Consequences of the combined deficiency in dystrophin and utrophin on the mechanical properties and myosin composition of some limb and respiratory muscles of the mouse

The mechanical properties and the myosin isoform composition were studied in three isolated muscles (EDL, soleus, diaphragm) of mutant mice lacking both dystrophin and utrophin (dko). They were compared with the corresponding muscles of the normal and the dystrophin-deficient (mdx) and the utrophin-deficient (uko) mice. In comparison with mdx muscles, dko muscles show a significant reduction of the normalized isometric force, confirmed by the reduced muscular activity of the whole animal. Kinetics parameters (twitch time-to-peak and half-relaxation time) were slightly reduced, and the maximal speed of shortening of soleus, Vmax, was reduced by 30%. The maximal power output (muW/mm3) was reduced by 50% in dko soleus. In the three muscles studied, the relative myosin heavy chains (MHC) composition showed a shift towards slower isoforms. dko EDL presented a dramatic decrease of the resistance ot tetanic contraction with forced lengthenings (eccentric contractions), while muscle lacking only utrophin (uko mutants) display a normal resistance to this exacting mechanical challenge. These experiments suggest that lack of both dystrophin and utrophin is very detrimental to the mice and that mechanical properties of the muscles may explain the overall phenotype. Moreover these results bring some support to the idea that the expression of utrophin in mdx muscle compensates, to some extent, for the lack of dystrophin.     

Molecular forms of acetylcholinesterase in dystrophic (mdx) mouse tissues

We analyzed the activity of acetylcholinesterase (AChE) and its molecular forms in the tissues of normal and dystrophic (mdx) mice, at different developmental stages. We studied the brain, the heart and the serum, in addition to four predominantly fast-twitch muscles (tibialis, plantaris, gastrocnemius and extensor digitorum longus (EDL)) and the slow-twitch, soleus muscle. We found no difference between mdx and control mice in the AChE activity of the brain and the heart. The skeletal muscles affected by the disease undergo active degeneration counterbalanced by regeneration between 3 and 14 weeks after birth. The distribution of AChE patches associated with neuromuscular junctions was abnormally scattered in mdx muscles, and in some cases (tibialis and soleus), the number of endplates was more than twice that of normal muscles. There were only minor differences in the concentration and pattern of AChE molecular forms during the acute phase of muscle degeneration and regeneration. After this period, however, we observed a marked deficit in the membrane-bound G4 molecular form of AChE in adult mdx tibialis, gastrocnemius and EDL but not in the plantaris or in the soleus, as compared with their normal counterparts. Whereas the amount of AChE markedly decreased in the serum of normal mice during the first weeks of life, it remained essentially unchanged in the serum of mdx mice. It is likely that this excess of AChE activity in serum originates from the muscles. A deficit in muscle G4 was also reported in other forms of muscular dystrophy in the mouse and chicken. Since it is not correlated to the acute phase of the disease in mdx and also occurs in genetically different dystrophies, it probably represents a secondary effect of the dystrophy.     

Insulin resistance and clinical evolution in infantile myotonic dystrophy

1. Extensor digitorum longus muscles of C57 BL/10 and mdx mice were overloaded by removing the synergist tibialis anterior muscle of 9-12-day-old animals. The effect of this operation on the weight, contractile properties and force of the extensor digitorum longus muscle was examined at two different ages, i.e. at 2-3 months (young group) and at 5-8 months (old group). The changes with age in both the control and overloaded muscles of normal and mdx mice are also described. The values obtained from the overloaded muscles were always compared with those for the control, unoperated extensor digitorum longus. 2. In the normal strain of mice the weight of the overloaded extensor digitorum longus muscle in the younger group was increased and it remained higher in the older animals. In the mdx mice the overloaded extensor digitorum longus muscles weighed more in the younger animals but not in the older group of mice. 3. The twitch and tetanic tensions of the overloaded muscles were slightly, but not significantly, increased in the younger group of mdx mice, whereas in the older animals there was a significant decrease in both twitch and tetanic tensions. 4. Thus the overloaded muscles from mdx mice progressively deteriorated with age. In both strains of mice the overloaded muscles become less fatigable with time.     

Recovery of muscle functions in rats following prolonged movement restraint

Experiments were carried out to measure time and force parameters of isometric contractions and structural parameters of muscles of hind limbs of rats which were exposed to hypokinesia for 130 days and then were kept under surveillance for 3 months. During recovery certain functions of the contractile system returned to the normal. At the same time M. soleus displayed persistent changes that were indicative of atrophic developments. Unlike M. soleus where delayed contraction (due to an increase in the time of relaxation) was followed by losses in force, fast muscles (M. ext. digit. longus and M. plantaris) showed recovery of force parameters. They also exhibited delayed contraction due to an increase in the time of tension development and half-relaxation. These differences are associated with dissimilar function and structure of muscular fibers. On the whole, the pattern of recovery of the motor functions reflects responses to an increased load of the neuromuscular system of animals which are kept in large cages after prolonged hypokinesia.     

High physiological levels of epinephrine do not enhance muscle glycogenolysis during tetanic stimulation

This study examined whether high physiological concentrations of epinephrine (EPI) would enhance muscle glycogenolysis during intense muscular contractions. Muscles of the rat hindlimb were perfused for 12 min at rest and 45 s of tetanic stimulation (1.0-Hz train rate, 100-ms train duration at 80 Hz) without EPI (control) or with 15 or 35 nM EPI. In the EPI groups the muscles were perfused with EPI for the last 2 min of rest perfusion and throughout stimulation. Glycogenolysis in the white gastrocnemius, red gastrocnemius, plantaris, and soleus muscles during stimulation was unaffected by the presence of EPI in the perfusion medium. In addition, muscle lactate and hindlimb lactate efflux were similar in EPI and control groups. It is concluded that EPI is not important for enhancing glycogenolysis in rat muscles composed predominantly of fast-twitch fibers during intense short-term tetanic stimulation.     

Skeletal muscle fatigue and physical endurance of young and old mice

In order to evaluate the role played by muscular and extramuscular factors in the development of fatigue in old age, the time course of fatigue in isolated skeletal muscles and spontaneous motor activity and endurance of whole animals were monitored using young (3-6 months) and old (34-36 months) CF57BL/6J mice. The isolated extensor digitorum longus (EDL) and soleus muscles from old mice had smaller (P < 0.05) mass and developed lower (P < 0.02) maximal tetanic tension at 100-Hz stimulation than the muscles of young mice. During stimulation at 30 Hz every 2.5 s, a 50% decline in original tetanic tension occurred by 109 s in young EDL and 129 s in old EDL, but by 482 s in young soleus and 1134 s (projected) in old soleus, indicating more (P < 0.05) resistance to fatigue in old than young soleus. However, the old mice showed significantly fewer (P < 0.002) spontaneous ambulatory movements than the young mice. On a treadmill with a belt speed of 10 m/min at an inclination of 0 degrees, the old mice could only run for 22 min compared to 39 min ran by young mice (P < 0.02). They took more rest periods (P< 0.02) than the young mice. In a quantitative swimming monitor, the old mice swam for a shorter (P < 0.05) time than young mice (20.4 min compared to 28.6 min). Integrated swimming activity at 20 min was smaller (P < 0.05) in old mice than in young mice (413 g/s compared to 628 g/s). Hence increased fatigue in old age is not caused by impairment of processes within the muscles, but by impairment of central or extramuscular processes.     

Does utrophin expression in muscles of mdx mice during postnatal development functionally compensate for dystrophin deficiency?

We correlated utrophin expression with the physiopathological course in mdx mice. Evolution of the pathology was assessed by monitoring expression of developmental MHC in mdx mice versus control. Utrophin expression is detected by dystrophin/utrophin cross-reacting antibodies and can only be evaluated in mdx mouse muscles (in absence of dystrophin). This protein was expressed at the periphery of all myotubes and myofibers during the first postnatal week. It began declining in fast muscles before the third week and disappeared from the soleus between the 3rd and the 4th week. The decrease was concomitant with a sudden degenerative/regenerative process affecting slow muscle earlier and more massively than fast muscles. The pathological process became stable in all muscle types (except the diaphragm), with greater utrophin expression in the soleus. These results in mdx mice along with observed utrophin expression in severely affected DMD patients suggest that overexpression of utrophin is not enough to explain the stability of regenerated fibers in mdx mice.     

Differing effects of antihypertensive agents on urinary albumin excretion

Skeletal muscles in an animal model of genetic hypertension (the spontaneously hypertensive rat. SHR) exhibit significant deficits in contractile performance. These deficits appear to be unrelated to the rise in blood pressure. Slow-twitch soleus muscles show a decrease in specific muscle tension and a reduced resistance to muscle fatigue during prolonged contractile activity. We tested the hypothesis that the reduced fatigue resistance occurs as a consequence of an impaired ability to maintain or restore Na+ and K+ balance across the sarcolemma during repeated contractions. This may result from a genetically based increase in the Na+ permeability of SHR muscles, coupled with a reduction Na+, K+ pump capacity as the animals mature. Soleus muscles in adult SHR exhibit a significant increase in intracellular Na+ content and a significant decrease in intracellular K+ content at rest. B6RB+ uptake in Na(+)-loaded hypertensive muscles is 45% less than predicted from the number of ouabain-binding sites available. Activation of Na+, K+ pumps using adrenaline or insulin produces a significantly smaller hyperpolarization in hypertensive soleus than in control muscles. Control soleus muscles are hyperpolarized for at least 10 min after a 4 min period of high-frequency activity, but hypertensive soleus muscles remain at resting polarity. Nonetheless, the number of ouabain-binding sites in hypertensive muscle is significantly greater than in control soleus, and binding affinities are similar. This apparent deficit in pump capacity might lead to a greater and more prolonged increase in extracellular K+ during repetitive contractions,and an associated decline in tension. Recently, we have been able to prevent the abnormal decrease in hypertensive soleus fatigue resistance by long-term treatment (8 weeks) with the Ca2+ blocker amlodipine. The therapy prevented or reversed the contractile deficits, but did not restore the responsiveness of the Na+, K+ pump to hormonal stimulation. The current data suggest that both a reduction in Na+, K(+)-pump capacity and changes in Ca2+ distribution play a role in the development of contractile deficits in hypertensive muscles.     

Appearance of complex branched muscle fibers is associated with a shift to slow muscle characteristics

To examine the effects of numerous complex branched fibers (CBF) on whole muscle contractile properties, we established a model of myopathic muscles containing a large number of CBF by repeated local injection of bupivacaine hydrochloride (Marcaine) into the plantaris (PLA) muscle. Marcaine injections were administered once weekly for 10 weeks into the right PLA muscles of Wistar male rats. The in situ contractile properties of Marcaine-injected PLA muscles (I-PLA) were examined under urethane anesthesia, and compared with the contralateral (control) PLA muscle (C-PLA). Numerical and morphological examination using the modified nitric acid fiber digestion method and scanning electron microscopy showed that Marcaine resulted in an 8-fold increase in the number of branched fibers in the I-PLA muscles and about 70% of these fibers were CBF. The latter were composed of ten or more muscle fibers fused together along with many thin and thick, long and short daughter branches. The time to peak tension of twitch and tetanus, and 1/2 relaxation time were significantly longer in I-PLA muscles, representing a shift to slow muscle characteristics. However, the total area of slow fibers/muscle cross-sectional area was similar in I-PLA and C-PLA muscles. Aggregates of same-type fibers (slow fibers) with small and large diameters were observed, reflecting an expected cross-sectional property of CBF. Our results suggest that the appearance of several CBF in a muscle is associated with a shift towards slower contractile properties in the affected muscle.     

Adrenergic and nitrergic responses of the rat isolated anococcygeus muscle to a new toxin (makatoxin I) from the venom of the scorpion Buthus martensi Karsch

Muscle fibre composition was compared among the proximal (25%), middle (50%) and distal (75%) regions of muscle to investigate whether denervation induces region-specific changes of fibre types in the soleus and plantaris muscles of rats. Decreases in mass were observed in both muscles after denervation. In the soleus muscle, denervation increased the percentage of type I fibres with a concomitant increase in the proportion of type IIC and IIA fibres. The extent of such transformations was greater in the proximal region than the middle and distal regions. In normal plantaris muscle, the middle region showed a higher proportion of type IIA fibres with a lower percentage of type IIB fibres reciprocally than other regions. These regional differences in fibre types were not detected in the 4-week denervated plantaris muscle. These findings suggest that denervation-induced transformations from type I to type II fibres begin in the proximal region in the soleus muscle of rats. In addition, regional differences in fibre types along the muscle length could be regulated by neuromuscular activity through normal innervation in the plantaris muscle.     

Frequency of tendon organ discharges elicited by the contraction of motor units in cat leg muscles

1. The responses elicited in individual tendon organs by the contraction of single motor units were studied in peroneus longus, peroneus brevis, tibialis anterior and soleus muscles. 2. No simple relation was found between the discharge frequency of a tendon organ and the tension produced in the muscle tendon by the contraction of individual motor units. 3. The sensitivity of a given tendon organ to contractile tension was not the same for each of the motor units which elicited its discharge. There was no correlation between the sensitivity of the receptor and the strength of the motor units. 4. Upon repetitive stimulation of a tendon-organ-activating motor unit at increasing rates, the frequency of the receptor sustained discharge reached a maximal value for rates of stimulation eliciting submaximal tetanic tension. Higher rates only produced an increase in the dynamic component of the tendon organ response. 5. These observations show that the contractile tension sensed by a tendon organ is not a simple fraction of the tension which appears at the muscle tendon. They might be accounted for as consequences of the fine structure of tendon organs and of variations in the number of muscle fibres contributed by different motor units to the bundle inserted on each receptor. The location of most tendon organs at musculo-aponeurotic junctions rather than in the tendon proper, could also be responsible for some of the observed discrepancies.     

Effects of long-term phasic electrical stimulation on denervated soleus muscle: guinea-pig contrasted with rat

Guinea-pig soleus muscles were denervated and electrically stimulated for periods of 43 to 66 days. Stimuli were in 1 s bursts of 40 Hz pulses, repeated every 5 min. Other guinea-pigs were denervated for 82 days without stimulation and, in a third group, the soleus muscle was necrotized and allowed to regenerate without reinnervation for 13-15 days. Isometric and isotonic recordings were made in vivo. Denervated guinea-pig muscles were embedded in epoxy resin for light and electron microscopy. Chronic stimulation of denervated guinea-pig soleus had no effects on the prolonged twitch or on reduced maximal shortening velocity, maximal rate of rise of tension and tetanic force. This contrasts with the slow-to-fast conversion produced by denervation and denervation-stimulation of rat soleus. Loss of force was much greater in rat than guinea-pig after denervation, and chronic stimulation increased force in rat to the same level as in guinea-pig after denervation (with or without stimulation). Eighty-day denervated guinea-pig soleus did not reveal those morphological signs of fibre breakdown and regeneration which are prominent in denervated rat soleus muscles. Those changes in rat resembled aneurally regenerated muscles in several aspects, especially the increased incidence of fibres with internal myo-nuclei which did not appear in guinea-pig soleus after denervation. Aneurally regenerated guinea-pig soleus became fast like aneurally regenerated rat muscle. Our data are compatible with the hypothesis that slow-to-fast transformation of denervated rat soleus is not directly brought about by chronic stimulation but by de-novo formation of fast-contracting regenerated fibres. The persistence of fibrillation in guinea-pig but not rat after denervation may account for the species difference.     

Skeletal muscle protein content and synthesis after voluntary running and subsequent unweighting

The effects of exercise training with or without subsequent unweighting on wet weight, protein content, and in vivo fractional protein synthesis were studied in soleus and plantaris muscles of juvenile female Sprague-Dawley rats under the following four conditions: normal weight bearing (N), voluntary-activity wheel running (WR) for up to 4 weeks, mechanical unweighting for 7 days via hindlimb suspension (HS), or wheel running followed by 7 days of hindlimb suspension (WR-HS). Fractional protein synthesis was determined by the 3H-phenylalanine flooding method. Increases (P < .05) in wet weight and protein content were detected in the soleus after just 1 week of running, with no increase in fractional protein synthesis. Two weeks of running were required for an increase in protein synthesis in this muscle. Significant increases in these parameters were first observed in the plantaris after 2 weeks of running. Maximal increases occurred by 3 weeks in both muscles. Reductions (P < .05) in soleus and plantaris parameters were observed in both HS and WR-HS groups compared with N and WR groups, respectively. However, protein content and fractional synthesis were maintained at significantly higher levels in WR-HS muscles compared with HS muscles. These results indicate that (1) wheel training represents a noninvasive method for inducing rapid hypertrophy of the skeletal muscles studied, in part by increasing fractional protein synthesis; (2) unweighting decreases protein content and synthesis to the same extent whether the muscles are trained; and (3) previously hypertrophied muscles display higher protein contents and fractional protein synthesis following unweighting compared with unweighted muscles of untrained animals.     

Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.     

Effects of chronic low frequency stimulation on contractile and elastic properties of hindlimb suspended rat soleus muscle

Chronic low frequency stimulation (10 Hz, 8 h x day[-1]) was used in this study to prevent the changes in the contractile and elastic properties of rat soleus muscles induced by 3 weeks of hindlimb suspension (HS). Results showed that electrostimulation was able to counteract in part the decrease in soleus muscle mass and tension output induced by unweighting. On the other hand, the increases in maximal shortening velocity and twitch speed following HS were not prevented by stimulation. Unweighting was responsible for an increase in series elastic compliance of soleus muscle. Chronic stimulation successfully counteracted this increase in series compliance probably by changing the properties of the tendon. The partial recovery of muscle mass and tension output as a result of stimulation enhanced the role of contractile activity in preventing muscle atrophy. Moreover, the inefficiency of the tonic activity imposed by stimulation in preventing the increase in twitch speed of soleus muscle during HS demonstrated the primacy of neuronal activity. Discrepant results concerning changes in contraction kinetics deduced from the twitch could have been due to the fact that such myograms also depend on the series compliance.     

The effect of ischemic preconditioning on the recovery of skeletal muscle following tourniquet ischemia

It has been well documented that ischemic preconditioning limits ischemic-reperfusion injury in cardiac muscle, but the ability of ischemic preconditioning to limit skeletal muscle injury is less clear. Previous reports have emphasized the beneficial effects of ischemic preconditioning on skeletal muscle structure and capillary perfusion but have not evaluated muscle function. We investigated the morphologic and functional consequences of ischemic preconditioning, followed by a 2-hour period of tourniquet ischemia on muscles in the rat hindlimb. The 2-hour ischemia was imposed without preconditioning, or was preceded by three brief (10 minutes on/10 minutes off) preischemic conditioning intervals. We compared muscle morphology, isometric contractile function, and muscle fatigue properties in predominantly fast-twitch, tibialis anterior muscles 3 (n = 8) and 7 (n = 8) days after ischemia-reperfusion. Two hours of ischemia, followed by reperfusion, results in a 20 percent reduction of muscle mass (p < 0.05) and a 33 percent reduction in tetanic tension (p < 0.05) when compared with controls (n = 8) at 3 days. The same protocol, when preceded by ischemic preconditioning, results in similar decreases in muscle mass and contractile function. Neuromuscular transmission was also impaired in both ischemic groups 7 days after ischemia. Nerve-evoked maximum tetanic tension was 69 percent of the tension produced by direct muscle stimulation in the ischemia group and 65 percent of direct tension in the ischemic preconditioning/ischemia group. In summary, ischemic preconditioning, using the same protocol reported to be effective in limiting infarct size in porcine muscle, had no significant benefit in limiting injury or improving recovery in the ischemic rat tibialis anterior. The value of ischemic preconditioning in reducing imposed ischemic-reperfusion-induced functional deficits in skeletal muscle remains to be demonstrated.     

The force-frequency relationship is altered in regenerating and senescent rat skeletal muscle

Maximal tetanic tension was elicited at 200, 150, and 150 Hz in control tibialis anterior muscles and at 150, 100, and 100 Hz in 14-day regenerating muscles of young (3 months), adult (18 months), and old (31 months) Fischer 344/Brown Norway F1 rats, respectively. In contrast to young rats, increasing stimulation frequency from 50 to 150 Hz did not elicit significantly greater tetanic tension in control or regenerating muscles of old rats. At higher stimulation frequencies, tetanic fade was prevalent in control and regenerating muscles of adult (250-300 Hz) and old rats (200-300 Hz), but was only present at 14 days of recovery in regenerating muscles of young rats (300 Hz). The decreased efficacy of rehabilitative and physical medicine procedures in adult and elderly patients who have suffered skeletal muscle injury could be explained, in part, by the postulate that tetanic fade is indicative of inadequate synaptic transmission.     

Effects of ovariectomy and hindlimb unloading on skeletal muscle

Female rats (7-8 mo old, n = 40) were randomly placed into the intact control (Int) and ovariectomized control (Ovx) groups. Two weeks after ovariectomy, animals were further divided into intact 2-wk hindlimb unloaded (Int-HU) and ovariectomized hindlimb unloaded (Ovx-HU). We hypothesized that there would be greater hindlimb unloading-related atrophy in Ovx than in Int rats. In situ contractile tests were performed on soleus (Sol), plantaris (Plan), peroneus longus (Per), and extensor digitorum longus (EDL) muscles. Body weight and Sol mass were approximately 22% larger in Ovx than in Int group and approximately 18% smaller in both HU groups than in Int rats (Ovx x HU interaction, P < 0.05), and there was a similar trend in Plan muscle (P < 0.07). There were main effects (P < 0.05) for both ovariectomy (growth) and hindlimb unloading (atrophy) on gastrocnemius mass. Mass of the Per and EDL muscles was unaffected by either ovariectomy or hindlimb unloading. Time to peak twitch tension for EDL and one-half relaxation times for Sol, Plan, Per, and EDL muscles were faster (P < 0.05) in Ovx than in Int animals. The results suggest that 1) ovariectomy led to similar increases of approximately 20% in body weight and plantar flexor mass; 2) hindlimb unloading may have prevented ovariectomy-related muscle growth; 3) greater atrophy may have occurred in Sol and Plan of Ovx animals compared with controls; and 4) removal of ovarian hormonal influence decreased skeletal muscle contraction times.     

Response of mouse plantaris muscle to functional overload: comparison with rat and cat

Functional overload (FO) of a muscle by removing its synergists results in a compensatory hypertrophy of the muscle. However, the extent of the response appears to be dependent, at least in part, on the activity and/or loading levels of the muscle following surgery. Thus, differences in the inherent physical activity levels across species may be an important factor to consider. In the present study, the effects of 8 weeks of FO on the isometric mechanical properties of the plantaris of mice (highly active) were determined and the findings compared with the results from previous studies performed on the plantaris of rats (highly active) and cats (less active). FO resulted in approximately a doubling of the mass, the physiological cross-sectional area and the maximum tetanic tension per unit cross-sectional area, was similar in the plantaris of control and FO mice. Isometric twitch speed properties were unaffected, but the tension enhancement in response to an increase in the rate of stimulation showed the pattern of a "faster" muscle following FO. The fatigue resistance of the plantaris in FO mice was significantly higher than in control mice. Although the degree of hypertrophy that occurred in the mouse plantaris was similar to that observed after FO in rats and in cats that are exercised intermittently at high intensities, there were differences in the mechanical properties that may be related to the adaptability of species and/or the behavioral responses to the overload.