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.     

Neuromuscular contacts of expanded motor units in rat soleus muscles are rescued by leupeptin

In the soleus muscle of the rat following section of the L5 ventral ramus (partial denervation) the remaining motor axons increase their territory by sprouting. Nerve sprouts are first seen two to three days after the operation, their number peaks at 10-14 days and subsequently remains at this level. The time course of the initial sprouting in partially denervated muscles is not altered by paralysing the muscles with alpha-bungarotoxin, and the initial extent of the sprouting is, if anything, greater in the paralysed muscles. However, unlike in controls, this level of sprouting is not maintained and neuromuscular contacts are lost when muscles recover from the paralysis. The loss of these contacts can be prevented by treatment of these partially denervated paralysed muscles with leupeptin, an inhibitor of calcium-activated neutral protease. Interestingly, more contacts are rescued when leupeptin is applied 10 days after alpha-bungarotoxin treatment, when sprouting has reached high levels, than at three days, when sprouting has just begun. The neuromuscular connections rescued by leupeptin are functional. Maximum tetanic tension produced by untreated soleus muscles two to five months after partial denervation is 66 +/- 9% of contralateral control muscles, but only 39 +/- 8% when the muscles were paralysed with alpha-bungarotoxin for 12-14 days after partial denervation. However, when partially denervated paralysed muscles were treated with leupeptin three and 10 days after alpha-bungarotoxin treatment their tension output is 74 +/- 3% and 81 +/- 8%, respectively. After partial denervation alone, motor units are twice their normal size. Short-term paralysis with alpha-bungarotoxin prevents this increase in motor unit territory. However, the application of leupeptin to the paralysed muscles rescues neuromuscular contacts, allowing motor unit size to remain expanded, at around 2-2.5-fold. Thus, following recovery from temporary paralysis with alpha-bungarotoxin, there is a sudden withdrawal of neuromuscular contacts and these can be rescued by treatment with leupeptin.     

Properties of isolated adult rat muscle fibres maintained in tissue culture

1. Adult rat skeletal muscles were dissociated by collagenase treatment and trituration, and the isolated muscle fibres were maintained in vitro for 2-3 weeks. At various stages, the fibres were examined physiologically and morphologically. 2. The isolated fibres underwent some changes characteristic of muscle denervated in vivo. For instance, input resistance increased and extrajunctional acetylcholine (ACh) receptors appeared. In addition, the beginning stages of apparent muscle fibre fragmentation were observed. 3. In other respects, the cultured isolated fibres behaved differently than in vivo denervated fibres. Fibrillation developed only occasionally in vitro. The onset of ACh supersensitivity was slower (6 days) than after denervation in vivo (2-3 days). Some fibres developed localized regions of destriation, which apparently was due to loss of in-register alignment of myofibrils.     

Recovery of muscle after different periods of denervation and treatments

Three aspects of reinnervation and recovery of skeletal muscle following various periods of denervation were investigated: (1) the effect of duration of denervation; (2) the effect of hyperthyroidism on recovery; and (3) whether the muscle or the nerve limits recovery. The rat medial gastrocnemius (MG) nerve was cut and then resutured after 0, 3, 7, 21, or 56 days. In a second group of animals, the MG muscle was denervated and, in addition, the animal received triiodothyronine (T3) supplementation during reinnervation. The third group of animals had the denervated MG muscle reinnervated by a larger number of newly transected foreign axons. The force produced by the reinnervated muscle depends on the period that the muscle was denervated. Recovery was impaired when the period of denervation exceeded 7 days. T3 treatment did not benefit the return of force production, nor did providing the muscle with a larger number of newly transected axons.     

Acetylcholinesterase mRNA level and synaptic activity in rat muscles depend on nerve-induced pattern of muscle activation

Acetylcholinesterase (AChE) mRNA levels are severalfold higher in fast rat muscles compared with slow. We hypothesized that AChE mRNA levels and AChE activity in the neuromuscular junction depend on a specific nerve-induced pattern of motor unit activation. Chronic low-frequency stimulation, mimicking the activation pattern in slow muscles, was applied to fast muscles in rats. Molecular forms of AChE were analyzed by velocity sedimentation, and AChE mRNA levels were analyzed by Northern blots. AChE mRNA levels in stimulated fast muscles dropped to 10-20% of control after 1 week and became comparable to those in slow soleus muscles. The activity of the junctional A12 AChE form in 35 d stimulated fast muscles decreased to 56% of control value, reaching that in the soleus muscle. Therefore, synaptic AChE itself depends on the muscle activation pattern. Complete inactivity after denervation also decreased the AChE mRNA level in fast muscles to <10% in 48 hr. In contrast, profuse fibrillations observed in noninnervated immature regenerating muscles maintain AChE mRNA levels at 80% of that in the innervated fast muscles. If protein synthesis was inhibited by cycloheximide, AChE mRNA levels in 3-d-old regenerating muscle, still containing myoblasts, increased approximately twofold. No significant increase after cycloheximide application was observed either in denervated mature fast muscles or in normal slow muscles. Low AChE mRNA levels observed in those muscles are probably not caused by decreased stability of AChE mRNA as demonstrated in myoblasts.     

The utility of K-correction to adjust for a defensive response set on the MMPI

We studied whether denervation affects the expression of tau, in particular phosphorylated tau, and how it is degraded in rat soleus muscles. Immunoblot analysis showed a high molecular weight, approximately 110 kDa (big tau), in normal muscle. Tau levels increased significantly in denervated muscles treated with chloroquine (a lysosomotrophic agent) and in untreated ones, as compared to levels of similarly treated contralateral, innervated muscles. Most of the tau in the innervated and denervated muscles was phosphorylated. Immunohistochemically, tau and beta-tubulin colocated in the sarcoplasm of innervated, saline-treated (intact) muscle, but the staining intensities were very weak. Both proteins, however, were expressed extensively in these areas in the denervated muscles from saline-treated rats. In the denervated muscle of chloroquine-treated rats there were numerous autophagic vacuoles in the sarcoplasm, and phosphorylated-tau accumulation was marked within these vacuoles, indicative that tau first was taken into autophagic, vacuoles by nonselective autophagy then degraded via the lysosomal as well as the nonlysosomal calpain system. Our findings suggest that phosphorylated big tau accumulates with beta-tubulin in denervated muscular atrophy, possibly in order to maintain or preserve the integrity of the muscle fiber during progressive atrophy or regeneration.     

Neurotrophic regulation of insulin-sensitive amino acid uptake in rat fast muscle

Our object was to determine how innervation regulates muscle insulin sensitivity. Insulin-stimulated uptake of the nonmetabolized amino acid, 2-amino-isobutyric acid, was used as a measure of insulin sensitivity in denervated rat extensor digitorum longus muscles retaining either a similar 2.5-cm ("proximal denervation') or a similar to 0.5-cm ("distal denervation') length of distal nerve stump. Because both muscles were inactive in the first 24 h, any difference in insulin sensitivity could be due only to some trophic influence of the distal nerve stump. Fifteen hours after either type of denervation, 2-aminoisobutyric acid uptake was refractory to insulin. However, at 24 h, insulin sensitivity of distally denervated muscles (with or without a second ipsilateral proximal denervation) was normal, whereas that of proximally denervated muscles was still relatively insensitive. In the absence of insulin, the two types of denervated muscles at 24 h showed no difference in 2-aminoisobutyric acid uptake. Finally, organ culture of paired muscles with or without long nerve stumps showed corresponding differences in insulin-stimulated 2-aminoisobutyric acid uptake after 48 h in vitro. Thus, a neurotrophic factor, independent of impulse activity, stretch, or changes in blood flow, regulates one type of muscle insulin sensitivity.     

A regenerative change during muscle adaptation to denervation in rats

The purpose of this study is to examine the cellular and molecular events coincident with muscle denervation, especially the regenerative changes seen following muscle denervation, the role of satellite cells in this process, and the possibility of apoptotic degeneration of myonuclei as a mechanism of myonuclei loss during muscle denervation atrophy. Myosin heavy chain (MHC) isoform expression during muscle denervation was examined using pyrophosphate acrylamide gel electrophoresis and immunohistochemistry. DNA fragmentation (apoptosis) in myonuclei of denervated fibers was investigated using agarose gel electrophoresis, the TUNEL technique and ELISA for cytoplasmic histone-associated DNA fragmentation. Immunohistochemistry for MyoD and BrdU was also performed. Following muscle denervation, embryonic MHC, which is not expressed in adult healthy muscles, was expressed in some denervated fibers as well as in small activated satellite cells; maximal expression was observed 2 to 3 weeks after denervation. Activation and proliferation of satellite cells were observed, while few typical regenerating fibers were identified. It is speculated that most activated satellite cells fused to the denervated maternal fibers in order to repair them instead of fusing to each other to form new fibers as a mechanism that compensates for the atrophic changes after denervation. Although DNA ladder formation was not observed with agarose gel electrophoresis, DNA fragmentation was detected by the TUNEL technique and ELISA, suggesting that apoptotic degeneration contributes to the loss of myonuclei associated with denervation atrophy.     

Mechanism of bone loss after liver transplantation: A histomorphometric analysis

This work analyzed the rat soleus muscle after single and recurrent contusions at different stages of regeneration. A noninvasive contusion was produced by a type of drop-mass equipment. The posterior region of the right hind limb received a trauma and both right and left soleus muscles were analyzed 1, 4, and 6 days after a single contusion (1x), and 6 and 30 days after periodic contusions (10x, one trauma per week for 10 weeks). Single contusion: there was no significant difference between right and left soleus muscle weight. All animals showed abundant signs of acute damage in the right soleus. AChE activity was identified in regeneration segments of the right soleus. Periodic contusions: there was an increase in the right soleus muscle weight (alpha = 5%) only in the animals evaluated 6 days after periodic contusions. The right soleus muscle showed a high incidence of chronic signs of damage, such as split fibers and a centralized nucleus, which predominated when compared with the acute signs. Right soleus muscles showed split fibers with AChE activity in both the proximal and middle regions. There was no difference in the incidence of muscle fiber types (I, II, and IIC) between right and left soleus muscles after periodic contusions. Skeletal muscle contusion is common in humans, especially in sport activities, where repetitive traumas are also frequent. The results of this work indicate that despite the regeneration process there is an important change in the morphological aspect of regenerated muscle fibers, which possibly affect muscle performance.     

Labelled decamethonium in cat muscle

1 Tritium-labelled decamethonium was infused intravenously in 12 cats at final rates of 1.3-4.2 nmol kg-1 min-1 to produce a steady plasma concentration which ranged between 0.21-1.3 mumol/l in different experiments. Muscle contractions were elicited by nerve stimulation and the potential at the end-plate regions of superficial fibres was recorded by extracellular electrodes. 2 Under these conditions, it was not possible to obtain a steady degree of neuromuscular block. The initial decrease in muscle contractions was followed by recovery towards the original value although the concentration of decamethonium in the plasma remained constant, or in some cases rose. The initial depolarization of the end-plate region also waned during the constant infusion of the drug. 3 Once the twitch tension had returned to control values during infusion of the drug, prolongation of the infusion for a total of four hours did not produce a secondary neuromuscular block. 4 Scintillation counting showed that during infusion of labelled decamethonium the radioactivity of the muscles increased progressively with time. The uptake was less in the soleus muscle than in the fast-contracting flexor longus digitorum and extensor longus digitorum muscles. Muscles which had been denervated 12-13 days previously showed a greater uptake of labelled drug than control muscles from the contralateral limb. 5 The labelled drug was localized by autoradiography of frozen sections of leg muscles following intra-arterial injection of decamethonium. Grain counts in individual fibres showed that small amounts of decamethonium had entered the muscle fibres along their entire length, and there was increased uptake of the drug into the cell in the region of the end-plate. 6 The mechanisms underlying the waning of the pharmacological response during constant application of depolarizing drugs are discussed.     

Myosin and troponin changes in rat soleus muscle after hindlimb suspension

We examined the myosin heavy-chain (MHC), troponin T (TnT), and troponin I (TnI) isoform composition in the rat soleus muscle after 21 days of hindlimb suspension using electrophoretic and immunoblotting analysis with specific monoclonal antibodies. The suspended soleus showed a shift in the MHC isoform distribution with a marked increase (from 1.0 to 33%) in the relative amount of type IIa and IIx MHC and a corresponding decrease in type I MHC. However, type IIb MHC, which represents a major component in fast-twitch muscles, was not detected in suspended soleus muscles. TnT and TnI isoform composition was also changed with the appearance of fast-type TnI and TnT bands. However, a high-mobility TnT band, which represents a major component in fast-twitch muscles, was not expressed in suspended soleus. These isoform transitions may be related to the increased maximal velocity of shortening and higher calcium sensitivity previously reported in the rat soleus after hindlimb suspension.     

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.     

Separation of biosynthetic oligosaccharide branch isomers using high-performance liquid chromatography on a porous two-dimensional graphite stationary phase

Tenascin is a large oligomeric glycoprotein of the extracellular matrix. Its location is innervated muscle tissues. We investigated immunohistologically, using two monoclonal antibodies (mah) against Tenascin, biopsied denervated human muscle of children and adults. Tenascin was present in the interstitial space among denervated muscle fibres. Accumulation of Tenascin in denervated adult muscle tissue was frequent, accumulation in denervated muscle tissue of children was sparse and weak. The two antibodies reacted correspondingly. Tenascin was not only found in the vicinity of atrophic muscle fibres, but also close to normally sized fibres, suggesting an early stage of denervation even before reduction of muscle fibre volume.     

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.     

Recovery of acetylcholine release and choline acetyltransferase activity after freezing-induced degeneration of rat soleus muscle

In order to study the effect of synaptic contact on the amounts of choline acetyltransferase (ChAT) and acetylcholine (ACh) in the nerve terminals and on their ability to release ACh, a freeze-thaw procedure was developed as a means to induce long lasting degeneration of rat soleus muscle. It was found that 4 days after the freeze thaw procedure the preparation did not contract upon direct electric stimulation and the level of creatine kinase (CK) was below detection. The preparation contained about 15% of the ChAT activity and 15% of the ACh content of the controls. The ACh release evoked by 50 mM KCl was 25% of controls, but it was, when expressed as a fraction of the ACh content, about twice as high as that in control muscles. At day 12, the preparation still did not contract and the level of CK was less than 5% of controls. The ChAT activity and the ACh content were 40% and 20% of controls, respectively. However, no release of ACh could be evoked by 50 mM KCl. At days 28 and 58 the preparation contracted upon stimulation of the nerve; the CK activity had recovered to about 20% and the ACh content to 40%, while the ChAT activity did not increase above 40%. The KCl-evoked ACh release had recovered to 20-30% of controls. The results indicate that freezing destroyed muscle cells and most intramuscular nerve branches. Subsequent regeneration of muscle fibres was slow, probably because freezing had killed many satellite cells in the muscle. Because the ChAT activity at day 12 had recovered when CK was almost absent and the preparation failed to contract, we conclude that there was expression of ChAT activity in 'nerve terminals' which do not make contact with regenerated muscle cells, although little if any ACh was released from these sites.     

Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

The phosphatase inhibitor okadaic acid (OA) was used to study the relationship between [Ca2+], rates of phosphorylation/dephosphorylation and the mechanical properties of smooth muscle fibres. Force/velocity relationships were determined with the isotonic quick release technique in chemically skinned guinea-pig taenia coli muscles at 22 degrees C. In the maximally thiophosphorylated muscle neither OA (10 microM) nor Ca2+ (increase from pCa 9.0 to pCa 4.5) influenced the force-velocity relationship. When the degree of activation was altered by varying [Ca2+] in the presence of 0.5 microM calmodulin, both force and the maximal shortening velocity (Vmax) were altered. At pCa 5.75, at which force was about 35% of the maximal at pCa 4.5, Vmax was 55% of the maximal value. When OA was introduced into fibres at pCa 6.0, force was increased from less than 5% to 100% of the maximal force obtained in pCa 4.5. The relationship between the degree of myosin light chain phosphorylation and force was similar in the two types of activation; varied [OA] at constant [Ca2+] and at varied [Ca2+]. The relation between force and Vmax when the degree of activation was altered with OA was almost identical to that obtained with varied [Ca2+]. The results show that Ca2+ and OA do not influence force or Vmax in the maximally phosphorylated state and suggest that the level of myosin light chain phosphorylation is the major factor determining Vmax. The finding that the relationship between force and Vmax was similar when activation was altered with OA and Ca2+ suggests, however, that alterations in the absolute rates of phosphorylation and dephosphorylation at a constant phosphorylation level do not influence the mechanical properties of the skinned smooth muscle fibres.     

Decreased insulin action on muscle glucose transport after eccentric contractions in rats

We have recently shown that eccentric contractions (Ecc) of rat calf muscles cause muscle damage and decreased glycogen and glucose transporter GLUT-4 protein content in the white (WG) and red gastrocnemius (RG) but not in the soleus (S) (S. Asp, S. Kristiansen, and E. A. Richter. J. Appl. Physiol. 79: 1338-1345, 1995). To study whether these changes affect insulin action, hindlimbs were perfused at three different insulin concentrations (0, 200, and 20,000 microU/ml) 2 days after one-legged eccentric contractions of the calf muscles. Compared with control, basal glucose transport was slightly higher (P < 0.05) in Ecc-WG and -RG, whereas it was lower (P < 0.05) at both submaximal and maximal insulin concentrations in the Ecc-WG and at maximal concentrations in the Ecc-RG. In the Ecc-S, the glucose transport was unchanged in hindquarters perfused in the absence or presence of a submaximal stimulating concentration of insulin, whereas it was slightly (P < 0.05) higher during maximal insulin stimulation compared with control S. At the end of perfusion the glycogen concentrations were lower in both Ecc-gastrocnemius muscles compared with control muscles at all insulin concentrations. Fractional velocity of glycogen synthase increased similarly with increasing insulin concentrations in Ecc- and control WG and RG. We conclude that insulin action on glucose transport but not glycogen synthase activity is impaired in perfused muscle exposed to prior eccentric contractions.     

A comparison of the electrophysiological effects of two organophosphates, mipafox and ecothiopate, on mouse limb muscles

Adult male albino mice were given single subcutaneous injections of either mipafox (110 mumol/kg) or ecothiopate (0.5 mumol/kg), two organophosphorus compounds (OPs). Acetylcholinesterase activity was measured in the soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles. At 7 and 28 days after dosing, in vitro electrophysiological measurements were carried out in the soleus and EDL. Action potentials and end-plate potentials were evoked at 30 Hz and recorded intracellularly from single muscle fibers. The amplitudes, time course, and latencies of these potentials were measured and the variability (jitter) of latencies was calculated. Recordings after mipafox were also made with 3-Hz stimulation. Acetylcholinesterase activity was inhibited by mipafox (65% in the soleus; 76% in the EDL) and ecothiopate (59% in the soleus; 42% in the EDL). Mipafox and ecothiopate both increased postjunctional (muscle action potential) jitter in the soleus and EDL at 7 days after dosing. Organophosphates caused an increase in end-plate potential amplitudes in the soleus. Mipafox caused an increase in prejunctional (end-plate potential) jitter at 28 days after dosing in both muscles. A single dose of ecothiopate also caused an increase in prejunctional jitter at 28 days in the soleus. The OP-induced increase in jitter was different at different frequencies of stimulation. The results show that there are electrophysiological changes in both muscles after administration of organophosphorus compounds. The slow-twitch soleus appears more sensitive to prejunctional changes caused by OPs than the fast-twitch EDL.     

Contractile properties of aged avian muscle after stretch-overload

The effects of ageing on muscle contractile adaptations to stretch-overload was examined in the anterior latissimus dorsi (ALD) muscle of 12 old (90 weeks of age) and 12 young adult (10 weeks of age) Japanese quails. A weight corresponding to 12% of the birds' body weight was attached to one wing for 30 days, while the contralateral wing served as the intra-animal control. In vitro contractile measurements were made at 25 degrees C by indirect stimulation of the ALD by its nerve (pulse 0.2 ms). Compared with young adult twitch characteristics, aged muscles had significantly greater contraction time (149 +/- 9 ms vs. 174 +/- 16 ms). Stretch-overload increased contraction time to 162 +/- 7 ms in young muscles and 215 +/- 14 ms in old muscles. Ageing and overload resulted in a greater fusing of twitches at stimulation frequencies of 5 and 10 Hz which resulted in a leftward shift of the force-frequency curve at these frequencies, relative to young adult control muscles. Maximal shortening velocity (Vmax) decreased from 2.6 +/- 0.3 to 1.2 +/- 0.1 muscle lengths/s in young muscles after overload. Vmax in old control muscles was similar to young muscles after stretch, but stretch further decreased Vmax in old muscles to 0.8 muscle lengths/s. Maximal tetanic force and specific force were similar in young and old muscles, both before and after stretch. These data indicate that ageing induces a slowing of both twitch contractile characteristics and shortening velocity in the ALD, without affecting maximal force capabilities.