Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells.

Little is known about the mechanism for the prolonged residence time of 25-hydroxyvitamin D (25OHD) in blood. Several lines of evidence led us to propose that skeletal muscle could function as the site of an extravascular pool of 25OHD. In vitro studies investigated the capacity of differentiated C2 murine muscle cells to take up and release 25OHD, in comparison with other cell types and the involvement of the membrane protein megalin in these mechanisms. When C2 cells are differentiated into myotubes, the time-dependent uptake of labeled 25OHD is 2-3 times higher than in undifferentiated myoblasts or nonmuscle osteoblastic MG63 cells (P < .001). During in vitro release experiments (after 25OHD uptake), myotubes released only 32% ± 6% stored 25OHD after 4 hours, whereas this figure was 60% ± 2% for osteoblasts (P < .01). Using immunofluorescence, C2 myotubes and primary rat muscle fibers were, for the first time, shown to express megalin and cubilin, endocytotic receptors for the vitamin D binding protein (DBP), which binds nearly all 25OHD in the blood. DBP has a high affinity for actin in skeletal muscle. A time-dependent uptake of Alexafluor-488-labeled DBP into mature muscle cells was observed by confocal microscopy. Incubation of C2 myotubes (for 24 hours) with receptor-associated protein, a megalin inhibitor, led to a 40% decrease in 25OHD uptake (P < .01). These data support the proposal that 25OHD, after uptake into mature muscle cells, is held there by DBP, which has been internalized via membrane megalin and is retained by binding to actin.

Mechanisms of stretch-induced muscle damage in normal and dystrophic muscle: role of ionic changes.

Muscle damage, characterized by prolonged weakness and delayed onset of stiffness and soreness, is common following contractions in which the muscles are stretched. Stretch-induced damage of this sort is more pronounced in the muscular dystrophies and the profound muscle damage observed in these conditions may involve similar pathways. It has been known for many years that damaged muscles accumulate calcium and that elevating calcium in normal muscles simulates many aspects of muscle damage. The changes in intracellular calcium, sodium and pH following stretched contractions are reviewed and the various pathways which have been proposed to allow ion entry are discussed. One possibility is that TRPC1 (transient receptor potential, canonical), a protein which seems to form both a stretch-activated channel and a store-operated channel, is the main source of Ca(2+) entry. The mechanisms by which the changes in intracellular ions contribute to reduced force production, to increased protein breakdown and to increased membrane permeability are considered. A hypothetical scheme for muscle damage which incorporates these ideas is presented.

Rises in whole muscle passive tension of mammalian muscle after eccentric contractions at different lengths.

This is a report of experiments carried out on the medial gastrocnemius muscle of the anesthetized cat, investigating the effects of eccentric contractions carried out at different muscle lengths on the passive and active length-tension relationships. In one series of experiments, the motor supply to the muscle was divided into three approximately equal parts; in the other, whole muscles were used. Fifty eccentric contractions were carried out over different regions of the active length-tension curve for each partial or whole muscle. Active and passive length-tension curves were measured before and after the eccentric contractions. When eccentric contractions were carried out at longer lengths, there was a larger shift of the optimum length for active tension in the direction of longer muscle lengths and a larger fall in peak isometric tension. Passive tension was higher immediately after the eccentric contractions, and if the muscle was left undisturbed for 40 min, it increased further to higher values, particularly after contractions at longer lengths. A series of 20 passive stretches of the same speed and amplitude and covering the same length range as the active stretches, reduced the passive tension which redeveloped over a subsequent 40-min period. It is hypothesized that there are two factors influencing the level of passive tension in a muscle after a series of eccentric contractions. One is injury contractures in damaged muscle fibers tending to raise passive tension; the other is the presence of disrupted sarcomeres in series with still-functioning sarcomeres tending to reduce it.

Effect of eccentric muscle contractions on Golgi tendon organ responses to passive and active tension in the cat.

To investigate the possibility of a peripheral contribution to the perturbations of force sensation reported to occur after eccentric exercise, responses to passive and active tension were recorded from Golgi tendon organs in the medial gastrocnemius muscle of the anaesthetised cat, before and after a series of eccentric contractions. After the eccentric contractions, nearly all tendon organs commenced firing at a shorter muscle length during slow passive stretch than before, probably because of a rise in whole muscle passive tension. There was a small drop in the sensitivity to incremental tension, but no mean change in tension threshold. Following the eccentric contractions, there was a small, but not significant, increase in tendon organ sensitivity to active tension, which was graded using a method of optimised, distributed stimulation of divided ventral roots. Sensitivity was estimated as the mean response over a range of tensions and as the change in discharge rate in response to incremental tension. The experiments provided the opportunity of comparing tendon organ sensitivities to graded passive and active whole muscle tension. In agreement with previous work in which whole muscle nerve stimulation was employed, little difference was found. It was concluded that the peripheral contribution to perturbations of force perception after eccentric exercise is likely to be small and that the centrally derived sense of effort plays the dominant role. Tendon organs appear to be remarkably reliable in signalling whole muscle tension, whether passive or active, and even after the muscle's force production has been disturbed by fatigue or eccentric exercise.

Passive mechanical properties of the medial gastrocnemius muscle of the cat.

1. This is a report on the history dependence of the passive mechanical properties of the medial gastrocnemius muscle of the anaesthetised cat. 2. The muscle was conditioned with an isometric contraction at the test length, or at 3 mm longer than the test length and then returned to the test length, where the level of resting tension was measured, as well as tension changes during a slow stretch. 3. The level of resting tension depended on the form of conditioning and, at the optimum length for active tension, the history-dependent component was 9 % of the total passive tension. 4. During a slow stretch, tension initially rose steeply up to a yield point, beyond which it rose more gradually. The shape of the tension rise depended on the form of conditioning. The level of tension at the yield point consisted of a stretch-dependent component, the 'short-range tension' plus the resting tension for that length. 5. The short-range tension increased with muscle length to peak close to the optimum for active tension. The slope of the tension rise during a stretch, the short-range stiffness, peaked at 2 mm beyond the optimum. 6. The short-range tension was small immediately after a conditioning contraction but grew in size as the interval was increased up to 60 s, with a time constant of 9.9 +/- 0.6 s. After a series of conditioning movements, it recovered more rapidly, with a time constant of 6.6 +/- 0.5 s. 7. The history-dependent changes in passive tension and the response to stretch are interpreted in terms of the presence, in sarcomeres of resting muscle fibres, of crossbridges between actin and myosin which have very slow formation rates, both at rest and during movements.

Changes in passive tension of muscle in humans and animals after eccentric exercise.

1. This is a report of experiments on ankle extensor muscles of human subjects and a parallel series on the medial gastrocnemius of the anaesthetised cat, investigating the origin of the rise in passive tension after a period of eccentric exercise. 2. Subjects exercised their triceps surae of one leg eccentrically by walking backwards on an inclined, forward-moving treadmill. Concentric exercise required walking forwards on a backwards-moving treadmill. For all subjects the other leg acted as a control. 3. Immediately after both eccentric and concentric exercise there was a significant drop in peak active torque, but only after eccentric exercise was this accompanied by a shift in optimum angle for torque generation and a rise in passive torque. In the eccentrically exercised group some swelling and soreness developed but not until 24 h post-exercise. 4. In the animal experiments the contracting muscle was stretched by 6 mm at 50 mm s(-1) over a length range symmetrical about the optimum length for tension generation. Measurements of passive tension were made before and after the eccentric contractions, using small stretches to a range of muscle lengths, or with large stretches covering the full physiological range. 5. After 150 eccentric contractions, passive tension was significantly elevated over most of the range of lengths. Measurements of work absorption during stretch-release cycles showed significant increases after the contractions. 6. It is suggested that the rise in passive tension in both human and animal muscles after eccentric contractions is the result of development of injury contractures in damaged muscle fibres.

Large-fiber mechanoreceptors contribute to muscle soreness after eccentric exercise.

Muscles subjected to eccentric exercise, in which the contracting muscle is forcibly lengthened, become sore the next day (delayed onset muscle soreness). In subjects who had their triceps surae of 1 leg exercised eccentrically by walking backwards on an inclined moving treadmill, mapping the muscle 48 hours later with a calibrated probe showed sensitive areas were localized but not restricted to the muscle-tendon junction. Injection of 5% sodium chloride into a sensitive site in the exercised leg did not produce more pain than injections into the unexercised leg, suggesting that nociceptor sensitization was not responsible. Applying controlled indentations to a sensitive area showed that the pain could be exacerbated by 20-Hz or 80-Hz vibration. In an unexercised muscle, vibration had the opposite effect; it reduced pain. Pain thresholds were measured before, during, and after a pressure block of the sciatic nerve. The block affected only large-diameter nerve fibers, as evidenced by disappearance of the H reflex and a weakened voluntary contraction, leaving painful heat and cold sensations unaltered. Pain thresholds increased significantly during the block. It is concluded that muscle mechanoreceptors, including muscle spindles, contribute to the soreness after eccentric exercise.

Tension changes in the cat soleus muscle following slow stretch or shortening of the contracting muscle.

1. The permanent extra tension after a stretch and the deficit of tension after a shortening in the soleus muscle of the anaesthetised cat were measured using distributed nerve stimulation across five channels. At low rates of stimulation the optimum length for a contraction was several millimetres longer than that when higher rates of stimulation were used, so that movements applied over the same length range could be on the descending limb of the full activation curve but on the ascending limb of the submaximal activation curve. 2. The extra tension after stretch and the depression after shortening were present only near the peak and on the descending limb of the length-tension curve. Effects on final tension of changing the speed and amplitude of stretches or shortenings were found to be small. 3. Statistical analysis showed that variations in the tension excess or deficit due to changing stimulus rate could be entirely attributed to the effect of stimulus rate on the length-tension relation, as when length was expressed relative to optimum for each rate, stimulus rate was no longer a significant determinant of the tension excess or deficit. 4. The extra tension after stretch and the depression after shortening disappeared if stimulation was interrupted and tension briefly fell to zero. 5. These effects were explained in terms of a non-uniform distribution of sarcomere length changes at long muscle lengths. During stretch some sarcomeres are stretched to beyond overlap while others lengthen hardly at all. During shortening some sarcomeres shorten much further than others. 6. These mechanisms have important implications for exercise physiology and sports medicine.

Damage to human muscle from eccentric exercise after training with concentric exercise.

1. It is known that a period of eccentric exercise provides protection against damage to muscle from subsequent eccentric exercise. Here we ask, does concentric exercise do the opposite, make muscle more prone to damage? 2. The triceps surae muscle group of one leg in each of eight human subjects was subjected to 30 min of concentric exercise per day, for 5 days. At the end of the training period there was a small but significant increase in passive torque in the exercised muscle (P < 0.05), with no changes in the untrained muscle. 3. After a single period of eccentric exercise, angle-torque curves for muscles of both legs shifted in the direction of longer muscle lengths, suggestive of an increase in series compliance. The shift in the concentrically trained muscle was significantly greater over the first 48 h post-exercise (P < 0. 05). 4. The volume of the trained leg increased significantly more than the untrained leg for five subjects over 72 h post-exercise (P < 0.05). Peak torque fell, passive stiffness increased and both muscles became sore, but with no significant differences between the two legs. 5. It is concluded that a period of concentric exercise increases the susceptibility of muscle to changes associated with the damage from eccentric exercise.