The effect of number of lengthening contractions on rat isometric force production at different frequencies of nerve stimulation.

AIM: To test the effect of 3, 10, 60 and 240 lengthening contractions (LC) on maximal isometric force of rat plantar flexor muscles at different stimulation frequencies. METHODS: Using a dynamometer and electrical nerve stimulation, maximally active skeletal muscles were stretched by ankle rotation to produce LC of the plantar flexor muscles in intact female rats. After the lengthening contraction protocols, maximal isometric force was measured at different frequencies of nerve activation to obtain frequency-dependent force deficits (weakness). RESULTS: The magnitude of the force deficit, measured 1 h after the protocols at 80 Hz, increased as a function of repetition number (three LC, 33.3 +/- 1.7%; 10 LC, 37.2 +/- 2.3%; 60 LC, 67.6 +/- 1.5%; 240 LC, 77.7 +/- 1.2%). Force deficits were also measured at each stimulation frequency tested (5:120 Hz). Using a ratio of isometric force at 20:100 Hz stimulation, the relative depression of force at low frequency was determined. The relative depression of isometric force at low frequency was most prominent during the early repetitions. CONCLUSION: As low-frequency force depression appears to result primarily from excitation-contraction (E-C) coupling failure, the early LC in a series of repeated contractions probably contribute most to damage of the cellular components involved in E-C coupling.

Force deficits after stretches of activated rat muscle-tendon complex with reduced collagen cross-linking.

The forces produced during stretches of passive and activated muscles, and isometric force deficits after stretching of activated muscles were examined in rat plantor flexor muscle-tendon complexes with reduced collagen cross-links (pyridinoline). Female Sprague-Dawley rats (n = 6, age 87 days) were injected twice daily for 43 days with beta-aminopropionitrile (BAPN, 333 mg/kg/day i.p.), an inhibitor of lysyl oxidase, which is responsible for the production of collagen cross-links. The relative weights of the plantar flexor muscles were similar for BAPN and saline-injected (control, C) rats (n = 6). Pyridinoline was lower in the tendon (22.9%), and in the plantaris (17.1%), and soleus (7.4%) muscles (P < 0.05), with no changes observed in collagen content (hydroxyproline), as determined by high-pressure liquid chromatography. At an ankle position of 90 degrees, groups had similar forces at 5, 10, 20, 40, 60 and 80 Hz before stretching. Forces at 40 degrees with stretches of the passive muscles (five times from 90 degrees to 40 degrees) were lower for all stretches in BAPN-injected rats (P < 0.05). Isometric force deficits resulting from stretches of activated muscles (80 Hz, 20 times from 90 degrees to 40 degrees, rest intervals 3 min) followed similar courses for BAPN-injected and C rats, and were 51.1 (2.4)% (C) and 54.7 (4.6)% (BAPN) before the last stretch. After 1 h of rest, isometric force deficits were 26% and 29% larger at 10 Hz and 5 Hz, respectively, in BAPN-treated rats (P < 0.05). The reduction in BAPN-injected collagen cross-linking of the skeletal muscle-tendon complex reduced the forces produced during stretches without muscle stimulation (i.e. passive stretch), and stretching of activated muscles produced larger isometric force deficits only at low stimulation frequencies.

Force deficits after repeated stretches of activated skeletal muscles in female and male rats.

Force deficits after stretches of activated plantar flexor muscles were measured in six male Sprague-Dawley rats (285 +/- 10 g, age 62 +/- 4 days, mean +/- SE) and compared with six age-matched (193 +/- 6 g, age 67 +/- 3 days) and six weight-matched female rats (273 +/- 7 g, age 141 +/- 9 days). Twenty stretches, imposed on isometric contractions at 90 degrees (0.2 ms pulse duration, 80 Hz, 5.4 +/- 0.3 V, duty cycle 0.006), were produced by ankle rotation from 90 to 40 degrees. Before the stretch protocol, weight-matched groups had similar isometric forces at an ankle position of 90 degrees at 5, 10, 20, 40, 60 and 80 Hz but forces were lower for age-matched females. For all groups, normalized force-frequency relationships were similar. During the stretch protocol, deficits for isometric force at 90 degrees and peak stretch force at 40 degrees with stretch number were similar for all groups. One hour after the stretches, isometric force deficits at 90 degrees at 40, 60 and 80 Hz were larger for females in weight-matched groups (e.g. 80 Hz, female: 47.8 +/- 1.7%; male: 41.1 +/- 1.7%; P < 0.05), perhaps because of a difference in age (P < 0.05). For age-matched groups, isometric force deficits at 90 degrees were similar at all frequencies. The susceptibility for force deficits by stretches of activated skeletal muscles was not gender-dependent for 2-month-old rats.

Recovery from 6 weeks of repeated strain injury to rat soleus muscles.

Recovery from chronic strain injury (50 strains daily, five times weekly for 6 weeks to hyperactive soleus muscles) was followed for 3 months in female rats after cessation of chronic hyperactivity induced by pretreatment of the plantar flexor muscles with tetanus toxin. After 6 weeks of repeated strains, muscle mass decreased by 62%, myofiber areas were reduced by 87%, and noncontractile tissue expanded dramatically by 222%. Collagen content increased by almost ninefold (control 40 +/- 3 microg/mg, chronic injury 392 +/- 53 microg/mg), whereas the molar ratio of collagen (pyridinoline) crosslinks to collagen remained the same (control 0.20 +/- 0.01, chronic injury 0.16 +/- 0.01). After 3 months of ambulation, muscle mass returned to normal but myofiber areas remained smaller by 21%, noncontractile tissue was still markedly elevated by 18% with increased collagen content (107 +/- 15 microg/mg), and the molar ratio of crosslinks to collagen increased by 75% during recovery. Thus, rat soleus muscles recovered very slowly and incompletely from chronic strain injuries that produced muscle fibrosis, highlighting the necessity of devising preventative strategies for repeated strain injuries.

Ethanol-mediated CYP1A1/2 induction in rat skeletal muscle tissue.

The causes of non-trauma-mediated rhabdomyolysis are not well understood. It has been speculated that ethanol-associated rhabdomyolysis may be attributed to ethanol induction of skeletal muscle cytochrome P450(s), causing drugs such as acetaminophen or cocaine to be metabolized to myotoxic compounds. To examine this possibility, the hypothesis that feeding ethanol induces cytochrome P450 in skeletal muscle was tested. To this end, rats were fed an ethanol-containing diet and skeletal muscle tissue was assessed for induction of CYP2E1 and CYP1A1/2 by immunohistochemical procedures; liver was examined as a positive control tissue. Enzymatic assays and Western blot analyses were also performed on these tissues. In one feeding system, ethanol-containing diets induced CYP1A1/2 in soleus, plantaris, and diaphragm muscles, with immunohistochemical staining predominantly localized to capillaries surrounding myofibers. Antibodies to CYP2E1 did not react with skeletal muscle tissue from animals receiving a control or ethanol-containing diet. However, neither skeletal muscle CYP1A1/2 nor CYP2E1 was induced when ethanol diets were administered by a different feeding system. Ethanol consumption can induce some cytochrome P450 isoforms in skeletal muscle tissue; however, the mechanism of CYP induction is apparently complex and appears to involve factors in addition to ethanol, per se.

Effect of resistance training on muscle fatigue and recovery in intact rats.

PURPOSE: To examine the effect of resistance training on muscle fatigue from intermittent contractions and subsequent recovery in intact rats. METHODS: By using electrical stimulation, plantar flexor muscles were trained with eccentric and concentric contractions (5 x 10 repetitions, 5 d x wk(-1) for 6 wk) during ankle rotations. By using nerve stimulation, concentric contractions (40) imposed on isometric contractions (stimulation time, 1.9 s; rest period, 13.6 s; intermittent contractions) induced fatigue. During recovery, equivalent contractions were used every 5 min for 30 min. RESULTS: Training increased isometric forces (19% and 23% at ankle positions of 1.57 and 0.70 rad), but muscle weights were not changed. After training, smaller declines in isometric (control, 68.9+/-1.4%; trained, 58.8+/-2.9%) and average concentric force (control, 71.6+/-0.7%; trained, 65.5+/-2.8%) occurred from fatigue. Recovery for 5 min returned isometric and average concentric force to 61.7+/-2.2% and 65.1+/-2.5% of initial values for controls and 76.9+/-2.2% and 77.1+/-2.2% after training. After recovery for 30 min, these forces were 87.6+/-0.7% and 89.2+/-1.1% of initial values for controls and recovered almost completely (94.2+/-1.3% and 94.6+/-1.6%) in trained muscles. During fatigue, the decline in force during successive concentric contractions was larger after training (from 19.7+/-1.1% to 50.1+/-2.0%; controls, from 19.9+/-2.0% to 41.7+/-1.4%). Recovery of this decline in force was training-independent and complete within 5 min. CONCLUSIONS: Rat plantar flexor muscles adapt to 6 wk of 5 d x wk(-1) resistance training with: 1) increased isometric force, 2) smaller losses in isometric and average concentric force during fatigue, 3) larger force decline during concentric contractions during fatigue, and 4) improved recovery following fatigue. Different mechanisms might account for the recovery of the average concentric force and the decline in force during concentric contractions.

Performance of plantar flexor muscles with eccentric and isometric contractions in intact rats.

PURPOSE: To examine the changes in performance of active plantar flexor muscles of rats by controlled dorsiflexion (i.e., stretching of muscles) at two angular velocities. METHODS: Repeated stretches (30) at two velocities of ankle rotation [slow stretch (0.87 rads x s(-1) (i.e., 50 degrees x (s-1))), fast stretch (10.47 rad x s(-1) (i.e., 600 degrees x s-1))] were superimposed on maximally active muscles from an ankle position of 1.57 rad to 0.70 rad (i.e., from 90 degrees to 40 degrees). Repeated isometric contractions (30) of the same duration (1,900 ms) and rest interval (3 min) were performed at 1.13 rad (i.e., 65 degrees). Performance was assessed by measuring the isometric torque at ankle positions of 1.57 and 0.70 rad, work during concentric contractions [range of motion 1.22 rad (i.e., 70 degrees)], and the time to produce 50% of the maximal isometric torque. RESULTS: Thirty isometric contractions resulted in a linear reduction in torque (total deficit of 13.8% at 1.57 rad), whereas for slow and fast stretches, half of the total, nonlinear deficit at 1.57 rad (about 30%) was completed after six stretches. Increases in half contraction times were larger for stretches than for isometric contractions. Reductions in isometric torque were greater at an ankle position of 1.57 rad than at 0.70 rad. One hour of rest after the repeated stretches and isometric contractions did not restore muscle performance. CONCLUSIONS: Isometric contractions of skeletal muscle can create a torque deficit which is much less than that after stretches. Repeated fast and slow stretches resulted in similar torque deficits which did not recover after a rest period of 1 h.

Force output during and following active stretches of rat plantar flexor muscles: effect of velocity of ankle rotation.

During the development of force deficits by repeated stretches, velocity-sensitive changes in the extra force produced during and after subsequent stretching has not been studied. In the present study, repeated dorsiflexion of the foot of rats with maximally contracting plantar flexor muscles was performed at two angular velocities [0.87 (slow muscle stretch) and 10.47rads(-1) (fast muscle stretch)] to examine the active force of the muscles during and following dorsiflexion. Dorsiflexion was performed 30 times with a rest period of 3min between the stretches to minimize muscle fatigue. The ability of rat plantar flexor muscles to produce additional force during the stretch was not velocity sensitive. In contrast, repeated dorsiflexion with fast muscle stretches, but not with slow muscle stretches, resulted in an increase in the force decay with time following the stretches (i.e. increased stress relaxation), as indicated by a change in the time constant of force decay during stress relaxation. Apparently, the stress-relaxation of rat plantar flexor muscles is sensitive to angular velocity of ankle movements; repeated fast, but not slow dorsiflexion, alters the stress relaxation process of active skeletal muscles exposed to stretches which create a force deficit. The change in time constant of force decay during stress relaxation in response to a series of repeated stretches might provide information on the sarcomere length distribution in skeletal muscles.

Isotonic dynamometry for the assessment of power and fatigue in the knee extensor muscles of females.

Impairments in muscle power production and recovery following short-duration intense activity could lead to decreased performance and risk of injury. We developed a power test for the knee extensor muscles using torque-velocity testing and moderate isotonic loads. Twenty-eight female volunteers performed three maximal efforts at each of four isotonic loads (27.1, 40.6, 54.2 and 67.8 N. m). If the calculated regression line for the torque-velocity data had an r2 >/= 0.95 (i.e. an acceptable test), maximal power (408 +/- 56 W) was computed from the data. Immediately after torque-velocity testing, the subjects repeated maximal effort knee extensions with 33.9 N. m for three bouts of 15 repetitions with 15 s of rest to produce muscle fatigue, defined as a decrease in power output during isotonic exercise. After a 4 min rest, the torque-velocity test was repeated and power calculated (345 +/- 48 W). For the group, the recovery of maximal power after the fatigue protocol was 85%. The extremes were represented by one subject who recovered only 70% of her maximal power and another who recovered completely (>98%). Physiological differences in muscle power following repeated exercise could have an impact on the outcome of therapeutic interventions for sports injuries, fatigue syndromes and occupational over-use conditions.

Changes in force by repeated stretches of skeletal muscle in young and old female Sprague Dawley rats.

We examined whether the decline in isometric force and peak stretch force during repeated stretches of activated plantar flexor muscles was larger in old (24 months, N=5) compared with young (4 months, N=5) female Sprague Dawley rats. Thirty stretches were imposed on isometric contractions (stimulation time 1.9 s, rest periods 180 s) by ankle rotation from 90 degrees to 40 degrees at 50 degrees x s(-1). Even though muscle weights were similar [2094 +/- 54 (young) vs 2033 +/- 73 mg (old) (mean +/- SE)], the isometric force (IF) at 90 degrees before stretch 1 and peak stretch force (PSF) at 40 degrees for stretch 1 were lower in old rats [18.9 +/- 0.5 vs 15.5 +/- 1.4 N (p=0.024) and 25.8 +/- 1.3 vs 20.5 +/- 0.9 N (p=0.017)]. After the stretches, the IF deficit was similar [38.5 +/- 3.0% (young) vs 39.8 +/- 3.4% (old)] and did not recover after 1 hour of rest [35.1+/- 4.1% (young) vs 36.9 +/- 4.3% (old)]. The decline in PSF was/also similar [36.0 +/- 5.2% (young) vs 26.6 +/- 1.8% (old)]. Skeletal muscles of 24-month-old female Sprague Dawley rats were weaker than 4-month-old rats but had similar susceptibility to develop IF deficits after stretches of activated muscles.

Force during stretches of rat skeletal muscles after hypertonia at short and long lengths.

Following injection of tetanus toxin into rat gastrocnemius muscle to produce hypertonia, plantar flexor muscles were allowed to shorten (S, n=5) without restraint or held lengthened (L, n=3) by splinting. Saline injected rats served as control (n=5). One week after injection, peak forces during 3 stretches with passive muscles and acute isometric force deficits produced by 15 stretches of electrically stimulated muscles were examined under pentobarbital anesthesia. Isometric force and mass of plantar flexors were similar in S rats but 16% lower in L rats compared to control. Peak passive forces were highest in S rats but not different between L rats and control. At the end of the stretch protocol, isometric force deficits were 26% larger in S rats compared to L rats and 17% smaller in L rats compared to control. Acute isometric force deficits produced by stretches of active skeletal muscles were dependent on the muscle length maintained during hypertonia. Our animal model could be used to test rehabilitation interventions during hypertonia of skeletal muscles.

Determination of fibrosis from cryostat sections using high performance liquid chromatography: skeletal muscle.

Analysis of hydroxyproline (collagen) and pyridinoline (collagen cross-links) in biopsies prepared for routine histological evaluation with OCT compound was performed. Frozen sections (250 microm-thick) were cut from cardiac muscle, diaphragm, liver, and soleus muscle from the rat. After removal of OCT compound by rinsing, the samples were dried, weighed and hydrolyzed in 6 N HCl. A portion of the hydrolysate was analyzed for hydroxyproline using high performance liquid chromatography with collagen type I as the standard. Collagen concentrations ranged from 6.6 microg/mg dry weight (liver) to 74.7 microg/mg dry weight (diaphragm). From the remainder of the hydrolyzate, pyridinoline cross-links of collagen were separated and analyzed similarly by high performance liquid chromatography. The concentration of pyridinoline ranged from 2.6 ng/mg dry weight (liver) to 35.6 ng/mg dry weight (diaphragm). These techniques were adequate to analyze both collagen and pyridinoline (i.e. collagen cross-links) in small biopsy samples (< 1 mg dry weight) routinely used in clinical pathology. The method proved useful in the quantitation of focal fibrosis in a partially denervated rat soleus. Denervation was confirmed using fast myosin immunohistochemistry which revealed large areas of small myofibres containing fast myosin. Collagen concentration increased by five-fold and collagen cross-links by more than 7-fold consistent with fibrotic changes known to occur with denervation.

Isometric and concentric performance of electrically stimulated ankle plantar flexor muscles in intact rat.

The relationship between muscle force and ankle position during isometric and pre-loaded slow concentric contractions (angular velocity, 0.52 rad s-1; range of motion, 1.22 rad) and the recovery of isometric force following concentric contractions at different velocities were determined for electrically stimulated plantar flexor muscles in intact rats. Pre-loaded refers to the isometric contraction which immediately precedes the concentric contraction. Ankle position was controlled by a dynamometer and force was recorded under the sole of the foot. The peak isometric force (19.2 N) was nearly constant at all ankle positions (range of motion, 1.57 rad). The muscle length and distal fibre length of gastrocnemius medialis at ankle positions between 0.79 rad and 2.01 rad were increased by 12.6 % and 20.3 %, respectively. During slow concentric contractions, the force progressively decreased (23.1+/-2.1 %); the force decreased by only 6.3 +/-0.9% during sustained isometric contractions of similar duration (3400 ms). The recovery of isometric force following concentric contractions with similar stimulation frequencies (80 Hz) was velocity dependent (i.e. more rapid at higher velocities). It is concluded that pre-loaded slow concentric contractions of the plantar flexor muscles in intact rats do not follow the same relationship as that of isometric force and ankle position. Our results in intact rats show that the force output of electrically stimulated ankle plantar flexor muscles measured under the sole of the foot can be used to study the physiological properties of skeletal muscle working in situ.

Cellular responses in exertion-induced skeletal muscle injury.

Muscle injury is a common result of muscle exertion caused by overload and over-activity. In this presentation, an attempt was made to discuss models of muscle injury which involve exertion but not excessive strain, although most functional activities of the extremities require some eccentric muscle actions. Muscle injury is characterized by cellular and extracellular matrix responses which appear to be common to all types of muscle trauma -- even in the absence of bleeding. Using tenotomy and functional over-load of the rat hindlimb muscles as examples, illustrations of several of these responses are presented and discussed.

Adaptation of rat gastrocnemius muscles to 2 weeks of centrifugation: myofibers and extracellular matrix.

BACKGROUND: The effect of 2 wk of exposure to centrifugation (2G) on gastrocnemius muscles of rats was investigated by morphometric and computer-assisted image analysis of muscle fiber areas and non-contractile tissue components (extracellular matrix). RESULTS: Muscle atrophy was seen in the myofibers from 2G rats which had decreased in cross-sectional area by 26%. In contrast, the non-contractile tissue component actually increased by 13%. These results were compared with soleus muscle atrophy seen following 2 wk of unloading by tail-suspension. In all cases, the extracellular matrix increased in proportion to the decrease in fiber area. A theoretical model was developed to assess the effect of changes in myofiber cross-sectional area on the relative content of the extracellular matrix. The experimental results from both rat gastrocnemius and soleus muscles were consistent with the model with slight variations due to the known differences in connective tissue content of different muscles in the rat. CONCLUSION: Thus, the gastrocnemius muscle atrophy seen after 2 wk of centrifugation results from loss of contractile and other myofiber specific proteins while the extracellular matrix remains relatively constant. The loss in myofiber content was greater than expected from changes in muscle wet weight and more than required to adapt to a decrease in body weight.

Dynamometer for rat plantar flexor muscles in vivo.

A dynamometer is designed and fabricated to measure the force output during static and dynamic muscle actions of the plantar flexor muscles of anaesthetised rats in vivo. The design is based on a computer-controlled DC servomotor capable of angular velocities in excess of 17.5 rad s-1. The system controls the range of motion, angular velocity and electrical stimulation of the muscles, while monitoring the force output at the plantar surface of the foot. The force output is measured by a piezo-electric load cell that is rated at 5 kg capacity. Angular velocity and position are measured by a DC tachometer and potentiometer, respectively. All measurement devices are linear (r2 = 0.9998). The design minimises inertial loading during high-speed angular motions, with a variation in force output of less than 0.2%. The dynamometer proves to be an accurate and reliable system for quantifying static and dynamic forces of rat plantar flexor muscles in vivo.

Fibrosis and intercellular collagen connections from four weeks of muscle strains.

The effect of repeated cycles of muscle strain was studied in the soleus muscle of female rats. Muscle strains were repeated 3X/week for 1 month using two different strain protocols. Striking changes, including marked variability in fiber size, evidence of degradation and regeneration, and an expanded extracellular matrix were pronounced in the fast-stretched muscles but not in the slow-stretched muscles. However, the slow-stretched muscles did contain struts of connective tissue joining adjacent myofibers. Therefore, repeated muscle strains at high strain rates produced morphological changes similar to many myopathies, including fibrosis, whereas adaptation occurred in response to the same number of strains at slow strain rates. Such diverse tissue responses have relevance to the understanding of the mechanisms of skeletal muscle dysfunction in cumulative trauma disorders and in the design of preventive actions and treatments.

Use of double labeling and photo CD for morphometric analysis of injured skeletal muscle.

We used computer-assisted analysis of myofiber cross-sectional areas to measure skeletal muscle responses to injury and disease. We developed a simple, inexpensive method for measuring myofiber size in human muscle samples using Kodak photo compact discs (CDs) as the image source. The photo CD serves as a permanent image storage medium and provides a high-resolution image that can be used to detect small myofibers. The use of double labeling for dystrophin and desmin allowed positive identification of both degenerating and regenerating fibers in a single biopsy specimen.

Changes in human skeletal muscle ultrastructure and force production after acute resistance exercise.

Muscle ultrastructure and contractile properties were examined before and after a single bout of resistance exercise (8 sets of 8 repetitions at 80% of 1 repetition maximum). Eight untrained males performed the concentric (Con) phase of arm-curl exercise with one arm and the eccentric (Ecc) phase with the other arm. Needle biopsies were obtained from biceps brachii before exercise (Base), immediately postexercise from each arm (post-Con and post-Ecc), and 48 h postexercise from each arm (48 h-Con and 48 h-Ecc). Electron microscopy was used to quantify the presence of disrupted fibers in each sample. Analysis of variance revealed a greater (P < or = 0.05) proportion of disrupted fibers in post-Con, post-Ecc, 48 h-Con, and 48 h-Ecc samples compared with Base. Significantly more fibers were disrupted in post-Ecc (82%) and 48 h-Ecc (80%) samples compared with post-Con (33%) and 48 h-Con (37%), respectively. Voluntary and evoked strength measurements recovered to Base values within 24 h in the Con arm but remained depressed (P < or = 0.05) for 72-96 h in the Ecc arm. These data indicate that both the raising and lowering phases of weightlifting produced myofibrillar disruption, with the greatest disruption occurring during the lowering phase.

Adaptation of rat soleus muscles to 4 wk of intermittent strain.

The effect of repeated strains on rat soleus muscles was investigated by stretching active muscles 3 times/wk for 4 wk with two different methods of stretching. The adaptation of myofibers and noncontractile tissue was followed by histochemical techniques and computer-assisted image analysis. Muscle hypertrophy was seen in the slow-stretched muscles, which increased in mass by 13% and increased in myofiber cross-sectional area by 30%. In the fast-stretched muscle, mass increased by 10% but myofiber cross-sectional area actually decreased. This decrease in mean fiber area was the result of a population of very small fibers (population A) that coexisted with slightly smaller normal-sized fibers (population B). Fibers in population A did not have the distribution expected from atrophy compared with atrophic fibers from unloaded muscles; they were much smaller. In addition, there was a 44% increase in noncontractile tissue in the fast-stretched muscles. Thus, soleus muscles subjected to repeated strains respond differently to slow and fast stretching. Slow stretching results in typical muscle hypertrophy, whereas fast stretching produces somewhat larger muscles but with a mixture of small and normal-sized myofibers accompanied by a marked proliferation of noncontractile tissue.