tested in small bundles of rat soleus fibers. Cobalt, at a concentration of 2 or 6 mM, reversibly depressed twitch and tetanic tension and the depression was much greater in solutions containing no added calcium ions. Magnesium caused much less depression of tension than cobalt. The depression of tension was not accompanied by membrane depolarization or a reduction in the amplitude of action potentials. A reduction caused by 6 mM cobalt in the amplitude of 40 or 80 mM potassium contractures was not accompanied by a comparable reduction in tension during 200 mM potassium contractures, and could be explained by a shift in the potassium contracture tension-voltage curve to more positive potentials (by +7 mV on average). Similar effects were not seen with 2 or 6 mM magnesium. At a concentration of 20 mM, both cobalt and magnesium depressed twitch and tetanic tension, cobalt having greater effect than magnesium. Both ions shifted the potassium contracture tension-voltage curve to the right by +5 to +10 mV, caused a small depression of maximum tension, and slowed the time course of potassium contractures. Cadmium (3 mM) depressed twitch, tetanic, and potassium contracture tension by more than 6 mM cobalt, but experiments were complicated by the gradual appearance of large contractures that became even larger, and sometimes oscillatory, when the solution containing cadmium was washed out. It was concluded that divalent cations affect both activation and inactivation of tension in a manner that cannot be completely explained by a change in surface charge.     

The effect of lactic acid on intracellular pH and adenosine output from superfused rat soleus muscle fibres

We investigated the effects of graded doses of lactic acid on the intracellular pH and adenosine output from superfused bundles of about 15 skeletal muscle fibres. Intracellular pH was determined using the fluorescent intracellular dye, 2',7'-bis-(2-carboxyethyl)-5-(and,6-) carboxyfluorescein (BCECF), and adenosine efflux was measured by HPLC. Intracellular pH was 7.07 +/- 0.05 under control conditions, which was around 0.35 units lower than extracellular pH, and adenosine output was 63 +/- 10 pmol/min/g. Lactic acid produced dose-dependent decreases in intracellular pH and dose-dependent increases in adenosine output: 10 mM lactic acid decreased intracellular pH to 6.57 +/- 0.04 and increased adenosine output to 159 +/- 34 pmol/min/g. The adenosine output and the intracellular pH were well correlated (r2 = 0.988; P < 0.01).     

Myotoxic effects of clenbuterol in the rat heart and soleus muscle.

Myocyte-specific necrosis in the heart and soleus muscle of adult male Wistar rats was investigated in response to a single subcutaneous injection of the anabolic beta(2)-adrenergic receptor agonist clenbuterol. Necrosis was immunohistochemically detected by administration of a myosin antibody 1 h before the clenbuterol challenge and quantified by using image analysis. Clenbuterol-induced myocyte necrosis occurred against a background of zero damage in control muscles. In the heart, the clenbuterol-induced necrosis was not uniform, being more abundant in the left subendocardium and peaking 2.4 mm from the apex. After position (2.4 mm from the apex), dose (5 mg clenbuterol/kg), and sampling time (12 h) were optimized, maximum cardiomyocyte necrosis was found to be 1.0 +/- 0.2%. In response to the same parameters (i.e., 5 mg of clenbuterol and sampled at 12 h), skeletal myocyte necrosis was 4.4 +/- 0.8% in the soleus. These data show significant myocyte-specific necrosis in the heart and skeletal muscle of the rat. Such irreversible damage in the heart suggests that clenbuterol may be damaging to long-term health.     

Histochemical profiles of rat soleus intrafusal fibres after chronic exercise.

Intrafusal fibres from the rat soleus were investigated for representative histochemical profiles in sedentary animals and animals chronically exercised for 17 weeks on a treadmill. The pattern of myosin adenosine triphosphatase (ATPase) activity in the polar region revealed three intrafusal fibre types: (1) myosin ATPase-dark (MD) fibres, alkali- and acid-stabile; (2) myosin ATPase-light (ML) fibres, alkali- and acid-labile; and (3) myosin ATPase-reversible (MR) fibres, alkali-stabile and acid-labile. The three fibre types were correlated with the level of reduced NADH diaphorase activity, with MR, ML and MD fibres staining dark, moderate and light, respectively. In the equatorial region the morphological features of representative ML and MD fibres revealed that they were nuclear bag fibres, while representative MR fibres were identified as nuclear chain fibres. The MR fibres in the exercised animals had higher levels of myosin ATPase alkaline stability and acid lability than MR fibres in the sedentary animals, suggesting the MR fibre profiles are selectively influenced by chronic exercise. The mean cross-sectional area of MR fibres from the exercised animals was significantly less than the MR fibres from the sedentary animals. In contrast to the effect of endurance training on NADH diaphorase activity in extrafusal muscle fibres, there was evidence of less activity in the MD fibres of the exercised animals.