Effect of hind-limb suspension on young and adult skeletal muscle. II. Dystrophic mice

Disuse atrophy induced by limb immobilization reportedly protects dystrophic mouse muscle from histopathological changes. This study was conducted to determine whether disuse atrophy induced by hind-limb suspension (HS) limits the histopathology and contractile abnormalities typically observed in the dystrophic mouse. Two weeks of hind-limb suspension were applied to dystrophic mice (line 129B6F1) at two ages, 4 weeks (6 mice) and 12 weeks (8 mice). Thirty-one untreated dystrophics served as controls. In general, HS exaggerated the dystrophic signs, especially in the younger mice; it reduced animal weight, muscle weight, maximum tetanic and twitch tensions, and rates of tetanic and twitch tension development. HS further slowed the contractile properties of soleus (SOL) and extensor digitorum longus (EDL) muscles, and increased their fatigue resistance. HS reduced the size of type I and IIA fibers in the 6-week SOL and EDL, but not in the 14-week muscles. HS produced a preferential atrophy of SOL type I fibers, with a parallel increase in type IIA fibers. However, it did not alleviate the fiber size variability, degree of necrosis, central nucleation, inflammation, or muscle fibrosis in dystrophic muscles. These data demonstrate that disuse by hind-limb suspension does not prevent the histopathological deterioration or loss of muscle function in 6- and 14-week dystrophic mice.     

Isometric contractile properties of fast- and slow-twitch muscles in normal and spastic mice

Isometric contractile properties of the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of 24 male C57 mice were studied in vitro at 35°C. Ten animals exhibited the hereditary movement disorder known as spasticity. EDL muscles of spastic and normal mice developed similar specific tensions and they had similar twitch tension:tetanic tension ratios. The time-to-peak tension and the time to half relaxation in the twitch of spastic EDL muscles were, on average, slightly longer. SOL muscles of spastic and normal mice also developed similar specific tensions and their twitch contractions had similar times to half-relaxation. The time-to-peak tension in the twitch and the twitch tension: tetanic tension ratio of spastic SOL muscles were, on average, higher than in normal SOL. The significance of our findings remains somewhat uncertain.     

Control of contractile properties within adaptive ranges by patterns of impulse activity in the rat

These experiments explore the relationship between patterned impulse activity and contractile properties of skeletal muscles. Soleus (SOL) and extensor digitorum longus (EDL) muscles of adult rats were denervated and stimulated directly from 4 to 15 weeks with the same number of pulse trains at different intratrain pulse frequencies (1-500 Hz), with different numbers of pulse trains (864-4,320,000 pulses/d) at the same intratrain pulse frequencies, or with different combinations of pulse trains at 10 and 100 Hz. Chronic stimulation of the denervated SOL resulted in twitch times-to-peak and half-relaxation times that varied in a graded manner between values longer than those in the normal SOL to values as fast as those in the normal EDL, depending upon the pattern used. Increasing pulse frequencies (constant number) resulted in faster twitches, lower twitch/tetanus ratios, increasing post-tetanic potentiations, and larger tetanic tensions. Increasing pulse numbers (constant frequencies) resulted in slower twitches, lower twitch/tetanus ratios, post-tetanic depressions, and higher fatigue indices. The effect of varying the pulse number on twitch parameters was greater at low frequencies (10-20 Hz) than at high frequencies (100 Hz). SOL muscles receiving pulse trains at both 10 and 100 Hz became much faster than muscles receiving pulse trains at 10 Hz only, even in the experiments where the stimulation pattern contained 9 times as many pulses at 10 as at 100 Hz. Chronic stimulation of both the denervated and the innervated EDL with large numbers of pulses at 10 or 15 Hz resulted in twitches that were only half as slow as those induced in the SOL by the same "slow" patterns. In addition, these patterns led to a marked decrease in maximum tetanic tension and a marked increase in twitch/tetanus ratio. During stimulation with a small number of pulses at 150 Hz, on the other hand, twitch speed, twitch/tetanus ratio, and maximum tetanic tension remained normal or almost normal. We conclude that the isometric twitch and related properties of the rat SOL muscle can be graded within wide "adaptive ranges" by varying either the number or the frequency of pulses. In the EDL, the corresponding adaptive ranges appear much narrower, suggesting that the EDL and the SOL contain intrinsically different muscle fibers.     

Long-term effects of estrogen on rat skeletal muscle

The long-term effects of estrogen on the development of rat extensor digitorum longus (EDL) and soleus (SOL) muscles were examined using physiological and histochemical methods. The rats were in three groups: group 1, ovariectomized; group 2, sham-operated; and group 3, ovariectomized followed by estradiol administration. Isometric twitch and tetanic tensions of both the EDL and SOL obtained from 10-week-old rats were evoked by electrical stimulation. The isometric twitch tensions of the EDL in groups 2 and 3 were significantly lower than in group 1, however, there was no significant differences in isometric twitch tensions of the SOL among the three groups. The isometric tetanic tensions of the EDL in groups 2 and 3 were also significantly lower than in group 1, and the isometric tetanic tension of the SOL in group 3 was significantly lower than in group 1. There were no changes in the total number of muscle fibers or in the ratios of fiber types. But the mean fiber diameter of all fiber types (particularly in types IIA and IIB) was significantly lower in group 3 than in groups 1 and 2. Therefore, the possibility that estrogen may play an inhibitory role in the development of skeletal muscle fibers has to be considered.     

Changes in isometric contractile properties of fast-twitch and slow-twitch skeletal muscle of C57BL/6J dy2J/dy2J dystrophic mice during postnatal development

Our primary aim was to determine if there exists a preferential involvement of the fast-twitch or slow-twitch skeletal muscle fibers in the dy2J/dy2J strain of murine dystrophy. The changes in the contractile properties of the slow-twitch soleus (SOL) and the fast-twitch extensor digitorum longus (EDL) muscles of normal and dystrophic mice were studied at 4, 8, 12, and 32 weeks of age. Isometric twitch and tetanus tension were decreased in the 4- and 8-week-old dystrophic EDL compared with controls, this situation being reversed in the older animals. At 12 weeks, the dystrophic EDL generated 15% more tetanic tension than normal EDL and by 32 weeks no significant difference was seen between normal and dystrophic EDL twitch or tetanus tension. By 8 weeks, dystrophic EDL exhibited a prolonged time-to-peak twitch tension (TTP) and half-relaxation time (1/2RT) of the isometric twitch which continued to 32 weeks. For the dystrophic SOL, decreased twitch and tetanus tension was observed from 4 to 32 weeks. At 8 and 12 weeks, TTP and 1/2RT of dystrophic SOL were prolonged. However, by 32 weeks there was no longer a significant difference seen in TTP or 1/2RT between normal and dystrophic SOL. Our results appear to indicate that a loss of the primary control which is determining the fiber composition of the individual muscles is occurring as the dystrophic process advances.     

Comparison of contractile properties between developing and regenerating soleus muscle: Influence of external calcium concentration upon the contractility

In newborn rat skeletal extensor digitorum longus (EDL) muscle, it has been found that an influx of calcium from the extracellular medium is necessary for contraction, in contrast to the situation observed in adult EDL muscle. The aim of the present study was to determine the influence of the extracellular calcium concentration ([Ca]o) upon the contractile responses elicited in developing as well as in regenerating (notexin-injected) soleus (SOL) muscle. A morphological study was performed to follow the steps of postnatal development and regeneration in SOL muscle. In nominally calcium-free solution, the amplitudes of the twitch and tetanic tensions were greatly reduced in 1–14-day-old developing SOL muscles, as well as in notexin-injected SOL muscles. With longer times after birth, twitch and tetanic tensions of SOL muscle were less affected by the absence of calcium. This contrasts with notexin-injected SOL muscle in which the amplitudes of the contractions remained strongly dependent on [Ca]o. The present finding suggests that some functional characteristics are different in regenerating muscle fibers and may be of interest in the evaluation of the contractile properties of muscles in which injections of genetically engineered or not autologous myoblasts or viral vector have been performed. © John Wiley & Sons, Inc.     

Contractile and fatigue properties of thyrotoxic rat skeletal muscle

The effects of thyrotoxicosis on the contractile properties and development of muscle fatigue in the slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles were examined in rats given 3 mg of L-thyroxine and 1 mg of L-triiodothyronine per kilogram of diet for 6 weeks. The hormone treatment produced significant decreases in the contraction time, one-half relaxation time, and twitch tension in the SOL, while the peak rate of tension development (+ dP/dt) and decline (? dP/dt) in this muscle were elevated. Additionally, the forcefrequency curve was shifted to the right and, thus, resembled the curve of a normal fast-twitch muscle. In contrast, the contractile properties of the fast EDL were relatively unaltered by the hormone administration. Thyrotoxicosis also changed the SOL response to contractile activity as twitch tension, + dP/dt, and ? dP/dt remained high, and a faster decline in muscle glycogen and an increase in lactate occurred compared to control muscles. These results clearly demonstrate a preferential effect of thyroid hormone on slow compared to fast skeletal muscle.     

Contractile properties, fiber types, and myosin isoforms in fast and slow muscles of hyperactive Japanese waltzing mice

This study focuses on the effects of neuromuscular hyperactivity on the contractile properties, fiber type composition, and myosin heavy chain (MHC) isoform expression of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles in Japanese waltzing mice (JWM) of the C57BL/6J-v2J strain. The same properties were studied in the homologous muscle of control CBA/J mice (CM). In comparison to CM, the JWM exhibited (i) longer activity periods, prolonged bouts of running and a higher food intake, (ii) slower twitch and tetanic contractions of both EDL and SOL muscles, decreased cold and post-tetanic potentiation of the EDL, as well as increased cold and post-tetanic depressions of the SOL. Electrophoretic analyses of MHC isoform revealed a shift toward slower isoforms in both EDL and SOL muscles of JWM as compared to the homologous muscles of CM, namely, a shift from the fastest MHCIIb to the MHCIId/x isoform in the EDL muscle and a shift from MHCIIa to MHCI in the SOL muscle. The latter also contained a higher percentage of type I fibers and displayed a higher capillary density than the SOL muscle of CM. These findings show that the inherently enhanced motor activity of the JWM leads to fiber type transitions in the direction of slower phenotypes. JWM thus represent a suitable model for studying fast-to-slow fiber transitions under the influence of spontaneous motor hyperactivity.     

Contrasting effects of suspension on hind limb muscles in the hamster

The contractile properties of plantaris and soleus muscles were studied in 46-day-old hamsters after 4 weeks of rear-end suspension; their controls were animals which had not been treated or else had been operated upon but not suspended. In the suspended hamsters the plantaris muscles, of which approximately 90% of fibers are normally type II, maintained the properties of fast-twitch muscles in terms of their contraction and half-relaxation times, maximum rates of rise of tetanic tension, and posttetanic potentiation of the twitch. The small reduction of mean tetanic tension, although not significant statistically, was compatible with relatively mild atrophy of the type II fibers; approximately half of the type I fibers appeared to have converted to type II. In contrast, the soleus muscles, normally slow-twitch with approximately 60% type I fibers, exhibited significant shortening of their contraction and half-relaxation times after suspension and the maximum rates of rise of tetanic tension were increased. These changes, and the greatly reduced twitch and tetanic tensions, were compatible with the finding of much greater atrophy of type I than type II fibers, together with the conversion of approximately 10% of fibers from type I to type II. Unlike other contractile variables, posttetanic depression of the soleus twitch was unaffected by suspension. Our findings have implications in relation to other models of disuse.     

Temperature dependence of contraction characteristics in developing rat muscles

Contractions of rat extensor digitorum longus (EDL, a fast muscle) and soleus (SOL, a slow muscle) muscles of different ages (1-4 weeks) were recorded in vitro with direct stimulation and at different temperatures (range 35-10 degrees C). Twitch tension in 4-week-old EDL muscle increased in cooling from 35 to 20 degrees C (cooling potentiation); the tension decreased in further cooling below 20 degrees C. This pattern of temperature dependence of twitch tension was seen in fast muscles of all ages (1-4 weeks). Twitch tension in 4-week-old SOL muscle decreased monotonically in cooling from 35 to 10 degrees C (cooling depression). This pattern of cooling depression was not clearly evident in younger SOL muscles. There was a marked hysteresis in the temperature dependence of twitch tension in the 1-week-old SOL muscles. Tetanic tension was depressed by low temperature in both EDL and SOL muscles at 1 week and at 4 weeks of age. Results show that the processes concerned with contractile activation are nearly fully developed in the fast muscle fibers at an early age (1 week), whereas they develop later in the slow muscle fibers.     

Influence of temperature on isometric tension development in mouse fast- and slow-twitch skeletal muscles

The influence of temperature (range 35 to 20°C) on the isometric contractile properties of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of the male C57 mice was studied in vitro, with direct stimulation. Cooling from 35 to 25°C resulted in an average twitch tension potentiation of 42% in EDL and 13% in SOL muscles. Further cooling to 20°C did not produce further potentiation in EDL muscles. The time-to-peak tension and the time to half-relaxation increased 2.4 to 3.2 times for a 10°C cooling in both muscles. The maximum tetanic tension was little changed in cooling from 35 to 30°C, but was depressed 16 to 19% in cooling to 20°C in both muscles. These results from mouse fast and slow muscles were compared with previously published data from muscles of the rat and the cat. The behaviors of the slow SOL muscles in the three species were found to be significantly different, but they could be explained on the basis of their muscle fiber-type composition.     

Effects of paraplegia produced by intrathecal 6-aminonicotinamide on motor units in the rat.

An intrathecal injection of 6-aminonicotinamide (6-AN) in adult rats produced a central myelopathy with destruction of neurons and gliosis. After a latent period of 24 to 36 h, the animals developed flaccid paralysis of the hind limbs, followed by irreversible hypertonic paraplegia. Motor nerve conduction velocities in the tibial nerve of 6-AN-treated animals (serial recordings 3–550 days) were the same as controls and teased nerve fibers to extensor digitorum longus (EDL) and soleus muscles showed no evidence of nerve regeneration. In vivo studies of contractile properties of 6-AN-treated soleus showed that the mean maximal twitch and tetanic tensions were greatly reduced compared to controls by 14 days. The same occurred in the 6-AN-treated EDL, but to a lesser degree. These changes in contractile properties persisted throughout the 570-day period of observation. There was no significant change in the percentage of fiber types in the soleus muscles after 6-AN. However, the EDL had an increased number of type I fibers by 30 days and this peaked at 90 days and was associated with a decrease in type IIC and IIA but not IIB fibers. Initially the type I fibers occurred at random, but by 2 to 3 months, they occurred in small groups of 5 to 20 fibers. The mean areas of all fibers were reduced as early as 2 weeks in both 6-AN-treated EDL and soleus muscles. These changes most likely resulted from the chronic hypertonic paraplegia with extensor posturing, followed by immobilization at knee and ankle joints and loss of some spinal motoneurons with peripheral sprouting, especially of type I motoneurons.     

Slowing of fast-twitch muscle in the dystrophic mouse

Isometric twitch tension was measured in fast-twitch and slow-twitch muscles of normal and dystrophic ( ) mice in vivo. In dystrophic mice more than 6 months old the fast-twitch extensor digitorum longus (EDL) showed a prolongation of the time to peak tension as well as the time to relax to one-half peak tension ( ) compared with age-matched controls. In younger dystrophic mice (4 to 6 weeks) the time to peak tension was prolonged but not significantly so. This apparent “slowing” of dystrophic fast-twitch muscle was accompanied by a reduction in both cooling potentiation and post-tetanic potentiation toward values typical of slow-twitch muscle. Slow-twitch soleus muscle (SOL) of old mice was almost unaffected by the dystrophic process with regared to its contractile characteristics. However, there appeared to be a slight, but significant “speeding” of young dystrophic SOL compared with age-matched control muscles. This was apparent in reduced times to peak tension and half-relaxation as well as an enhanced cooling potentiation. We suggest that the altered contractile characteristics result from a change in some intrinsic property of the muscle fibers rather than from extrinsic factors such as the additional perimysial connective tissue seen in these muscles.     

Changes in number and distribution of orthogonal arrays during postnatal muscle development

Quantitative freeze-fracture electron microscopy was utilized to study the changes in number and distribution of orthogonal arrays (aggregates of 6-7 nm particles) of the sarcolemmas of the fast twitch extensor digitorum longus (EDL) and slow twitch soleus muscles during the first few weeks of postnatal development in the rat. In the adult rat, orthogonal arrays are present in high densities in the fast twitch type II fibers but only in low densities in slow twitch type I fibers. In this study, the changing histochemical profiles of fiber types in the EDL and soleus muscles were also determined for the first month of postnatal development and correlated with the changing number and distribution of orthogonal arrays during the same time frame. At day 3 postpartum, EDL and soleus fibers possessed few orthogonal arrays. The developing EDL fibers rapidly acquired additional orthogonal arrays until the approximate adult number and distribution were attained at postpartum day 25. In contrast, the slow twitch soleus fibers rapidly acquired orthogonal arrays and type IIA fibers until day 35 when both were in excess of adult values. Subsequently, the number of arrays and type IIA fibers declined to normal adult ranges. We suggest that the patterns of development of orthogonal arrays and fiber types are different in the EDL and soleus because the types of innervating motor units are different in the two muscles. The EDL is innervated almost entirely by fast motor units throughout early development and maturity. The soleus, however, is initially innervated by a more heterogeneous population of motor neurons. Thus, during the period of polyneuronal innervation which occurs normally during the first weeks of postnatal development, many individual soleus fibers may possess simultaneous innervation by axons from different motor neuron types. These dual influences may be responsible for the irregular pattern of development of orthogonal arrays and type IIA fibers in developing soleus fibers. Later, as the adult pattern of monosynaptic innervation is developed, expected adult values of orthogonal arrays and fiber types are attained.     

Effects of age on enzyme-histochemical fibre spectra and contractile properties of fast- and slow-twitch skeletal muscles in the rat

Enzyme-histochemical fibre spectra and contractile properties were studied in fast-twitch (extensor digitorum longus (EDL) or tibialis anterior (TA)) and slow-twitch (soleus (S)) muscles of young adult (6 months) and old (20-24 months) male albino rats. It was found that ageing affected fibre size, fibre type proportions, and contractile properties of muscle tissue in both qualitative and quantitative terms and that these age-related alterations differed between fast- and slow-twitch muscles. In the fast-twitch TA and EDL, no differences were observed in either the total number of fibres, the cross-sectional area or the absolute and relative numbers of different muscle fibre types and subtypes between young adult and old animals. In the slow-twitch S, on the other hand, both the total number of muscle fibres and the average cross-sectional fibre area were smaller in the old animals. The fibre loss and fibre atrophy were most pronounced in type II fibres, especially type IIA. In TA, twitch force was higher and tetanus force was unaltered in the old as compared with the young adult animals, resulting in an increased twitch:tetanus ratio in old age. In S, on the other hand, both these forces were lower in the old animals and the twitch:tetanus ratio was accordingly unchanged with age. When the tetanus force was related to age-related differences in total muscle fibre cross-sectional area, no differences were found in the maximum force-generating capacity of maintained contractile material in either fast- or slow-twitch muscles between the two age groups. Probable mechanisms underlying the above alterations are discussed.     

Recovery from fatigue in fast and slow single intact skeletal muscle fibers from aging mouse

In the present work, we studied the recovery from fatigue (RF) of single intact fast- and slow-twitch muscle fibers from young (age 5--7 months) and old (age 22--24 months) mice. To examine whether differences in RF underlie decreases in muscle strength and endurance with aging, we performed in vitro experiments in manually dissected extensor digitorum longus (EDL) and soleus muscle fibers. We measured the recovery of the maximum force every 5 min for a total period of 30 min after inducing fiber fatigue. Fibers were classified, according to the fatigue index, into the following three groups: 0.75--0.99, 0.5--0.74, and <0.5. Although the tetanic tension of EDL and soleus fibers from young and old mice recovered significantly, no statistically significant difference in tension or recovery time was observed between age groups. These data support the concept that the reported decline in muscle force and endurance with aging is not related to changes in RF of individual muscles fibers.     

Effect of muscle creatine content manipulation on contractile properties in mouse muscles

The effects of muscle creatine manipulation on contractile properties in oxidative and glycolytic muscles were evaluated. Whereas control mice (NMRi; n = 12) received normal chow (5 g daily), three experimental groups were created by adding creatine monohydrate (CR group; 5%, 1 week; n = 13); beta-guanidinoproprionic acid, an inhibitor of cellular creatine uptake (beta-GPA group; 1%, 2 weeks; n = 12); or CR following beta-GPA (beta-GPA+CR group; n = 11). Total creatine (TCr) and the contractile properties of incubated soleus and extensor digitorum longus (EDL) muscles were determined. For the soleus, compared with control, TCr increased in the CR group (+25%), decreased in beta-GPA group (-50%), and remained stable in the beta-GPA+CR group, whereas, for the EDL, TCr was similar in the CR, and lower in the beta-GPA (-40%) and beta-GPA+CR (-15%) groups. None of the experimental groups (CR, beta-GPA, or beta-GPA+CR) showed changes in peak tension (P(peak)), time to peak tension, or relaxation in soleus or EDL during twitch or tetanic stimulation. For the soleus, fatigue reduced P(peak) to approximately 60% of initial P(peak); 5 min of recovery restored P(peak) to values approximately 15% higher in CR than in controls. P(peak) recovery was not affected by beta-GPA or beta-GPA+CR in the soleus or any treatment in the EDL. Thus, peak tension recovery is enhanced by creatine intake in oxidative but not glycolytic muscles. This may be implicated in the beneficial action of creatine loading.     

Functional effects of uridine triphosphate on the atrophied soleus muscle of rat after unloading

The purposes of the study were to determine the effects of a pyrimidine nucleotide, the uridine triphosphate (UTP), on the contractile and histochemical properties of the soleus (SOL) muscle following disuse atrophy due to hindlimb unloading (HU) hypokinesia. UTP was injected either during the HU period (2 weeks) or later during the recovery period. In this latter condition, contractile and histochemical properties were studied after 5, 8, 11, and 15 days of spontaneous recovery. HU induced decreases in the SOL weight, force output (twitch and tetanic tensions), time to peak tension during the twitch, and the percentage of type I fibers. The injection of UTP during the HU period did not counteract the modification in speed-related properties, but the decrease in force output was partly counteracted and the proportion of type II C fibers was increased. When UTP was injected during the recovery periods, force-related properties recovered more rapidly. These results suggest that UTP may reduce the loss of force induced by atrophy. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 172–178, 1997.     

Properties of extensor digitorum longus muscle and skinned fibers from adult and aged male and female Actn3 knockout mice

Absence of α-actinin-3, encoded by the ACTN3 "speed gene," is associated with poorer sprinting performance in athletes and a slowing of relaxation in fast-twitch muscles of Actn3 knockout (KO) mice. Our first aim was to investigate, at the individual-fiber level, possible mechanisms for this slowed relaxation. Our second aim was to characterize the contractile properties of whole extensor digitorum longus (EDL) muscles from KO mice by age and gender. We examined caffeine-induced Ca(2+) release in mechanically skinned EDL fibers from KO mice, and measured isolated whole EDL contractile properties. The sarcoplasmic reticulum of KO muscle fibers loaded Ca(2+) more slowly than that of wild-types (WTs). Whole KO EDL muscles had longer twitch and tetanus relaxation times than WTs, and reduced mass and cross-sectional area. These effects occurred in both male and female mice, but they diminished with age. These changes in KO muscles and fibers help to explain the effects of α-actinin-3 deficiency observed in athletes.     

Contractile and histochemical properties of sliced muscle grafts regenerating in normal and denervated rat limbs.

Rat extensor digitorum longus muscles were transversely sliced into 7–8 segments. The muscle slices were autografted back into their original beds. In one series the recipient limbs were normal and in the other, limbs were denervated. At postoperative intervals of 7, 14, 30, and 60 days, the contractile properties (Latency period, contraction and half relaxation times, time parameters of contraction of twitch and tetanus, and twitch and tetanic tension) and histochemical properties (succinic dehydrogenase and myofibrillar ATPase) were analyzed. Sliced grafts regenerating in normally innervated legs followed a typical conversion from slow to fast contraction times, whereas regenerates in denervated limbs remained slow. Histochemically, innervated regenerates developed a heterogeneous pattern of muscle fiber type staining during the second month, whereas different histochemical types of muscle fibers did not appear in noninnervated regenerates. As in ontogeny, denervation retards or prevents the full structural and functional differentiation of regenerating muscle fibers.