Mechanical and energetic properties of dystrophic (mdx) mouse muscle

The mechanical and energetic properties of extensor digitorum longus (EDL) and soleus muscles of X chromosome-linked muscular dystrophic mutant (mdx) mice aged 4-6 weeks were studied and compared with those of the muscles of normal mice. Maximum tetanic tension, the speed of contraction of relaxation, and the heat production of mdx soleus muscles were not significantly different from those of the normal muscles. However, in mdx EDL muscles, the tension and heat production were significantly reduced, and relaxation was prolonged. To study the cause of these changes in mdx EDL muscles, tension and heat production were measured at various muscle lengths greater than optimum for tension. Both the amount of twitch heat and the heat rate for a tetanus were linearly related to the tension and had non-zero intercepts at zero tension, the activation heat. The twitch activation heat and the tension-related heat in tetani of mdx EDL muscles were not different from those in normal muscles. On the other hand, the tetanus activation heat of mdx EDL muscles was significantly smaller than that of normal muscles. Assuming that the degenerated fibers do not contribute to the active force produced, these results suggest that the amount of Ca2+ released in a contraction is not significantly different between normal and mdx muscles, but the Ca-ATPase activity of the salcoplasmic reticulum is reduced in mdx EDL, which could cause the slowing of relaxation.     

Neurotrophic regulation of muscle cholinesterase: effects of botulinum toxin and denervation

1. In order to determine the role of acetylcholine (ACh) transmission in neurotrophic regulation of muscle cholinesterase (ChE), the effects of botulinum toxin treatment were compared with those of denervation, in terms of two fractions of muscle ChE. The sternocleidomastoid muscles of rats were either denervated or injected with botulinum toxin. After 7 days the muscles were frozen, and sequential sections were assayed for acetylcholinesterase activity. By this method, the ChE activity of end-plates (EPChE) and non-end-plate regions (background ChE, BChE) were separately determined.2. The background ChE was reduced to the same extent by botulinum toxin and denervation. This is interpreted to mean that the neurotrophic regulation of BChE may be wholly cholinergic.3. The EPChE was significantly reduced by both treatments, but the effect of botulinum toxin was only half as great as that of denervation. This indicates that EPChE is only partially regulated by ACh transmission. The larger effect of denervation suggests that some non-cholinergic influence may be operating in this situation as well. Disruption of the structural integrity of the end-plate, with consequent loss of binding sites for the ChE, may account for the additional effect of denervation.     

Control of ACh sensitivity by muscle activity in the rat

1. Rat soleus and extensor digitorum longus (EDL) muscles were examined following complete blockade of sciatic nerve impulses with anaesthetics or diphtheria toxin for periods up to 14 days.2. Muscles showed atrophy equivalent to that seen after similar periods of denervation.3. Nerve blockade appeared to have little or no effect on neuromuscular transmission when tested by stimulation beyond the block. Normal spontaneous miniature end-plate potentials were present.4. Nerve impulse blockade caused the entire muscle membrane to become sensitive to iontophoretically applied acetylcholine.5. The increase in sensitivity in soleus could be prevented by chronic nerve stimulation distal to the region of block.6. Tenotomy, of 5-12 days duration, which produced atrophy, had no effect on the sensitivity of soleus to acetylcholine.7. Chronic direct stimulation of denervated soleus or EDL muscles could prevent the usual denervation supersensitivity, or cause it to decline towards normal once it had appeared. However, the sensitivity of the end-plate region remained normal.     

Chronic effects of botulinum toxin on neuromuscular transmission and sensitivity to acetylcholine in slow and fast skeletal muscle of the mouse

1. A sublethal dose of botulinum toxin (type A) was injected into the muscles of one hind limb of the mouse causing local paralysis.

2. Neuromuscular transmission and muscle sensitivity to acetylcholine (ACh) were studied in vitro in soleus and extensor digitorium longus (EDL) from 6 hr to 4 months after the injection of toxin.

3. Both soleus and EDL failed to respond to nerve stimulation within 6 hr of the injection of toxin.

4. In muscle fibres in which neuromuscular transmission was blocked, subthreshold end-plate potentials (e.p.p.s) were recorded. The amplitude of the e.p.p.s increased during recovery from the effects of the toxin and both muscles contracted in response to nerve stimulation after 2-3 weeks.

5. For about 2 months muscles fatigued more rapidly than normal during repetitive nerve stimulation because of the low quantal content of e.p.p.s.

6. Supersensitivity to ACh developed in 3-5 days and persisted after the return of neuromuscular transmission. Muscle sensitivity to ACh became normal when the rate of fatigue during nerve stimulation was normal.

     

Effects of an anabolic hormone on striated muscle growth and performance

Chronic administration of an anabolic hormone, nandrolone phenylpropionate, in sedentary female rats for 6 weeks gave a 20% increase in body weight and the same proportional increase in all muscles sampled (heart, diaphragm, soleus, TA, EHP and EDL), such that the muscle/body weight ratio was unchanged. Cardiac muscle was unresponsive to treatment. Acute stimulation of EDL via lateral popliteal nerve gave similar values for contraction time, 1/2 relaxation time and twitch: tetanus ratio in both groups suggesting no slowing of the muscle. Fatigue resistance of EDL was improved with 0.29±0.029 vs. 0.46±0.071 of maximum isometric twitch tension being developed after 10 min repetitive stimulation at 4 Hz. This improved endurance was not accompanied by any increase in strength and could not be explained on the basis of cellular hypertrophy, but appears to reflect an increased aerobic capacity of skeletal muscle. The proportion of FOG fibres in EDL increased, 38±1.1% vs. 46±1.1%, and this was paralleled in the other skeletal muscles. Specific hypertrophy of FOG and FG fibres could be conclusively demonstrated in soleus and TA, respectively.     

Differential expression of tenascin after denervation, damage or paralysis of mouse soleus muscle

Summary The expression of the extracellular matrix molecule tenascin was studied by immunocytochemistry and Western blotting in soleus muscles of adult mice after nerve damage (denervation), muscle injury (induced by enforced running or freezing) and functional block of synaptic transmission (botulinum toxin). Enhanced expression of tenascin in the extracellular spaces around focally damaged muscle fibres was found already 10 h after onset of running on a motor-driven treadmill which causes muscle injury in soleus muscle. Tenascin expression reached a peak at 2–3 days post-exercise, after which it declined gradually and became undetectable by two weeks after injury. Similarly, cryo-damage of soleus musclesin situ led to upregulation of tenascin. Chronic muscle denervation after sciatic nerve transection caused a persistent (studied up to 31 days) expression of tenascin at denervated endplates and in intramuscular nerve branches but not in other tissue compartments. Local application of botulinum toxin Type A, which results in muscle inactivity but not in tissue degeneration, however, did not induce tenascin expression 12 h to 12 days post-injection. Expression of tenascin after denervation and muscle damage, but its absence after paralysis, were verified by SDS-PAGE and Western blot analysis. Independent of the type of injury (muscle, nerve or both) the known major isoforms of mouse tenascin, as judged by M_r comparison, were re-expressed, with no preponderance of individual M_r forms. These results show that tenascin expression in adult muscles is induced by both axon and muscle fibre damage but not by muscle inactivity. In contrast, NCAM, in accordance with previous observations, showed enhanced expression both as a result of inactivity and in association with tissue repair.     

Contractile properties of aneurally regenerated compared with denervated muscles of rat

Summary The time course of aneural regeneration in slow-twitch soleus muscles of young adult rats was studied and compared with the changes following denervation in soleus and fast extensor digitorum longus muscles. Regeneration was induced by auto-grafting after treatment with bupivacaine; isometric contractions were recorded from 5 to 70 days later. Force was detected at 5 days; at 12 days force was maximal (at least 20% of original) and thereafter fell exponentially. Force varied normally with total fibre area, except at 5 and 71 days when force generating capacity was low. Contraction and relaxation in the twitch were longer than normal (maximally at 5 days), and were closer to denervated soleus than EDL; in contrast, the maximal rate of rise of force was as high as that of denervated EDL and much higher than in denervated soleus. It is suggested that the muscle was fundamentally fast contracting, but the twitches were probably slow because of greater than normal activation following a single stimulus — a hypothesis supported by twitch: tetanus ratios that were higher than in denervated muscles. Tetanic force was much more sensitive than normal to changes of muscle length from optimum, despite the fact that the lengths of regenerated muscles were similar to those of contralateral muscles.The properties of denervated soleus gradually approached those of regenerated soleus, probably because of replacement of original fibres by regenerated ones.     

Effect of implantation of an extra nerve on the recovery of neuromuscular transmission from botulinum toxin.

1. The common peroneal nerve was implanted into soleus in the mouse and 2 weeks later a sublethal dose of botulinum toxin injected causing a block of neuromuscular transmission at the terminals of the soleus nerve. Most muscle fibres became innervated by the common peroneal nerve. 2. Recovery of neuromuscular transmission at the soleus nerve terminals was delayed in the common peroneal nerve implanted muscles. 3. Stimulation of the soleus nerve after botulinum-evoked subthreshold end-plate potentials (e.p.p.s) in virtually every fibre tested in unoperated muscles. In common peroneal nerve-implanted muscles stimulation of the soleus nerve failed to evoke e.p.p.s in about 40% of fibres tested and where e.p.p.s were recorded their amplitudes were generally smaller. 4. When the common peroneal nerve was cut 2 months after botulinum, neuromuscular transmission at soleus nerve terminals occurred after 4 weeks. When the common peroneal nerve was cut 6 months after botulinum, transmission was found at soleus nerve terminals within 1 week. 5. Recovery of transmission at soleus nerve terminals from the effects of botulinum toxin is delayed if the muscle fibres become innervated by the common peroneal nerve and a proportion of soleus nerve terminals cease to release acetylcholine (ACh) until after the peroneal nerve has been cut.     

Inhibition of cell division and the development of denervation hypersensitivity in skeletal muscle

1. It was found that in 3 day denervated soleus and EDL muscles nuclear division takes place. This can be stopped by administration of either 5-fluoro-uracil or vincristine. 2. The increased sensitivity to acetylcholine following denervation developed at a time when rapid cell division takes place. Both 5-fluoro-uracil and vincristine prevent the development of denervation hypersensitivity both in soleus and EDL muscles. 3. Muscle injury causes some cell division locally, but some phagocytic cells that infiltrate the damaged muscle appear to be blood-borne as their appearance within the muscle is not entirely prevented by either 5-fluoro-uracil or vincristine. Correspondingly the development of increased sensitivity that follows muscle injury is only partly prevented by administration of cytotoxic drugs. 4. It is suggested that the presence of dividing cells is causally related to the development of denervation hypersensitivity.     

Slow skeletal muscles of the mouse have greater initial efficiency than fast muscles but the same net efficiency

The aim of this study was to determine whether the net efficiency of mammalian muscles depends on muscle fibre type. Experiments were performed in vitro (35°C) using bundles of muscle fibres from the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles of the mouse. The contraction protocol consisted of 10 brief contractions, with a cyclic length change in each contraction cycle. Work output and heat production were measured and enthalpy output (work + heat) was used as the index of energy expenditure. Initial efficiency was defined as the ratio of work output to enthalpy output during the first 1 s of activity. Net efficiency was defined as the ratio of the total work produced in all the contractions to the total, suprabasal enthalpy produced in response to the contraction series, i.e. net efficiency incorporates both initial and recovery metabolism. Initial efficiency was greater in soleus (30 ± 1%; n = 6) than EDL (23 ± 1%; n = 6) but there was no difference in net efficiency between the two muscles (12.6 ± 0.7% for soleus and 11.7 ± 0.5% for EDL). Therefore, more recovery heat was produced per unit of initial energy expenditure in soleus than EDL. The calculated efficiency of oxidative phosphorylation was lower in soleus than EDL. The difference in recovery metabolism between soleus and EDL is unlikely to be due to effects of changes in intracellular pH on the enthalpy change associated with PCr hydrolysis. It is suggested that the functionally important specialization of slow-twitch muscle is its low rate of energy use rather than high efficiency.     

Contractile and histochemical properties of regenerating cross-transplanted fast and slow muscles in the rat

Summary The soleus (SOL) or extensor digitorum longus (EDL) muscles of month-old rats were denervated for 14 days and then cross-transplanted so that the fast muscle was placed into the bed of the slow muscle and vice versa. At 17, 30, 60, and 90 days the transplants were tested for certain contractile and histochemical properties. By 90 days the cross-transplanted SOL showed complete conversion of the full contraction time and nearly complete conversion of the half relaxation time to those of the normal EDL. In contrast, the contraction and relaxation times of the cross-transplanted EDL became considerably slowed, but did not attain the values of the normal SOL. Histochemical staining for ATPase and SDH activity demonstrated similar transformations of fiber types. The degree of transformation of twitch and histochemical characteristics in cross-transplanted muscles was greater than the values reported after cross-innervation of the same muscles. The cross-transplantation model has certain advantages over nerve cross-union experiments because the cross-transplanted muscle is placed in the normal functional environment of the other muscle.Supported by grants from the Muscular Dystrophy Associations of America and a scientific exchange between the Academies of Sciences of Czechoslovakia and the United States.     

The Effects of Dietary Creatine Supplements on the Contractile Properties of Rat Soleus and Extensor Digitorum Longus Muscles

Daily creatine supplements (0.258 g kg(-1) ) were administered to adult male Wistar rats (n = 7) in the drinking water. Age matched rats (n = 6) acted as controls. After 5-6 days, contractile properties were examined in soleus and extensor digitorum longus (EDL) muscle strips in vitro at 30 degrees C. In soleus muscles, creatine supplements decreased the half-relaxation time of the isometric twitch from 53.6 +/- 4.3 ms in control muscles to 48.4 +/- 5.5 ms but had no effect on twitch or tetanic tension or on twitch contraction time. In EDL muscles twitch tension, tetanic tension, twitch contraction and half-relaxation times were all unaffected by creatine supplements. Creatine supplements increased the fatigue resistance of the soleus muscles but had no effect on that of the EDL muscles. After a 5 min low-frequency fatigue test, tension (expressed as a percentage of initial tension) was 56 +/- 3 % in control soleus muscles, whereas that in the creatine-supplemented muscles was 78 +/- 6 % (P < 0.01). In the EDL muscles, the corresponding values were 40 +/- 2 % and 41 +/- 9 %, respectively. The force potentiation which occurred in the EDL muscles during the initial 20-30 s of the fatigue test was 170 +/- 10 % of initial tension in the control muscles 24 s after the initial stimulus train but was reduced (P < 0.01) to 130 +/- 20 % in the creatine-supplemented muscles. In conclusion, soleus muscle endurance was increased by creatine supplements. EDL endurance was unaffected but force potentiation during repetitive stimulation was decreased. Experimental Physiology (2001) 86.2, 185-190.     

Correlation between shortening velocity,force—velocity relation and histochemical fibre-type composition in rat muscles

Summary Isometric and isotonic contractions of three muscles in the rat hind leg (soleus, extensor digitorum longus (EDL) and peroneus longus (PL)) were recordedin situ at 35° C and with nerve stimulation. Additionally, the histochemical muscle fibre-type composition of the three muscles was determined by the method of Guth and Samaha (1970). The data obtained from soleus and EDL muscles were similar to those reported in previous studies. On the basis of twitch contraction time, rate of rise of tetanic tension and maximum shortening velocity, the contraction speed of EDL was 2–3 times higher than in soleus. In the PL muscle, the twitch contraction time, rate of tension rise and shortening velocity were 17 ms, 30Po/s and 12 muscle fibre lengths/s, respectively; the data showed that the contraction speed of PL muscle was intermediate between that of the soleus and EDL muscles. In the case of soleus, more than 75% of the cross-sectional area was occupied by type 1 (slow) fibres; in both EDL and PL muscles more than 90% of the area was occupied by type 2 (fast fibres). However, the two fast muscles (EDL and PL) had different proportions of type 2B fibres; the area occupied by the type 2B fibre complement was less than 5% in PL, whereas it was around 70% in EDL muscle. The differences in shortening velocity and force—velocity relation among the three muscles could be explained on the basis of their respective muscle fibre-type compositions.     

Effects of growth hormone on skeletal muscle. II. Studies on regeneration and denervation in adult rats

The effects of human recombinant growth hormone (rhGH) on regenerating skeletal muscle after ischaemic necrosis and on denervated skeletal muscle were studied in normal adult rats. One group of rats was treated with 4 IE rhGH daily by subcutaneous injections, while control rats were injected with saline. The treatment with rhGH resulted in increased levels of insulin-like growth factor-I (IGF-I) in serum. Ischaemic necrosis was achieved in the extensor digitorum longus (EDL) muscle by cutting the supplying vessels and nerve fascicles at the entrance into the muscle. The wet weight and DNA: protein ration in the regenerating muscle were determined 2 and 4 weeks after the operation. The weight of the regenerating muscles in the rats treated with rhGH during the period of study was larger than in the control rats, while the DNA:protein ratio did not differ significantly between the groups. Denervation of the EDL and soleus muscles followed by subsequent reinnervation was obtained by freezing the sciatic nerve with a forceps chilled in liquid nitrogen. Rats treated with rhGH during the period of denervation and reinnervation, i.e. during the 4 weeks after the freezing of the sciatic nerve, revealed increased weight of both the reinnervated and normal muscles compared to corresponding muscles of control rats. Denervation of the EDL and soleus muscles without subsequent reinnervation was achieved by cutting the sciatic nerve at the level of the thigh. Four weeks after denervation the muscles showed atrophy, mainly affecting type 2 fibres in the EDL muscle and both type 1 and type 2 fibres in the soleus muscle.     

The use of local anaesthetics to produce prolonged motor nerve block in the study of denervation hypersensitivity

1. Silastoseal cuffs containing 25% and 45% lignocain, 25% marcain and 25% NaCl were placed onto the sciatic nerve. The effects of this on the sciatic nerve as well as the soleus and extensor digitorum longus (EDL) muscles was studied. 2. Cuffs containing local anaesthetics caused both early and late phases of paralysis, cuffs containing NaCl caused only the late phase of paralysis, showing that the late phase of paralysis was not due to the anaesthetic properties of the drugs. The paralysis produced by these procedures was irreversible. 3. Responses to indirect and direct stimulation of soleus and EDL muscles were compared to assess the degree of denervation. In case of cuffs containing 25% marcain and 45% lignocain more than half of the muscle fibres were denervated. 4. Acetylcholine sensitivity was assesses in all experiments. Soleus and EDL muscles from all animals that had cuffed nerves containing either local anaesthetics or 25% NaCl were hypersensitive to ACh. The degree of hypersensitivity could be correlated to the degree of "denervation" as assessed by comparing the directly and indirectly elicited twitch tensions. 5. It was found that the sciatic nerve undergoes degenerative changes when exposed to cuffs containing either local anaesthetics or 25% NaCl.     

The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor end-plates in slow and fast skeletal muscle of the mouse

1. A sublethal dose of tetanus toxin was injected into the muscles of one hind leg of the mouse and caused local tetanus which persisted for 4 weeks.2. Neuromuscular transmission was studied in vitro in nerve-muscle preparations of soleus, a slow muscle, and extensor digitorum longus (EDL), a fast muscle, from 1 day to 6 months after the injection of toxin.3. Soleus failed to respond to nerve stimulation, became supersensitive to acetylcholine and showed spontaneous fibrillations for several weeks before returning to normal. EDL did not show these changes. A higher dose of tetanus toxin, lethal within 24 hr, caused paralysis of EDL as well as soleus.4. In muscle fibres in which neuromuscular transmission was blocked spontaneous miniature end-plate potentials (m.e.p.p.s) were recorded. The frequency of m.e.p.p.s was increased by repetitive nerve stimulation but not by raising the external potassium concentration.5. The amplitude of spontaneous m.e.p.p.s showed a skew distribution because of a disproportionate number of potentials of less than 0.2 mV.6. Raising the external calcium concentration did not restore neuromuscular transmission.7. Histological examination of soleus showed atrophy of muscle fibres with normal preterminal axons. There was sprouting from motor nerve terminals and subsequently new motor end-plates were formed. These changes were not found in EDL.8. The results indicate that, in the mouse, tetanus toxin causes a presynaptic block of neuromuscular transmission and ;functional denervation' of muscle. Slow muscle is more sensitive to the effects of the toxin than fast.     

Differentiation of fast and slow muscles in the rat after neonatal denervation: A physiological study

Summary Whether an intact innervation is essential for postnatal muscle differentiation was examined in the rat by recording physiological contraction parameters. Muscles in one leg were denervated neonatally (within 24 h of birth) and, between 3–28 days after the operation, their contractions were compared with those of the contralateral control muscles. Experiments were performed on the extensor digitorum longus (edl, a fast muscle) and the soleus (a slow muscle) muscles and contractions were recordedin vitro, at 35 C and with direct stimulation. When compared with the control muscles, 3–4-day-old neonatally denervated fast and slow muscles had longer twitch contractions, higher twitch/tetanus ratios and certain other specific differences in their contraction parameters. These denervation-induced changes in neonatal muscles were essentially similar to those produced 3–7 days after denervation in the differentiated (4-week-old) fast muscle. Despite differences in their absolute values, the contraction parameters of neonatally denervated and control edl muscles changed similarly during development, indicating that postnatal differentiation of fast muscle fibres is independent of a neuronal influence. In the case of the neonatally denervated soleus muscle, the developmental changes in contraction parameters, i.e. shortening of the twitch duration, increase of rate of rise and rate of relaxation in the tetanus and increase of the maximum shortening velocity, were more pronounced than in the control slow muscle; also, there were similarities with the pattern of fast muscle differentiation. Thus, muscle fibre differentiation in soleus becomes altered towards that of a fast muscle after neonatal denervation.     

Effect of short time denervation on intracellular elemental content and fibre atrophy pattern of slow and fast twitch rat muscle

Fast twitch extensor digitorum longus (EDL) and slow twitch soleus (S) muscles were studied after 5 and 16 days of denervation. Elemental contents of single muscle fibres were obtained with energy dispersive X-ray microanalyses (XRMA) applied on cryosections visualised in the scanning or scanning-transmission mode of electron microscopy. Cross sections in series with those produced for analytical electron microscopy were stained for enzyme histochemical fibre typing. A similar increase of Na and Cl was observed in EDL and S fibres after 16 days of denervation and a decrease of K was observed as well in EDL. This time after denervation is known to correspond to the maximal acetylcholine receptor synthesis extrajunctionally. There were no changes of elemental content after 5 days and thus the early membrane changes as i.a. fall in resting membrane potential and increased permeability for Na as well as increased endocytosis did not correspond to changes detectable by XRMA. Time course of atrophy differed between histochemical fibre types in EDL. In S all the muscle fibres atrophied earlier than in EDL and without any difference between fibre types. Then changes in elemental composition of muscle fibres after denervation do not seem to be related to degree and time course of muscle fibre atrophy.     

Activation of glycogen phosphorylase by electrical stimulation of isolated fast-twitch and slow-twitch muscles from rat

The influence of muscle contraction, induced by electrical stimulation, on the activity of glycogen phosphorylase, the contents of high-energy phosphates, hexose-monophosphates and lactate have been studied in isolated extensor digitorum longus (EDL) and soleus muscles from rats. The activity of phosphorylase a + b was about nine times higher in fast twitch muscles (EDL) than in slow-twitch soleus and remained unchanged during the stimulation. A pronounced increase of phosphorylase a occurred during the stimulation in EDL muscle. Stimulation with a frequency of 50 Hz for 10 s and 2 Hz for 90 s resulted in a 44-fold and five-fold increase in phosphorylase a, respectively. In contrast, stimulation of soleus muscle resulted in only a minor increase of phosphorylase a. The rate of glycogenolysis increased in both muscles during the stimulation but the increase was four to five times higher in the EDL than in soleus muscle. The content of phosphocreatine (PCr) before stimulation was much higher in EDL than in soleus but similar after the stimulation. This resulted in a three- to four-fold higher release of inorganic phosphate (Pi) in EDL than in soleus during contraction. Pi has previously been shown to be present in a limiting amount for the activity of phosphorylase and the increase during contraction is of importance for increasing the glycogenolytic rate. It is concluded that the higher glycogenolytic capacity in fast-twitch muscles compared to slow-twitch muscles is due to: (1) higher content of phosphorylase a + b, (2) higher degree of transformation of the enzyme into the a form during contraction, and (3) higher content of PCr, which liberates a large amount of Pi during contraction.