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Transcutaneous and epidural electrical spinal cord stimulation techniques are becoming more valuable as electrophysiological and clinical tools. Recently, we observed selective activation of proximal and distal motor pools during epidural spinal stimulation. In the present study, we hypothesized that the characteristics of recruitment curves obtained from leg muscles will reflect a relative preferential activation of proximal and distal motor pools based on their arrangement along the lumbosacral enlargement. The purpose was to describe the electrophysiological responses to transcutaneous stimulation in leg muscles innervated by motoneurons from different segmental levels. Stimulation delivered along the rostrocaudal axis of the lumbosacral enlargement in the supine position resulted in a selective topographical recruitment of proximal and distal leg muscles, as described by threshold intensity, slope of the recruitment curves, and plateau point intensity and magnitude. Relatively selective recruitment of proximal and distal motor pools can be titrated by optimizing the site and intensity level of stimulation to excite a given combination of motor pools. The slope of the recruitment of particular muscles allows characterization of the properties of afferents projecting to specific motoneuron pools, as well as to the type and size of the motoneurons. The location and intensity of transcutaneous spinal electrical stimulation are critical to target particular neural structures across different motor pools in investigation of specific neuromodulatory effects. Finally, the asymmetry in bilateral evoked potentials is inevitable and can be attributed to both anatomical and functional peculiarities of individual muscles or muscle groups., (Copyright © 2015 the American Physiological Society.)

The present study investigated the mechanical role of the dorsoventral curvature of the Achilles tendon in the conversion of the shortening of the plantarflexor muscles into ankle joint rotation. Dynamic, sagittal-plane magnetic resonance spin-tagged images of the ankle joint were acquired in six healthy subjects during both passive and active plantarflexion movements driven by a magnetic resonance compatible servomotor-controlled foot-pedal device. Several points on these images were tracked to determine the 1) path and deformation of the Achilles tendon, 2) ankle's center of rotation, and 3) tendon moment arms. The degree of mechanical amplification of joint movement was calculated as the ratio of the displacements of the calcaneus and myotendinous junction. In plantarflexion, significant deflection of the Achilles tendon was evident in both the passive (165.7 ± 7.4°; 180° representing a straight tendon) and active trials (166.9 ± 8.8°). This bend in the dorsoventral direction acts to move the Achilles tendon closer to the ankle's center of rotation, resulting in an ∼5% reduction of moment arm length. Over the entire range of movement, the overall displacement of the calcaneus exceeded the displacement of the myotendinous junction by ∼37%, with the mechanical gains being smaller in dorsi- and larger in plantarflexed joint positions. This is the first study to assess noninvasively and in vivo using MRI the curvature of the Achilles tendon during both passive and active plantarflexion movements. The dorsoventral tendon curvature amplifies the shortening of the plantarflexor muscles, resulting in a greater displacement of the tendon's insertion into the calcaneus compared with its origin.

Muscle fiber deformation is related to its cellular structure, as well as its architectural arrangement within the musculoskeletal system. While playing an important role in aponeurosis displacement, and efficiency of force transmission to the tendon, such deformation also provides important clues about the underlying mechanical structure of the muscle. We hypothesized that muscle fiber cross section would deform asymmetrically to satisfy the observed constant volume of muscle during a contraction. Velocity-encoded, phase-contrast, and morphological magnetic resonance imaging techniques were used to measure changes in fascicle length, pinnation angle, and aponeurosis separation of the human gastrocnemius muscle during passive and active eccentric ankle joint movements. These parameters were then used to subsequently calculate the in-plane muscle area subtended by the two aponeuroses and fascicles and to calculate the in-plane (dividing area by fascicle length), and through-plane (dividing muscle volume by area) thicknesses. Constant-volume considerations of the whole-muscle geometry require that, as fascicle length increases, the muscle fiber cross-sectional area must decrease in proportion to the length change. Our empirical findings confirm the definition of a constant-volume rule that dictates that changes in the dimension perpendicular to the plane, i.e., through-plane thickness, (-6.0% for passive, -3.3% for eccentric) equate to the reciprocal of the changes in area (6.8% for passive, 3.7% for eccentric) for both exercise paradigms. The asymmetry in fascicle cross-section deformation for both passive and active muscle fibers is established in this study with a ∼22% in-plane and ∼6% through-plane fascicle thickness change. These fiber deformations have functional relevance, not only because they affect the force production of the muscle itself, but also because they affect the characteristics of adjacent muscles by deflecting their line of pull.

Tendinous tissues respond to chronic unloading with adaptive changes in mechanical, elastic, and morphological properties. However, little is known about the changes in the detailed structures of the entire tendinous tissue and whether the change in tendon stiffness is related to morphology. We investigated changes in dimensional (volume, cross-sectional area, segmented lengths) and elastic (Young's modulus) properties of the Achilles tendon and distal aponeurosis in response to chronic unilateral lower limb suspension (ULLS) using velocity encoded phase contrast (VE-PC) and three-dimensional morphometric magnetic resonance imaging (MRI). Five healthy subjects underwent ULLS for 4 wk. Axial morphometric MRI was acquired along the entire length from the calcaneous to the medial gastrocnemius insertion. An oblique sagittal VE-PC MRI was also acquired. The Young's modulus could be calculated from this cine dynamic sequence of velocity encoded images from the slope of the stress-strain curve during the submaximal isometric plantar flexion. After 4 wk of ULLS, we found significant (46.7%) decrease in maximum plantar flexion torque. The total volumes of entire tendinous tissue (determined as the sum of the Achilles tendon and distal aponeurosis) increased significantly by 6.4% (11.9 vs. 12.7 ml) after ULLS. In contrast, Young's modulus decreased significantly by 10.4% (211.7 vs. 189.6 MPa) for the Achilles tendon and 29.0% for the distal aponeurosis (158.8 vs. 113.0 MPa) following ULLS. There was no significant correlation between relative change in volume and Young's modulus with 4 wk of ULLS. It is suggested that, although tendon hypertrophy can be expected to adversely affect tendon stiffness, the absence of any significant correlation between the magnitude of tendon hypertrophy and reduced Young's modulus indicates that dimensional factors were not critical to the elastic properties.

Velocity-encoded phase-contrast magnetic resonance (MR) imaging techniques and a computer-controlled MR-compatible foot pedal device were used to investigate the medial gastrocnemius muscle and aponeurosis deformations during passive and active eccentric movements of the plantarflexors. Intrafascicular strain, measured as the ratio of strain in the fascicle segment at its insertion to strain at its origin, was nonuniform along the proximodistal axis of the muscle (P < 0.01), progressively increasing from the proximal to distal direction. The high intrafascicular strain regions appeared to correlate with the muscle regions that are likely to encounter high stress concentrations, i.e., the regions where the muscle physiological cross section decreases close to the tendons. The architectural gear ratio, i.e., the mechanical amplification ratio of fascicle length displacement to that of tendon/aponeuroses in a pennate muscle, also exhibited significant regional differences, with the highest ratios in the proximal region of the muscle accompanied by a higher initial pennation angle and a larger range of fascicular rotation about the origin. Values close to unity in the distal region of the muscle suggest that the aponeurosis separation may decrease in this region. Fascicle length and pennation angle changes were significantly influenced by force generation in the muscle, probably due to a shortening of the loaded muscle fibers relative to a passive condition. Overall, our data illustrate significant proximodistal intramuscular heterogeneity as supported by a regionally variable end-to-end strain ratio of fascicles and angle changes in the medial gastrocnemius muscle during passive and active ankle movements. These observations emphasize the need to reassess current conceptual models of muscle-tendon mechanics.

The objective of this study was to measure and monitor changes in Achilles tendon mechanical properties and force production capability of triceps surae muscles after 4 wk of limb suspension and 6 wk of physical rehabilitation. Five healthy volunteers underwent unilateral lower limb suspension followed by weekly physiotherapy. A velocity-encoded, phase-contrast magnetic resonance imaging (VE-PC-MRI) technique was used to estimate the tendon strain as a function of force produced during the submaximal isometric contractions. After limb suspension, triceps surae muscle strength decreased to 53.2 +/- 15.6% (mean +/- SD) of the presuspension level (P < 0.05). Young's modulus, estimated from the slope of the tendon stress-strain relationship, decreased by 17.1% (from 140.50 +/- 29.33 to 119.95 +/- 36.07 MPa, P < 0.05), while the tendon transition point, reflecting the "toe region," increased by 55.7% (from 2.2 +/- 1.0% to 3.4 +/- 1.24%). Muscle strength, tendon stiffness, and transition point recovered to presuspension levels by the end of 6 wk of rehabilitation. Calcaneus movement was significant during the "isometric" contraction, accounting for 52.13 +/- 7.63% of the tendon displacement. Tendon cross-sectional area determined from anatomic magnetic resonance axial images remained unchanged, suggesting that the altered tendon elastic modulus and transition point were largely due to material deterioration. The increase in the transition point following chronic unloading as measured by the VE-PC-MRI technique has not been previously reported and offers new insights into the biomechanical changes that may occur in the tendon crimp structure.

The behavior of the entire medial gastrocnemius (MG) superficial and deep aponeurosis structure was investigated with velocity-encoded phase-contrast, spin-tag, and three-dimensional morphometric magnetic resonance imaging. The displacements and strain of both these aponeuroses, muscle length, and the cross-sectional segment length of the deep aponeurosis were measured during isometric plantarflexion at 20% and 40% of maximal voluntary contraction (MVC). The length of the entire MG shortened during 20% and 40% MVC. All regions of interest in both aponeuroses moved proximally. Positive strain (lengthening) occurred in both ends of the deep aponeurosis and in the proximal region of the superficial aponeurosis. In contrast, negative strain (shortening) was observed in the middle region of the deep aponeurosis and in the distal region of the superficial aponeurosis. Consistent with this shortening of the deep aponeurosis length along the proximal-distal axis was expansion of the aponeuroses in the medial-lateral and anterior-posterior directions in the cross-sectional plane. It is concluded that at low to moderate force levels of isometric contraction, regional differences in strain occur along the proximal-distal axis of both aponeuroses, and some regions of both aponeuroses shorten.

Anabolic and catabolic markers of muscle protein metabolism were examined in inactivity-induced atrophying muscles with and without daily short-duration, high-resistance isometric contractions. Inactivity was achieved via spinal cord isolation (SI), which results in near inactivity of the hindlimb musculature without compromising the motoneuron-muscle connectivity. Adult rats were assigned to a control (Con) or SI group in which one limb was stimulated (SI-Stim, 5 consecutive days of brief bouts of high-load isometric contractions) while the other served as a SI control (SI). Both the medial gastrocnemius (MG) and soleus weights (relative to body weight) were approximately 71% of Con in the SI, but maintained at Con in the SI-Stim group. Activity of the IGF-1/phosphatidylinositol 3-kinase (PI3K)/Akt pathway of protein synthesis was similar among all groups in the MG. Expression of atrogin-1 and muscle RING finger-1 (MuRF-1), markers of protein degradation, were higher in the MG and soleus of the SI than Con and maintained at Con in the SI-Stim. Compared with Con, the anti-growth factor myostatin was unaffected in the MG and soleus in the SI but was lower in the MG of the SI-Stim. These results demonstrate that upregulation of specific protein catabolic pathways plays a critical role in SI-induced atrophy, while this response was blunted by 4 min of daily high-resistance electromechanical stimulation and was able to preserve most of the muscle mass. Although the protein anabolic pathway (IGF-1/PI3K/Akt) appears to play a minor role in regulating mass in the SI model, increased translational capacity may have contributed to mass preservation in response to isometric contractions.

The efficacy of high-load, short-duration isometric contractions, delivered as one vs. two sessions per day, on blunting inactivity-induced adaptations in the medial gastrocnemius (MG) were compared. Adult rats were assigned to a control (Con) or spinal cord-isolated (SI) group where one limb was stimulated (SI-Stim) while the other served as a SI control (SI-C). One bout of stimulation (BION microstimulator) consisted of a 100-Hz, 1-s stimulus, delivered every 30 s for 5 min with a 5-min rest period. This bout was repeated six times consecutively (SI-Stim1) or with a 9-h rest interval after the third bout (SI-Stim2) for 30 consecutive days. MG weights (relative to body weight) were 63, 72, and 79% of Con in SI-C, SI-Stim1, and SI-Stim2, respectively. Mean fiber size was 56% smaller in SI-C than in Con, and it was 19 and 31% larger in SI-Stim1 and SI-Stim2, respectively, compared with SI-C. Maximum tetanic tension was 42, 60, and 73% of Con in SI-C, SI-Stim1, and SI-Stim2, respectively. Specific tension was 77% of Con in SI-C, and at Con levels in both SI-Stim groups. SI increased the percent IIb myosin heavy chain composition (from 49 to 77%) and IIb+ fibers (from 63 to 79%): these adaptations were prevented by both Stim paradigms. These results demonstrate that 1) brief periods of high-load isometric contractions are effective in reducing inactivity-induced atrophy, functional deficits, and phenotypic adaptations in a fast hindlimb extensor, and 2) the same amount of stimulation distributed in two compared with one session per day is more effective in ameliorating inactivity-related adaptations.

The in vivo strain properties of human skeletal muscle-tendon complexes are poorly understood, particularly following chronic periods of reduced load bearing. We studied eight healthy volunteers who underwent 4 wk of unilateral lower limb suspension (ULLS) to induce chronic unloading. Before and after the ULLS, maximum isometric ankle plantar flexion torque was determined by using a magnetic resonance (MR)-compatible dynamometry. Volumes of the triceps surae muscles and strain distribution of the soleus aponeurosis and the Achilles tendon at a constant submaximal plantar flexion (20% pre-maximal voluntary contraction) were measured by using MRI and velocity-encoded, phase-contrast MRI techniques. Following ULLS, volumes of the soleus and the medial gastrocnemius and the maximum isometric ankle plantar flexion (maximum voluntary contraction) decreased by 5.5+/-1.9, 7.5+/-2.7, and 48.1+/-6.1%, respectively. The strain of the aponeurosis along the length of the muscle before the ULLS was 0.3+/-0.3%, ranging from -1.5 to 2.7% in different locations of the aponeurosis. Following ULLS, the mean strain was -6.4+/-0.3%, ranging from -1.6 to 1.3%. The strain distribution of the midregion of the aponeurosis was significantly influenced by the ULLS, whereas the more distal component showed no consistent changes. Achilles tendon strain was not affected by the ULLS. These results raise the issue as to whether these changes in strain distribution affect the functional properties of the triceps surae and whether the probability of strain injuries within the triceps surae increases following chronic unloading in those regions of this muscle complex in which unusual strains occur.

Bioassayable growth hormone (BGH) in rats is released in large quantities from the pituitary in response to the activation of large, proprioceptive afferent fibers from fast and mixed fiber-type hindlimb musculature. We hypothesized that hindlimb unloading (HU) of adult male rats would 1) reduce the basal levels of plasma BGH, and 2) abolish stimulus-induced BGH release. Rats were exposed to HU for 1, 4, or 8 wk. Plasma and pituitaries were collected under isoflurane anesthesia for hormone analyses. Additionally, at 4 and 8 wk, a subset of rats underwent an in situ electrical stimulation (Stim) of tibial nerve proprioceptive afferents. Basal plasma BGH levels were significantly reduced (-51 and -23%) after 1 and 8 wk of HU compared with ambulatory controls (Amb). Although Amb-Stim rats exhibited increased plasma BGH levels (88 and 143%) and decreased pituitary BGH levels (-27 and -22%) at 4 and 8 wk, respectively, stimulation in HU rats had the opposite effect, reducing plasma BGH (-25 and -33%) and increasing pituitary BGH levels (47 and 10%) relative to HU alone at 4 and 8 wk. The 22-kDa form of GH measured by immunoassay and the plasma corticosterone, T3, T4, and testosterone levels were unchanged by HU or Stim at all time points. These data suggest that BGH synthesis and release from the pituitary are sensitive both to chronically reduced neuromuscular loading and to acute changes in neuromuscular activation, independent of changes in other circulating hormones. Thus BGH may play a role in muscle, bone, and metabolic adaptations that occur in response to chronically unloaded states.

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.

The effects of tendon vibration on bioassayable growth hormone (BGH) secretion from the pituitary gland were investigated in anesthetized adult male rats. The tendons from predominantly fast-twitch ankle extensor muscles (gastrocnemius and plantaris) or a predominantly slow-twitch ankle extensor (soleus) were vibrated by using a paradigm that selectively activates group Ia afferent fibers from muscle spindles. The lower hindlimb was secured with the muscles near physiological length, and the tendons were vibrated for 15 min at 150 Hz and a displacement of 1 mm. Control rats were prepared similarly, but the tendons were not vibrated. Compared with control, vibration of the tendons of the fast ankle extensors markedly increased (160%), whereas vibration of the slow soleus decreased (68%), BGH secretion. Complete denervation of the hindlimb had no independent effects on the normal resting levels of BGH, but it prevented the effects of tendon vibration on BGH secretion. The results are consistent with previous findings showing modulation of BGH release in response to in vivo activation or in situ electrical stimulation of muscle afferents (Bigbee AJ, Gosselink KL, Grindeland RE, Roy RR, Zhong H, and Edgerton VR. J Appl Physiol 89: 2174-2178, 2000; Gosselink KL, Grindeland RE, Roy RR, Zhong H, Bigbee AJ, and Edgerton VR. J Appl Physiol 88: 142-148, 2000; Gosselink KL, Grindeland RE, Roy RR, Zhong H, Bigbee AJ, Grossman EJ, and Edgerton VR. J Appl Physiol 84: 1425-1430, 1998). These data provide evidence that this previously described muscle afferent-pituitary axis is neurally mediated via group Ia afferents from peripheral skeletal muscle. Furthermore, these data show that activation of this group Ia afferent pathway from fast muscles enhances, whereas the same sensory afferent input from a slow muscle depresses, BGH release.

To determine whether long-term reductions in neuromuscular activity result in alterations in metabolic capacity, the activities of oxidative, i.e., succinate dehydrogenase (SDH) and citrate synthase (CS), and glycolytic, i.e., alpha-glycerophosphate dehydrogenase (GPD), enzyme markers were quantified in rat soleus muscles 1, 3, and 6 mo after a complete spinal cord transection (ST). In addition, the proportional content of lactate dehydrogenase (LDH) isozymes was used as a marker for oxidative and glycolytic capacities. The myosin heavy chain (MHC) isoform content of a fiber served as a marker of phenotype. In general, MHC isoforms shifted from MHC1 toward MHC2, particularly MHC2x, after ST. Mean SDH and CS activities were higher in ST than control at all time points. The elevated SDH and CS activities were indicative of an enhanced oxidative capacity. GPD activities were higher in ST than control rats at all time points. The increase in activity of SDH was larger than GPD. Thus the GPD-to-SDH (glycolytic-to-oxidative) ratio was decreased after ST. Compared with controls, total LDH activity increased transiently, and the LDH isozyme profile shifted from LDH-1 toward LDH-5, indicative of an enhanced glycolytic capacity. Combined, these results indicate that 1) the metabolic capacities of soleus fibers were not compromised, but the interrelationships among oxidative and glycolytic capacity and MHC content were apparently dissociated after ST; 2) enhancements in oxidative and glycolytic enzyme activities are not mutually exclusive; and 3) chronic reductions in skeletal muscle activity do not necessarily result in a reduced oxidative capacity.

It is becoming increasingly apparent that precise knowledge of the anatomic features of muscle, aponeurosis, and tendons is necessary for understanding how a muscle-tendon complex generates force and accomplishes length changes. This report presents both anatomic and functional data from the human soleus muscle acquired by using magnetic resonance imaging. The results show a strong relationship between the complex three-dimensional structure of the muscle-tendon system and the intramuscular distribution of tissue velocities during in vivo isometric contractions. The proximal region of the muscle is unipennate, whereas the midregion has a radially bipennate hemicylindrical structure, and the distal region is quadripennate. Tissue velocity mapping shows that the highest velocity regions overlay the aponeuroses connected to the Achilles tendon. These are located on the anterior and posterior surfaces of the muscle. The lowest velocities overlay the aponeuroses connected to the origin of the muscle and are generally located intramuscularly.

The distribution of strain along the soleus aponeurosis tendon was examined during voluntary contractions in vivo. Eight subjects performed cyclic isometric contractions (20 and 40% of maximal voluntary contraction). Displacement and strain in the apparent Achilles tendon and in the aponeurosis were calculated from cine phase-contrast magnetic resonance images acquired with a field of view of 32 cm. The apparent Achilles tendon lengthened 2.8 and 4.7% in 20 and 40% maximal voluntary contraction, respectively. The midregion of the aponeurosis, below the gastrocnemius insertion, lengthened 1.2 and 2.2%, but the distal aponeurosis shortened 2.1 and 2.5%, respectively. There was considerable variation in the three-dimensional anatomy of the aponeurosis and muscle-tendon junction. We suggest that the nonuniformity in aponeurosis strain within an individual was due to the presence of active and passive motor units along the length of the muscle, causing variable force along the measurement site. Force transmission along intrasoleus connective tissue may also be a significant source of nonuniform strain in the aponeurosis.

The goal of this study was to use the model of spinal cord isolation (SI), which blocks nearly all neuromuscular activity while leaving the motoneuron muscle-fiber connections intact, to characterize the cellular processes linked to marked muscle atrophy. Rats randomly assigned to normal control and SI groups were studied at 0, 2, 4, 8, and 15 days after SI surgery. The slow soleus muscle atrophied by approximately 50%, with the greatest degree of loss occurring during the first 8 days. Throughout the SI duration, muscle protein concentration was maintained at the control level, whereas myofibrillar protein concentration steadily decreased between 4 and 15 days of SI, and this was associated with a 50% decrease in myosin heavy chain (MHC) normalized to total protein. Actin relative to the total protein was maintained at the control level. Marked reductions occurred in total RNA and DNA content and in total MHC and actin mRNA expressed relative to 18S ribosomal RNA. These findings suggest that two key factors contributing to the muscle atrophy in the SI model are 1). a reduction in ribosomal RNA that is consistent with a reduction in protein translational capacity, and 2). insufficient mRNA substrate for translating key sarcomeric proteins comprising the myofibril fraction, such as MHC and actin. In addition, the marked selective depletion of MHC protein in the muscles of SI rats suggests that this protein is more vulnerable to inactivity than actin protein. This selective MHC loss could be a major contributor for the previously reported loss in the functional integrity of SI muscles. Collectively, these data are consistent with the involvement of pretranslational and translational processes in muscle atrophy due to SI.

We examined the expression of several molecular markers of protein balance in response to skeletal muscle atrophy induced by spinal cord isolation (SI; i.e., a complete transection of the spinal cord at both a midthoracic and a high sacral level plus complete deafferentation between the two transection sites). This treatment nearly eliminates neuromuscular activity (activation and loading) of the hindlimb muscles while maintaining neuromuscular connectivity. SI was associated with a reduced transcriptional activity (via pre-mRNA analyses) of myosin heavy chain (MHC) and actin. In addition, there was an increased gene expression of enzyme systems impacting protein degradation (calpain-1; plus enzymes associated with polyubquitination processes) that could further contribute to the protein deficits in the SI muscles via degradative pathways. IGF-I receptor and binding protein-5 mRNA expression was induced throughout the 15-day period of SI, whereas IGF-I mRNA was induced at 8 and 15 days. These responses occurred in the absence of an upregulation of translational regulatory proteins (p70 S6 kinase; eukaryotic 4E binding protein 1) to compensate for the decreased protein translational capacity. These data collectively demonstrate that 1). the molecular changes accompanying SI-induced muscle atrophy are not necessarily the reverse of those occurring during muscle hypertrophy, and 2). the rapid and marked atrophy that defines this model of muscle inactivity is likely the result of multifactorial processes affecting transcription, translation, and protein degradation.

The effects of a complete spinal cord transection (ST) on the mechanical properties of the rat soleus were assessed 3 and 6 mo post-ST and compared with age-matched controls. Maximal tetanic force was reduced by approximately 44 and approximately 25% at 3 and 6 mo post-ST, respectively. Similarly, maximum twitch force was reduced by approximately 29% in 3-mo and approximately 17% in 6-mo ST rats. ST resulted in faster twitch properties as evidenced by shorter time to peak tension (approximately 45%) and half-relaxation time (approximately 55%) at both time points. Maximum shortening velocity was significantly increased in ST rats whether measured by extrapolation from the force-velocity curve (approximately twofold at both time points) or by slack-test measurements (over twofold at both time points). A significant reduction in fatigue resistance of the soleus was observed at 3 (approximately 25%) and 6 mo (approximately 45%) post-ST. For the majority of the speed-related properties, no significant differences were detected between 3- and 6-mo ST rats. However, the fatigue resistance of the soleus was significantly lower in 6- vs. 3-mo ST rats. These data suggest that, between 3 and 6 mo post-ST, force-related properties tended to recover, speed-related properties plateaued, and fatigue-related properties continued to decline. Thus some specific functional properties of the rat soleus related to contractile force, speed, and fatigue adapted independently after ST.

Plasma growth hormone (GH) measured by immunoassay [immunoassayable GH (IGH)] and by tibial bioassay [bioassayable GH (BGH)] increases in humans in response to exercise. In rats, however, IGH does not change in response to exercise. The objective of this study was to determine the BGH response to an acute exercise bout in rats. The rats ran on a treadmill at a rate of 27 m/min for 15 min, after which plasma and pituitary hormones, including IGH and BGH, and plasma metabolites were measured. Plasma and pituitary IGH were unchanged from control groups after the acute exercise bout, whereas plasma BGH was increased by 300% and pituitary BGH was decreased by 50%. Plasma thyroxine and corticosterone levels were significantly increased after a single exercise bout, but plasma testosterone, 3,5, 3'-triiodothyronine, glucose, lactate, and triglyceride concentrations were unchanged. Given previous results from in situ nerve stimulation studies (Gosselink KL, Grindeland RE, Roy RR, Zhong H, Bigbee AJ, Grossman EJ, and Edgerton VR. J Appl Physiol 84: 1425-1430, 1998), these in vivo results are consistent with the rapid BGH response during exercise being induced by the activation of muscle afferents.

Immunoassayable and bioassayable growth hormone responses to vibration-induced activation of muscle spindle afferents of the soleus (Sol) or tibialis anterior (TA) muscles were studied in 10 men. Subjects were supine while a 10-min vibration stimulus (100 Hz; 1.5-mm amplitude) was applied to the muscle, with each of the muscles tested on separate days. Blood samples were collected before, during, immediately after, and after 5 and 10 min of vibration. Plasma growth hormone concentrations were determined by radioimmunoassay (IGH) for all sampling periods and by bioassay (BGH; measurement of tibial epiphysial cartilage growth in hypophysectomized rats) for samples obtained before and immediately after vibration. Plasma IGH concentrations were similar at all time points during the Sol or TA experiments. After 10 min of muscle vibration, mean plasma BGH was elevated 94% [1,216 +/- 148 (SD) to 2, 362 +/- 487 microg/l; P = 0.0001] for TA and decreased 22% (1,358 +/- 155 to 1,058 +/- 311 microg/l; P = 0.09) for Sol. These data demonstrate that activation of TA muscle spindle afferents increases circulating BGH but not IGH. The absence of a similar vibration-induced BGH response for the Sol indicates a differential regulation of BGH release by these two predominantly slow muscles, perhaps related to their respective flexor and extensor functions. These data indicate that a muscle afferent-pituitary axis modulates the release of BGH, but not IGH, from the pituitary in humans and that this axis is muscle specific, similar to that observed in rats.

Earth's gravity has had a significant impact on the designs of the neuromotor systems that have evolved. Early indications are that gravity also plays a key role in the ontogenesis of some of these design features. The purpose of the present review is not to assess and interpret a body of knowledge in the usual sense of a review but to look ahead, given some of the general concepts that have evolved and observations made to date, which can guide our future approach to gravitational biology. We are now approaching an era in gravitational biology during which well-controlled experiments can be conducted for sustained periods in a microgravity environment. Thus it is now possible to study in greater detail the role of gravity in phylogenesis and ontogenesis. Experiments can range from those conducted on the simplest levels of organization of the components that comprise the neuromotor system to those conducted on the whole organism. Generally, the impact of Earth's gravitational environment on living systems becomes more complex as the level of integration of the biological phenomenon of interest increases. Studies of the effects of gravitational vectors on neuromotor systems have and should continue to provide unique insight into these mechanisms that control and maintain neural control systems designed to function in Earth's gravitational environment. A number of examples are given of how a gravitational biology perspective can lead to a clearer understanding of neuromotor disorders. Furthermore, the technologies developed for spaceflight studies have contributed and should continue to contribute to studies of motor dysfunctions, such as spinal cord injury and stroke. Disorders associated with energy support and delivery systems and how these functions are altered by sedentary life styles at 1 G and by space travel in a microgravity environment are also discussed.

The release of a bioassayable form of growth hormone (BGH), distinct from growth hormone as measured by immunoassay (IGH), from the rat pituitary into the blood is differentially regulated by afferent input from fast and slow skeletal muscles. Specifically, activation of low-threshold fast muscle afferents for 15 min increased plasma BGH by 217 and 295% and decreased pituitary BGH by 68 and 45% in male and female rats, respectively. In contrast, activation of slow muscle afferents inhibited BGH release, decreasing plasma BGH by approximately 60% and increasing pituitary BGH by 30-50% in male rats. Female rats from which food had been withheld for approximately 12 h had elevated basal plasma BGH levels, which then were decreased by 81% after slow muscle nerve stimulation. Plasma IGH concentrations were unchanged after any nerve stimulation condition. These results demonstrate that regulation of BGH release can be differentially mediated through low-threshold afferent inputs from fast or slow skeletal muscle. Furthermore, the results indicate that BGH responses are independent of gender or feeding status.

After 2 or 4 mo of bed rest (6 degrees head-down tilt) and 1 mo of ambulation, there was a tendency toward a higher percentage of fibers expressing fast myosin heavy chain (MHC) isoforms and a de novo appearance of fibers coexpressing type I+IIa+IIx and IIa+IIx MHC in human soleus fibers. After 2 and 4 mo of bed rest, the mean size of type I fibers decreased by 12 (P > 0.05) and 39%, respectively. Because myonuclear number/mm of fiber length was unchanged, myonuclear domain was smaller after bed rest than before. The mean size and myonuclear domain of type I fibers were largest after 1 mo of recovery. The effects of wearing an antigravity device (Penguin suit), which had a modest but continuous resistance at the knee and ankle (Penguin-1) or knee resistance without loading on the ankle (Penguin-2), for 10 consecutive h/day were determined during 2 mo of bed rest. Mean fiber sizes in Penguin-1, but not Penguin-2, group were maintained at or above pre-bed-rest levels, whereas neither group showed phenotype changes. Myonuclear domain in type I fibers was larger in Penguin-1 and smaller in Penguin-2 group post- compared with pre-bed rest, indicating that a single daily 10-h bout of modest muscle loading can prevent bed-rest-induced soleus fiber atrophy but has minimal effect on myosin phenotype. The specific adaptive cellular strategies involved may be a function of the duration and magnitude of the adaptive stimulus as well as the immediate activity history of the fiber before the newly changed functional demands.

We have reported that bed rest suppressed the release of bioassayable growth hormone (BGH) that normally occurs after an acute bout of unilateral plantar flexor exercise (G. E. McCall, C. Goulet, R. E. Grindeland, J. A. Hodgson, A. J. Bigbee, and V. R. Edgerton. J. Appl. Physiol. 83: 2086-2090, 1997). In the present study, the effects of spaceflight on the hormonal responses to this exercise protocol were examined. Four male astronauts on the National Aeronautics and Space Administration Shuttle Transport System (STS-78) mission completed the exercise protocol before, during, and after a 17-day spaceflight. The maximal voluntary contraction torque output at the onset of exercise was similar on all test days. Before spaceflight, plasma BGH increased 114-168% from pre- to postexercise. During spaceflight and after 2 days recovery at normal gravity (1 G), the BGH response to exercise was absent. After 4 days of recovery, this response was restored. Plasma concentrations of immunoassayable growth hormone were similar at all time points. The preexercise plasma immunoassayable insulin-like growth factor I (IGF-I) levels were elevated after 12 or 13 days of microgravity, and a approximately 7% postexercise IGF-I increase was independent of this spaceflight effect. The suppression of the BGH response to exercise during spaceflight indicates that some minimum level of chronic neuromuscular activity and/or loading is necessary to maintain a normal exercise-induced BGH release. Moreover, these results suggest that there is a muscle afferent-pituitary axis that can modulate BGH release.

The effects of 10 wk of functional overload (FO), with and without daily treadmill endurance training, on the cross-sectional area, myonuclear number, and myonuclear domain size of mechanically isolated single fiber segments of the adult rat plantaris were determined. The fibers were typed on the basis of high-resolution gel electrophoresis for separation of specific myosin heavy chain (MHC) isoforms and grouped as type I(+) (containing some type I MHC with or without any combination of fast MHCs), type IIa(+) (containing some type IIa with or without some type IIx and/or IIb but no type I MHC), and type IIx/b (containing only type IIx and/or IIb MHCs). Type I(+) fibers had a higher myonuclear number than did both fast types of fibers in the control and FO, but not in the FO and treadmill trained, rats. All fiber types in both FO groups had a significantly larger (36-90%) cross-sectional area and a significantly higher (61-109%) myonuclear number than did control. The average myonuclear domain size of each fiber type was similar among the three groups, except for a smaller domain size in the type IIx/b fibers of the FO compared with control. In general, these data indicate that during hypertrophy the number of myonuclei increase proportionally to the increase in fiber volume. The maintenance of myonuclear domain size near control values suggests that regulatory mechanisms exist that ensure a tight coupling between the quantity of genetic machinery and the protein requirements of a fiber.

There have been no systematic comparisons of skeletal muscle adaptations in response to voluntary wheel running under controlled loading conditions. To accomplish this, a voluntary running wheel for rats and mice was developed in which a known load can be controlled and monitored electronically. Five-week-old male Sprague-Dawley rats (10 rats/group) were assigned randomly to either a 1) sedentary control group (Control); 2) voluntary exercised with no load (Run-No-Load) group; or 3) voluntary exercised with additional load (Run-Load) group for 8 wk. The load for the Run-Load group was progressively increased to reach approximately 60% of body weight during the last week of training. The proportions of fast glycolytic (FG), fast oxidative glycolytic (FOG), or slow oxidative (SO) fibers in the plantaris were similar in all groups. The absolute and relative plantaris weights were greater in the Run-Load group compared with the Control and Run-No-Load groups. The mean fiber cross-sectional areas of FG, FOG, and SO fibers were 20, 25, and 15% greater in the Run-Load than in Control rats. In addition, these fiber types were 16, 21, and 12% larger in Run-Load than in Run-No-Load rats. The muscle weights and mean cross-sectional areas of each fiber type were highly correlated with the average running distances and total work performed in the Run-Load, but not the Run-No-Load, group. The slope of the relationship between fiber size and running distance and total work performed was significant for each fiber type but was higher for FG and FOG fibers compared with SO fibers. These data show that the load on a rat running voluntarily can determine the magnitude of a hypertrophic response and the population of motor units that are recruited to perform at a given loading condition.

There are forms of growth hormone (GH) in the plasma and pituitary of the rat and in the plasma of humans that are undetected by presently available immunoassays (iGH) but can be measured by bioassay (bGH). Although the regulation of iGH release is well documented, the mechanism(s) of bGH release is unclear. On the basis of changes in bGH and iGH secretion in rats that had been exposed to microgravity conditions, we hypothesized that neural afferents play a role in regulating the release of these hormones. To examine whether bGH secretion can be modulated by afferent input from skeletal muscle, the proximal or distal ends of severed hindlimb fast muscle nerves were stimulated ( approximately 2 times threshold) in anesthetized rats. Plasma bGH increased approximately 250%, and pituitary bGH decreased approximately 60% after proximal nerve trunk stimulation. The bGH response was independent of muscle mass or whether the muscles were flexors or extensors. Distal nerve stimulation had little or no effect on plasma or pituitary bGH. Plasma iGH concentrations were unchanged after proximal nerve stimulation. Although there may be multiple regulatory mechanisms of bGH, the present results demonstrate that the activation of low-threshold afferents from fast skeletal muscles can play a regulatory role in the release of bGH, but not iGH, from the pituitary in anesthetized rats.

The purpose of this study was to determine the effects of functional overload (FO) combined with growth hormone/insulin-like growth factor I (GH/IGF-I) administration on myonuclear number and domain size in rat soleus muscle fibers. Adult female rats underwent bilateral ablation of the plantaris and gastrocnemius muscles and, after 7 days of recovery, were injected three times daily for 14 days with GH/IGF-I (1 mg/kg each; FO + GH/IGF-I group) or saline vehicle (FO group). Intact rats receiving saline vehicle served as controls (Con group). Muscle wet weight was 32% greater in the FO than in the Con group: 162 +/- 8 vs. 123 +/- 16 mg. Muscle weight in the FO + GH/IGF-I group (196 +/- 14 mg) was 59 and 21% larger than in the Con and FO groups, respectively. Mean soleus fiber cross-sectional area of the FO + GH/IGF-I group (2,826 +/- 445 microm2) was increased compared with the Con (2,044 +/- 108 microm2) and FO (2,267 +/- 301 microm2) groups. The difference in fiber size between the FO and Con groups was not significant. Mean myonuclear number increased in FO (187 +/- 15 myonuclei/mm) and FO + GH/IGF-I (217 +/- 23 myonuclei/mm) rats compared with Con (155 +/- 12 myonuclei/mm) rats, although the difference between FO and FO + GH/IGF-I animals was not significant. The mean cytoplasmic volume per myonucleus (myonuclear domain) was similar across groups. These results demonstrate that the larger mean muscle weight and fiber cross-sectional area occurred when FO was combined with GH/IGF-I administration and that myonuclear number increased concomitantly with fiber volume. Thus there appears to be some mechanism(s) that maintains the myonuclear domain when a fiber hypertrophies.

Hormonal responses to muscle activity were studied in eight men before (-13 or -12 and -8 or -7 days), during (2 or 3, 8 or 9, and 13 or 14 days) and after (+2 or +3 and +10 or +11 days) 17 days of bed rest. Muscle activity consisted of a series of unilateral isometric plantar flexions, including 4 maximal voluntary contractions (MVCs), 48 contractions at 30% MVC, and 12 contractions at 80% MVC, all performed at a 4:1-s work-to-rest ratio. Blood was collected before and immediately after muscle activity to measure plasma growth hormone by radioimmunoassay (IGH) and by bioassay (BGH) of tibia epiphyseal cartilage growth in hypophysectomized rats. Plasma IGH was unchanged by muscle activity before, during, or after bed rest. Before bed rest, muscle activity increased (P < 0.05) BGH by 66% at -13 or -12 days (2,146 +/- 192 to 3,565 +/- 197 microg/l) and by 92% at -8 or -7 days (2,162 +/- 159 to 4,161 +/- 204 microg/l). After 2 or 3 days of bed rest, there was no response of BGH to the muscle activity, a pattern that persisted through 8 or 9 days of bed rest. However, after 13 or 14 days of bed rest, plasma concentration of BGH was significantly lower after than before muscle activity (2,594 +/- 211 to 2,085 +/- 109 microg/l). After completion of bed rest, muscle activity increased BGH by 31% at 2 or 3 days (1,807 +/- 117 to 2,379 +/- 473 microg/l; P < 0.05), and by 10 or 11 days the BGH response was similar to that before bed rest (1,881 +/- 75 to 4,160 +/- 315 microg/l; P < 0.05). These data demonstrate that the ambulatory state of an individual can have a major impact on the release of BGH, but not IGH, in response to a single bout of muscle activity.

In the present study of rats, we examined the role, during 2 wk of hindlimb suspension, of growth hormone/insulin-like growth factor I (GH/IGF-I) administration and/or brief bouts of resistance exercise in ameliorating the loss of myonuclei in fibers of the soleus muscle that express type I myosin heavy chain. Hindlimb suspension resulted in a significant decrease in mean soleus wet weight that was attenuated either by exercise alone or by exercise plus GH/IGF-I treatment but was not attenuated by hormonal treatment alone. Both mean myonuclear number and mean fiber cross-sectional area (CSA) of fibers expressing type I myosin heavy chain decreased after 2 wk of suspension compared with control (134 vs. 162 myonuclei/mm and 917 vs. 2,076 micron2, respectively). Neither GH/IGF-I treatment nor exercise alone affected myonuclear number or fiber CSA, but the combination of exercise and growth-factor treatment attenuated the decrease in both variables. A significant correlation was found between mean myonuclear number and mean CSA across all groups. Thus GH/IGF-I administration and brief bouts of muscle loading had an interactive effect in attenuating the loss of myonuclei induced by chronic unloading.

The effects of growth hormone (GH) or insulin-like growth factor I (IGF-I) with or without exercise (ladder climbing) in countering the effects of unweighting on fast muscles of hypophysectomized rats during 10 days of hindlimb suspension were determined. Compared with untreated suspended rats, muscle weights were 16-29% larger in GH-treated and 5-15% larger in IGF-I-treated suspended rats. Exercise alone had no effect on muscle weights. Compared with ambulatory control, the medial gastrocnemius weight in suspended, exercised rats was larger after GH treatment and maintained with IGF-I treatment. The combination of GH or IGF-I plus exercise in suspended rats resulted in an increase in size of each predominant fiber type, i.e., types I, I + IIa and IIa + IIx, in the medial gastrocnemius compared with untreated suspended rats. Normal ambulation or exercise during suspension increased the proportion of fibers expressing embryonic myosin heavy chain in hypophysectomized rats. The phenotype of the medial gastrocnemius was minimally affected by GH, IGF-I, and/or exercise. These results show that there is an IGF-I, as well as a GH, and exercise interactive effect in maintaining medial gastrocnemius fiber size in suspended hypophysectomized rats.

The effects of 1 and 10 wk of functional overload (FO) of the rat plantaris with (FOTr) and without daily endurance treadmill training on its myosin heavy chain (MHC) composition were studied. After 1 and 10 wk of FO, plantaris mass was 22 and 56% greater in FO and 37 and 94% greater, respectively, in FOTr rats compared with age-matched controls. At 1 wk, pure type I and pure type IIa MHC fibers were hypertrophied in FO (39 and 44%) and FOTr (70 and 87%) rats. By 10 wk all fiber types comprising >5% of the fibers sampled showed a hypertrophic response in both FO groups. One week of FO increased the percentage of hybrid (containing both type I and type IIa MHC) fibers and of fibers containing embryonic MHC. By 10 wk, the percentage of pure type I MHC fibers was approximately 40% in both FO groups compared with 15% in controls, and the percentage of fibers containing embryonic MHC was similar to that in controls. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed an increase in type I MHC and a decrease in type IIb MHC in both FO groups at 10 wk, whereas little change was observed at 1 wk. These data are consistent with hypertrophy and transformation from faster to slower MHC isoforms in chronically overloaded muscles. The additional overload imposed by daily endurance treadmill training employed in this study (1.6 km/day; 10% incline) results in a larger hypertrophic response but appears to have a minimal effect on the MHC adaptations.

The effects of 14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044) on myosin heavy chain (MHC) isoform content of the rat soleus muscle and single muscle fibers were determined. On the basis of electrophoretic analyses, there was a de novo synthesis of type IIx MHC but no change in either type I or IIa MHC isoform proportions after either SF or HS compared with controls. The percentage of fibers containing only type I MHC decreased by 26 and 23%, and the percentage of fibers with multiple MHCs increased from 6% in controls to 32% in HS and 34% in SF rats. Type IIx MHC was always found in combination with another MHC or combination of MHCs; i.e., no fibers contained type IIx MHC exclusively. These data suggest that the expression of the normal complement of MHC isoforms in the adult rat soleus muscle is dependent, in part, on normal weight bearing and that the absence of weight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.

The effects of 14 days of spaceflight on myonuclear number, fiber size, and myosin heavy chain (MHC) expression in isolated rat soleus muscle fiber segments were studied. Single soleus muscle fibers from rats flown on the Spacelab Life Sciences-2 14-day mission were compared with those from age-matched ground-based control rats by using confocal microscopy and gel electrophoresis. Spaceflight resulted in a significant reduction in the number of fibers expressing type I MHC and an increase in the number of fibers expressing type IIx or IIa MHC. Space-flight also resulted in an increase in the percentage of fibers coexpressing more than one MHC and in the reexpression of the neonatal isoform of MHC in some fibers. Fiber cross-sectional area was significantly reduced in pure type I MHC-expressing fibers and in fibers coexpressing type I+II MHC but not in fibers expressing one or more type II MHC in the flight rats. The number of myonuclei per millimeter was significantly reduced in type I MHC-expressing fibers from the flight rats but was not significantly different in type I+II and type II MHC-coexpressing fibers. Fibers expressing neonatal MHC were similar in size to control fibers but had significantly fewer myonuclei per millimeter than flight fibers not expressing neonatal MHC. In type I MHC-expressing fibers, the reduction in fiber cross-sectional area was greater than the reduction in myonuclear number; thus the average cytoplasmic volume per myonucleus was significantly lower in flight than in control fibers. The reduction in both myonuclear number and fiber size of fibers expressing type I MHC after 14 days of spaceflight supports the hypothesis that changes in the number of myonuclei may be a contributing factor to the reduction in fiber size associated with chronic unloading of the musculature.

The effects of insulin-like growth factor (IGF-I) or growth hormone (GH) with and without exercise on predominantly slow muscles of hypophysectomized hindlimb-suspended (HS) rats were determined. HS resulted in a 21, 23, and 30% decrease in soleus, adductor longus, and vastus intermedius masses, respectively, compared with ambulatory rats. Compared with values in HS rats, IGF-I increased the vastus intermedius mass and GH or exercise alone increased both the soleus and vastus intermedius masses. There was a strong interactive effect between GH, but not IGF-I, and exercise in all three muscles of HS rats. The soleus fiber type distribution of HS rats was not affected by any treatment. HS resulted in a 24, 18 (P > 0.05), 32, and 20% (P > 0.05) decrease in the size of soleus fibers containing type I, IIa, I + IIa, and IIa + IIx myosin heavy chains, respectively, compared with ambulatory hypophysectomized rats. Hormone or exercise alone had no effect on fiber size in HS rats. However, all fiber sizes (except for type IIa + IIx in IGF-I with exercise rats) were larger in HS rats treated with GH or IGF-I and exercise than those in HS rats. These data indicate an interactive effect of both GH and IGF-I with exercise in maintaining fiber size of chronically non-weight-bearing predominantly slow muscles. Furthermore, the results suggest that the myosin heavy-chain phenotype in rats deficient in all pituitary factors is unresponsive to short-term administration of either GH or IGF-I or to exercise or HS.

To determine whether the adaptations in myosin heavy chain (MHC) isoform expression after functional overload (FO) are accompanied by commensurate adaptations in protein isoforms responsible for relaxation [sarco(endo)plasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (SERCA) and phospholamban (PHL)] in single muscle fibers, the isoforms of MHC and SERCA and the presence or absence of PHL were determined for cat plantaris fibers 3 mo after FO. In control plantaris the relative MHC isoform composition was 23% type I, 21% type IIa, and 56% type IIb. FO resulted in a shift toward slower isoforms (33% type I, 44% type IIa, and 23% type IIb). In the deep region of the plantaris the proportions of type I MHC and hybrid MHC fibers (containing type I and II MHCs) were 40 and 200% greater in FO cats, respectively. FO resulted in a 47% increase in the proportion of fibers containing only the slow SERCA isoform (SERCA2) and a 41% increase in the proportion of fibers containing PHL. The proportions of fibers containing type I MHC, SERCA2, and PHL in control and FO plantaris were linearly correlated. These data show that adaptations in MHC isoform expression are accompanied by commensurate adaptations in sarcoplasmic reticulum protein isoforms in single muscle fibers after FO.

Reported specific tension measurements for human skeletal muscle vary widely. This variability could be due, at least in part, to the determination of the physiological cross-sectional area (PCSA) of the muscles. In the present study, serial magnetic resonance images were taken every 10 mm along the lower leg of 8 male subjects to calculate the volume and subsequently the PCSAs of the individual muscles producing plantar flexor and dorsiflexor torques. Maximum plantar flexor and dorsiflexor voluntary isometric torques were determined at ankle joint angles of 90, 100, 110, and 120 degrees. Peak tendon force estimated from torque and moment arm measurements was more than fourfold higher in the plantar flexors (3,623 +/- 136 N) than in the dorsiflexors (832 +/- 19 N). PCSAs were about eight- and threefold higher than the anatomic cross-sectional areas at the level of maximum girth of the calf for the plantar flexor and dorsiflexor groups, respectively. Mean muscle volume and PCSA were 4.6 and 12 times larger in the plantar flexors compared with the dorsiflexors, respectively. The PCSAs of both plantar flexors (r = 0.92) and dorsiflexors (r = 0.80) were highly correlated with the tendon tension of the respective muscle groups. The maximum specific tension was more than twofold higher in dorsiflexors than in plantar flexors. These data suggest that factors other than PCSA contribute to the force output potential of ankle plantar flexors and dorsiflexors in humans.

Biopsies from the vastus lateralis muscle were obtained from three astronauts before and after two 5-day flights and from five astronauts before and after one 11-day flight (space shuttle flights: STS-32, -33, and -34). Muscle fibers from two separate samples from each biopsy were classified as type I and II or as type I, IIA, and IIB by using qualitative myofibrillar adenosinetriphosphatase (ATPase) staining. Cross-sectional area (CSA), number of capillaries per fiber, and the activities of succinate dehydrogenase (SDH), alpha-glycerophosphate dehydrogenase (GPD), and myofibrillar ATPase were determined from one sample of fibers of each myofibrillar ATPase type. Postflight biopsies had 6-8% fewer type I fibers than preflight. Mean fiber CSAs were 16-36% smaller after the 11-day flight with the relative effect being type IIB > IIA > I. Mean fiber CSAs were 11 and 24% smaller in type I and II fibers after 5 days of flight. Myofibrillar ATPase activities increased in type II but not in type I fibers after flight, whereas SDH activity was unaffected in either fast or slow fibers. GPD activity in type I fibers was approximately 80% higher (P > 0.05) postflight compared with preflight. Myofibrillar ATPase/SDH ratios in type II fibers were higher after than before flight, suggesting that some fast fibers were more susceptible to fatigue after flight. The GPD/SDH ratios were elevated in some type I fibers after spaceflight. The number of capillaries per fiber was 24% lower after than before flight, whereas the number of capillaries per unit CSA of muscle tissue was unchanged. These data suggest that adaptations in the size, metabolic properties, and vascularity of muscle fibers can occur rapidly in the space environment. These adaptations were qualitatively similar to those observed in animals after actual or simulated spaceflight conditions for short periods.

The influence of microgravity on the myosin phenotype of skeletal muscle fibers in the vastus lateralis of eight crew members was studied before and after 5-day (n = 3) and 11-day (n = 5) spaceflights (space shuttle flights: STS-32, -33 and -34). Single-fiber electrophoresis analyses showed that the proportion of fibers expressing only slow (type I) myosin heavy chain (MHC) in the vastus lateralis was significantly lower after than before 11 days of spaceflight. Although the family of type II MHC isoforms was elevated post- compared with preflight, the distribution among the isoforms of type II MHC was not statistically different. Based on monoclonal and polyclonal antibodies specific for three adult MHC isoforms and single-fiber electrophoresis, approximately 3% of the fibers analyzed coexpressed all three adult MHC isoforms. The results from immunohistochemical staining with two different sets of antibodies indicate a reduction in the percentage of fibers expressing type I MHC as a result of spaceflight. The mean difference, however, was significant only when the fibers were categorized simply as type I or II. These changes appeared to be highly individualized among the astronauts. These results suggest that a rapid change in MHC isoform expression can occur in some muscle fibers after a relatively brief exposure to spaceflight.

Although a mammalian skeletal muscle fiber may contain thousands of myonuclei, the importance of this number or the potential to modulate it in adult muscle has not been clearly demonstrated. Using immunohistochemistry and confocal microscopy, we examined the plasticity of myonuclear number and fiber size in isolated fast and slow fiber segments from adult cat hindlimb muscles in response to chronic alterations in neuromuscular activity and loading. Compared with slow fibers in the soleus of control cats, myonuclear number in presumably transformed fast fibers was 32% lower and fiber size was decreased 73% after elimination of neuromuscular activation for 6 mo by spinal isolation. Slow fibers in the soleus of spinal-isolated cats had smaller cross-sectional areas, whereas myonuclear number was not significantly different than that in the control cats. Myonuclear number in fast plantaris fibers was more than threefold higher and fiber size was 2.8-fold higher after 3 mo of functional overload compared with the plantaris of control cats. Compared with control slow plantaris fibers, myonuclear number and fiber size also increased in overloaded slow plantaris fibers. These results demonstrate that changes in myonuclear number are associated with changes in myosin type and suggest that modulations in the amount of available DNA may be a factor in regulating cytoplasmic volume of muscle fibers in response to chronic changes in neuromuscular activity.

The effect of a midthoracic spinal cord transection (ST) on myosin heavy chain (MHC) isoform expression in the rat soleus muscle was studied. Electrophoretic analyses demonstrated that 15 days after ST there were significant proportional increases in type IIx, decreases in type IIa, and no change in type I MHC composition. Thirty days after ST, some type IIb MHC was expressed, there were further proportional increases in type IIx, an increase in type IIa (compared with 15-day ST), and a decrease in type I MHC. At both time periods after ST, many fibers expressed multiple MHCs, as demonstrated by immunohistochemistry where a battery of monoclonal antibodies specific to MHC isoforms was used. Fibers were observed containing types I and II together or multiple type II MHC isoforms. These data suggest that the expression of the normal complement of MHC isoforms in the adult rat soleus muscle is dependent, in part, on normal neuromuscular activation.

The contraction times (CTs) of functionally isolated motor units (MUs) in the soleus (SOL) and medial gastrocnemius (MG) muscles were determined in cats that had been spinalized at ages 2 (n = 15) or 12 (n = 9) wk and then either subjected to exercise on a treadmill or simply given manipulative care of the hindlimbs. The MUs were tested approximately 12 wk after the low-thoracic cordotomy, and comparisons were made with data from control animals. The CT of 50.9 ms obtained for SOL units (n = 163) in the spinal cats was 22% shorter than the mean of 65.0 ms for MUs (n = 57) from control cats (n = 4). Contrary to expectation, the CT in animals spinalized at 12 wk was significantly shorter than that in the 2-wk group. The CT for MG units (n = 105) in spinal cats was also significantly shorter (11%) than that in controls cats (n = 66, 6 cats), and those units identified by their high fatigue index as being of slow or fatigue-resistant type had a shorter CT than units with a low index. No distinction in CT of exercised and nonexercised groups was detected for either muscle. These findings are discussed in relation to the bearing influences of supraspinal and segmental origin have on CT duration in SOL and MG muscles during growth of the kitten. A slight, significant decrease (6%) in the fatigue index of SOL MUs (n = 144) was detected, but the values remained high (mean 0.87).

The objectives of the present study were to determine the size and enzyme properties of soleus fibers of rats subjected to a 4-day spaceflight (National Aeronautics and Space Administration, STS-41) and the effects of exogenous growth hormone (GH) on the atrophic response of the muscle. Four groups of rats were studied: 1) control (Con), 2) Con plus GH treated (Con + GH), 3) flight (Fl), and 4) F1 plus GH treated (Fl + GH). Cross-sectional area and the activities of succinate dehydrogenase and myofibrillar adenosinetriphosphatase (ATPase) were determined in fibers identified in frozen serial cross sections. Fibers were categorized immunohistochemically as slow, fast, or slow-fast on the basis of their reaction with slow and fast myosin heavy-chain (MHC) monoclonal antibodies. Fibers also were categorized as light or dark on the basis of their staining for ATPase at pH 8.6. After the 4-day flight, mean body weight was significantly decreased compared with control. The absolute and relative (muscle wt/body wt) soleus weights were significantly smaller in the Fl and Fl + GH rats compared with their respective ground-based controls. In both flight groups, the cross-sectional area of the light ATPase fibers was significantly smaller (approximately 30%) than control. Three of 11 flight rats had a higher proportion of fibers expressing both slow and fast MHCs than expected on the basis of the fiber type distribution in the 11 control rats. Mean fiber succinate dehydrogenase and ATPase activities were similar among the four groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The adaptation of single fibers in medial gastrocnemius (MG), a fast-twitch extensor, and tibialis anterior (TA), a fast-twitch flexor, was studied after 14 days of spaceflight (COSMOS 2044) or hindlimb suspension. Cross-sectional area (CSA) and succinate dehydrogenase (SDH), alpha-glycerophosphate dehydrogenase (GPD), and myofibrillar adenosinetriphosphatase (ATPase) activities were determined in fibers identified in frozen serial cross sections. Fibers were categorized as light, dark, or intermediate on the basis of myosin ATPase staining and alkaline preincubation and immunohistochemically as reacting with slow, fast, or both slow and fast myosin heavy chain monoclonal antibodies. Because there was a close relationship between these two means of categorizing fibers, all fibers were categorized on the basis of the immunohistochemical reaction. The percentage of slow- and fast-twitch fibers of the MG and TA were unchanged in either group. Mean fiber size of all fibers, irrespective of type, was unaffected in either muscle after flight or suspension. The fibers that expressed both fast and slow myosin heavy chains were smaller than control in the MG of both experimental groups. Compared with control, the SDH and total SDH activities in the MG were significantly less in suspended rats, with the fast-twitch fibers showing the largest difference. The ATPase activity in the MG was higher in flight than in control or suspended rats. There were no significant effects of flight on fibers of the TA. In contrast, the TA in suspended rats had higher GPD activities than either control or flight rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of spaceflight on the distribution of succinate dehydrogenase (SDH) activity throughout the cross section of fibers in the soleus was studied in five male rats and in five rats maintained under ground-based simulated flight conditions (control). The flight (COSMOS 1887) was 12.5 days in duration, and the animals were killed approximately 2 days after return to 1 G. Fibers were classified as slow-twitch oxidative or fast-twitch oxidative-glycolytic in histochemically prepared tissue sections. The distribution of SDH activity throughout the cross section of 20-30 fibers (each type) was determined using quantitative histochemical and computer-assisted image analysis techniques. In all the fibers, the distribution of SDH activity was significantly higher in the subsarcolemmal than in intermyofibrillar region. After spaceflight the entire regional distribution of SDH activity was significantly altered in the slow-twitch oxidative fibers. The fast-twitch oxidative-glycolytic fibers of the spaceflight muscles exhibited a significantly lower SDH activity only in their subsarcolemmal region. These data suggest that when determining the influence of spaceflight on muscle fiber oxidative metabolism enzymes, it is important to consider the location of the enzyme throughout the cross section of a fiber. Furthermore the functional properties of the soleus that depend on the metabolic support of mitochondria in the subsarcolemmal region may be primarily affected by exposure to microgravity.

The present study was designed to determine whether the degree and kind of adaptation of a muscle fiber to a functional overload (FO) are determined by properties that are intrinsic to that fiber. The study also addresses the question of the capability of fibers to maintain a normal level of coordination of proteins per fiber as fiber volume changes dramatically. The plantaris muscle of six adult female cats was overloaded for 12 wk by bilateral synergist removal. Plantaris muscle fiber mean size doubled after FO, although some very small fibers that stained dark for adenosinetriphosphatase (ATPase) were observed in some of the FO muscles. There appeared to be no change in total succinate dehydrogenase activity per fiber. A reduction in succinate dehydrogenase activity per unit volume was observed in a substantial number of fibers, reflecting a disproportionate increase in fiber volume relative to mitochondrial volume. In contrast, total alpha-glycerophosphate dehydrogenase activity and actomyosin ATPase activity increased as fiber size increased, whereas there was no change in alpha-glycerophosphate dehydrogenase and ATPase activities per unit volume. Control and FO muscle fibers generally expressed either a fast or slow myosin heavy chain type, but in some cases FO muscle fibers expressed both fast and slow myosin heavy chains. The persistence of variability in fiber sizes and enzyme activities in fibers of overloaded muscles suggests a wide range in the adaptive potential of individual fibers to FO. These data indicate that a severalfold increase in cell size may occur without significant qualitative changes in the coordination of protein regulation associated with metabolic pathways and ATP utilization.

Spaceflight or hindlimb suspension results in a loss of mass and alterations of the metabolic and contractile protein profiles of skeletal muscles toward that resembling faster muscles. Given the influence of motoneurons on muscle properties, ventral horn cells of the lumbosacral enlargement of the spinal cord were studied to determine whether similar adaptations were present. Three groups of rats (5 per group) were studied: control, 14-day spaceflight, and 14-day hindlimb suspension. Spinal cords were quick-frozen, and the succinate dehydrogenase (SDH) activity and cross-sectional area of the soma of ventral horn cells were measured using a computer-enhanced image-processing system. Briefly, the optical density for SDH activity was determined after 8 min of incubation in a medium that gave a steady-state enzymatic reaction. Soma sizes were determined in cells with a visible nucleus. Although there were no significant differences among the three groups in mean cross-sectional area and SDH activity, the population distributions of both variables shifted significantly. In the flight rats, there was a shift toward smaller cells. Compared with control, the population distribution of SDH activities in the flight rats shifted toward higher activities, whereas the distribution shifted toward lower activities in the suspended rats. When considering the interactive effects within individual cells, there was a higher percentage of small cells with high SDH activities in the flight than in the control or suspended rats. These contrasting effects of spaceflight and hindlimb suspension suggest that the changes observed in ventral horn cells were due to factors other than simply the absence of weight support.

Spaceflight causes considerable atrophy in hindlimb muscles of the rat. The purpose of this study was to investigate the effect of a 14-day spaceflight (COSMOS 2044) on selected morphological and metabolic properties of single muscle fibers in a nonhuman primate, Macaca mulatta. Biopsies were taken from the soleus (Sol), medial gastrocnemius (MG), and tibialis anterior (TA) muscles of two rhesus monkeys 107 days before flight and 24 h after return from flight. Muscle biopsies were taken from two independent sites in each muscle by use of a small (3-mm OD) Bergstrom biopsy needle. The biopsies weighed 8-14 mg and contained 100-200 fibers, of which an average of 40 fibers were acceptable for metabolic and size analyses. The 14-day spaceflight had little effect on fiber size in the Sol and MG muscles, whereas there appeared to be a slight decrease in size in the TA. In each of the flight animals, the mean fiber size in the postflight biopsies increased relative to preflight values. An increase in fiber size over the same period of time was also observed in four control monkeys that were the same age and approximately the same weight as the flight monkeys. The relative increase in size was related to the body weight of the monkey at the time of the pre- and postflight biopsies. The mean fiber succinate dehydrogenase activity appeared to decrease in the MG, whereas there was no apparent effect of spaceflight on the Sol and TA muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological and enzymatic responses in fibers expressing fast, slow, or both types of myosin heavy chain (MHC) were studied in rats after 14 days of spaceflight (COSMOS 2044) or hindlimb suspension. Although the percentage of slow-twitch fibers was unchanged, a higher percentage of fibers that expressed both slow and fast MHC was observed in flight and suspended rats than in synchronous ground-based controls. The soleus was 25 and 34% smaller than control after 14 days of flight and suspension, with the reduction in fiber cross-sectional area (CSA) being greater in slow- than in fast-twitch fibers in both experimental groups. The activities of succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) were not significantly affected by flight or suspension. The total SDH activity (i.e., SDH activity x CSA) decreased significantly in the slow-twitch fibers of the flight and the fast-twitch fibers of the suspended rats, in large part due to fiber atrophy. A shift in MHC expression in 14 and 9% of the fibers in flight and suspended rats occurred without a change in myosin adenosinetriphosphatase activity. The SDH and GPD activities of the fibers that expressed both slow and fast MHC were slightly higher than the slow-twitch fibers and slightly lower than the fast-twitch fibers. These data indicate that events were initiated within 14 days of spaceflight or suspension that began to reconfigure the protein profiles of 9-14% of the slow-twitch fibers from typical slow-twitch toward those of fast-twitch fibers, while all fibers were dramatically losing total protein.(ABSTRACT TRUNCATED AT 250 WORDS)