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Changes in the forms of the components of the troponin complex during regeneration of injured skeletal muscle.

Using the immunoperoxidase technique, antibodies to the fast components of the troponin complex stained all regenerating cells after localized alcohol injury to rat skeletal muscle. Antibodies to slow troponin components stained only some of these cells. About 6 weeks after injury with the nerve intact, the fast and slow forms of the troponin components were located in different cells. During the later stages of regeneration, staining for myosin ATPase correlated with the staining with antibodies to fast and slow troponin components. A similar staining pattern was also observed in the early stages of regeneration of muscle denervated at the time of injury. In this case, antibodies to fast skeletal muscle troponin components continued to stain all the cells 10 weeks after injury. Injured denervated muscle cells stained equally dark by myosin ATPase after preincubation at pH 9.4 over this period. None of the regenerating myotubes in denervated muscle stained for myosin ATPase after preincubation at pH 4.3.

Animals↗

Insulin resistance of denervated rat muscle: a model for impaired receptor-function coupling.

The effect of short-term denervation on the response to insulin was studied in isolated rat soleus and extensor digitorum longus (EDL) muscles 6 and 24 h after severing one sciatic nerve. Impaired insulin sensitivity and response occurred within 6 h postdenervation in solei. After 24 h, EDL of fed and fasted rats and solei of fed rats showed no stimulation of glycogen synthesis even with supraphysiological doses, whereas solei of fasted rats showed markedly decreased sensitivity and response to insulin. Insulin resistance of glycogen synthesis represented impaired stimulation of glucose transport and impaired glucose-independent activation of glycogen synthase by insulin. Changes in initial glycogen content of muscles did not correlate with insulin resistance. Insulin binding after denervation showed only minimum impairment and did not account for the marked insulin resistance. The response of denervated solei to epinephrine was unimpaired. Insulin resistance, which develops early after denervation in red and white muscles, represents primarily a defect in receptor-function coupling, suggesting that in muscle, nervous stimuli and/or contractile activity modulate signal transmission by the occupied insulin receptor.

Animals↗

Arteriole constriction following ischemia in denervated skeletal muscle.

Arteriolar diameters and flow patterns were determined in the isolated rat cremaster muscle following 4 hr of total ischemia. Two groups of six rats each were studied--an innervated group and a denervated group. The microcirculation was observed using intravital microscopy, and the images were recorded on videotape. The whole arterial tree was scanned, and the smallest vessel diameter for each arterial segment was measured. Preischemic mean arteriole diameters were significantly larger in the denervated group, compared to the innervated group. Following ischemia, during the 3 hr of reperfusion, the main (AI) cremaster arteriole from both the innervated and denervated groups constricted to 50 to 70 percent of pre-ischemic diameters. However, the actual diameters (as distinct from percentage change) were not significantly different between the innervated and denervated groups. There were significant differences in flow patterns. Some A2 and A3 vessels became temporarily nonflowing vessels (either open but without low, or closed by severe spasm). These poorly-functioning vessels were significantly more common in the denervated group. Furthermore, this phenomenon of temporary no-flow persisted longer in the denervated group over the 3-hr reperfusion period. These observations suggest that the ability to recover quickly from an ischemic insult is less in tissues that have been denervated.

Animals↗

Alanine and aspartate aminotransferases in normal and denervated skeletal muscle.

Activities of alanine and aspartate aminotransferases are maintained during the first 5 weeks of growth, but decrease subsequently in normal chick gastrocnemii. In sciatectomized muscles, a 5-fold elevation in these enzymes reveals increased utilization of amino acids as a compensatory metabolic support during denervation atrophy.

Alanine↗

Cyclic adenosine monophosphate accumulation and beta-adrenergic binding in unweighted and denervated rat soleus muscle.

Unweighting, but not denervation, of muscle reportedly "spares" insulin receptors, increasing insulin sensitivity. Unweighting also increases beta-adrenergic responses of carbohydrate metabolism. These differential characteristics were studied further by comparing cyclic adenosine monophosphate (cAMP) accumulation and beta-adrenergic binding in normal and 3-day unweighted or denervated soleus muscle. Submaximal amounts of isoproterenol, a beta-agonist, increased cAMP accumulation in vitro and in vivo (by intramuscular [IM] injection) to a greater degree (P less than .05) in unweighted muscles. Forskolin or maximal isoproterenol had similar in vitro effects in all muscles, suggesting increased beta-adrenergic sensitivity following unweighting. Increased sensitivity was confirmed by a greater receptor density (Bmax) for [125I]iodo-(-)-pindolol in particulate preparations of unweighted (420.10(-18) mol/mg muscle) than of control or denervated muscles (285.10(-18) mol/mg muscle). The three dissociation constant (Kd) values were similar (20.3 to 25.8 pmol/L). Total binding capacity (11.4 fmol/muscle) did not change during 3 days of unweighting, but diminished by 30% with denervation. This result illustrates the "sparing" and loss of receptors, respectively, in these two atrophy models. In diabetic animals, IM injection of insulin diminished cAMP accumulation in the presence of theophylline in unweighted muscle (-66% +/- 2%) more than in controls (-42% +/- 6%, P less than .001). These results show that insulin affects cAMP formation in muscle, and support a greater in vivo insulin response following unweighting atrophy. These various data support a role for lysosomal proteolysis in denervation, but not in unweighting, atrophy.

Animals↗

Differential expression of nicotinic acetylcholine receptor genes in innervated and denervated chicken muscle.

We have studied the mRNAs encoding all four subunits of the acetylcholine receptor in 13-day embryonic, innervated and denervated chicken pectoral muscle. In all three states the transcript sizes of the alpha, beta-, gamma- and delta-subunit mRNAs were approximately 3.2, 2.8, 1.8 and 1.9 kb respectively. Denervation was found to result in a large increase in the steady-state levels of each mRNA compared with those in innervated muscle. This increase was 8- to 9-fold for the beta and delta subunits and approximately 12-fold for the alpha subunit. The evidence obtained shows a coordinate regulation of the acetylcholine receptor genes in response to denervation. Interestingly, no gamma-subunit transcript was detected in innervated muscle, while significant levels were detected in embryonic and denervated tissue. This evidence suggests that there exists in a non-mammalian muscle acetylcholine receptor an additional subunit, analogous to the bovine epsilon subunit, and shows that the extra-junctional receptor of adult denervated muscle is produced by the same set of mRNAs which produce the major form of the receptor in embryonic muscle.

Animals↗

Calcium binding protein changes of sarcoplasmic reticulum from rat denervated skeletal muscle.

Two Ca2+ sequestering proteins were studied in fast-twitch (EDL) and slow-twitch (soleus) muscle sarcoplasmic reticulum (SR) as a function of denervation time. Ca2+-ATPase activity measured in SR fractions of normal soleus represented 5% of that measure in SR fractions of normal EDL. Denervation caused a severe decrease in activity only in fast-twitch muscle. Ca2+-ATPase and calsequestrin contents were affected differently by denervation. In EDL SR, Ca2+-ATPase content decreased progressively, whereas in soleus SR, no variation was observed. Calsequestrin showed a slight increase in both muscles as a function of denervation time correlated with increased 45Ca-binding. These results indicate first that Ca2+-ATPase activity in EDL was under neural control, and that because of low Ca2+-ATPase activity and content in slow-twitch muscle no variation could be detected, and secondly that greater calsequestrin content might represent a relative increasing of heavy vesicles or decreasing of light vesicles as a function of denervation time in the whole SR fraction isolated in both types of muscles.

Animals↗

Changes in protein levels of elongation factors, eEF1A-1 and eEF1A-2/S1, in long-term denervated rat muscle.

PURPOSE: This study was designed to determine whether the quantitative relationship between the levels of the eEF1A-1(developmental) and eEF1A-2/S1 (adult) isoforms of peptide elongation factor remains stable after denervation of skeletal muscle or whether in response to denervation the relative amount of the developmental form would increase. In normal postnatal rat muscle, eEF1A-2/S1 is the dominant form represented, and levels of eEF1A-1 are extremely low. METHODS: One hind limb in young adult rats was permanently denervated. Denervated and corresponding contralateral control muscles were removed for biochemical and morphological analysis from 2 days to 25 months after denervation. RESULTS: By one month after denervation, relative levels of eEF1A-1 rose dramatically in relation to those of eEF1A-2/S1, and they remained high throughout the remainder of the 25-month denervation period. Ultrastructural analysis showed a complex mix of muscle fiber atrophy, dying muscle nuclei and muscle fibers, and newly forming muscle fibers in the same tissue. CONCLUSIONS: As during muscle regeneration, levels of the developmental eEF1A-1 isoform of peptide elongation factor greatly increased relative to those of the adult eEF1A-2/S1 adult isoform following denervation in rat muscles. However, in contrast to regeneration, the elevated level of eEF1A-1 did not return to the basal minimal level. Since switching from eEF1A-1 to eEF1A-2/S1 is an indicator that terminal differentiated is completed, the failure of eEF1A-1 to return to basal level may be indicative of the persistence of an unstable tissue environment that includes muscle fiber atrophy, degeneration and neomyogenesis. The specific cellular basis for the increase in eEF1A-1 could not be determined from this study.

Animals↗

Evidence for active chloride accumulation in normal and denervated rat lumbrical muscle.

Intracellular Cl- activity (aiCl) was measured with Cl(-)-sensitive microelectrodes in normal and denervated rat lumbrical muscle. In normal muscle bathed in normal Krebs solution, aiCl lay close to that predicted by the Nernst equation. The addition of 9-anthracene carboxylic acid, which blocks Cl- conductance, caused aiCl to increase far above that predicted by a passive distribution. Furosemide (10 microM) reversibly blocked this accumulation. After muscle denervation, aiCl progressively increased for 1-2 wk. The rise occurred in two stages. The initial stage (1-3 d after denervation) reflected passive Cl- accumulation owing to membrane depolarization. At later times, aiCl continued to increase, with no further change in membrane potential, which suggests an active uptake mechanism. This rise approximately coincided with the natural reduction in membrane conductance to Cl- that occurs several days after denervation. Na+ replacement, K+ replacement, and furosemide each reversibly blocked the active Cl- accumulation in denervated muscle. Quantitative estimates suggested that there was little difference between Cl- flux rates in normal and denervated muscles. The results can be explained by assuming that, in normal muscle, an active accumulation mechanism operates, but that Cl- lies close to equilibrium owing to the high membrane conductance to Cl-. The rise in aiCl after denervation can be accounted for by the membrane depolarization, the reduction in membrane Cl- conductance, and the nearly unaltered action of an inwardly directed Cl- "pump."

Animals↗

[Synthetical treatment for facial paralysis using static and dynamic techniques].

OBJECTIVE: To investigate a synthetical technique combining static and dynamic states for treating facial paralysis. METHODS: From October 1993 to November 2005, 93 patients (26 males, 67 females; aged 9-69 years; illness course, 6 months to 24 years) with facial paralysis were admitted for treatment, of whom 48 were unknown in their pathological origins, 32 had a history of intracranial or ear operation, 13 had their facial paralysis related to trauma. We applied synthetical techniques for treating facial paralysis, including denerved muscle (extensor hallucis brevis and extensor digitorum brevis) free graft using an operating or chemical technique, temporal muscular flap suspending, treatment of eye closure by uplifting zygomo-malar tissues and temporal muscular flap suspending, brow lifting by the Buried Guiding Suture Appliance, Botox injection, and the nerve amputation of normal side for mandibular marginal ramus paralysis. RESULTS: After the treatment for 82 patients with the method of the denerved muscle free graft combining femoral muscular flap suspending, 3 patients with the method of uplifting zygomo-malar tissues combining temporal muscular flap suspending for eye closure, 15 patients with the method of the Buried Guiding Suture for eyebrow lifting, 4 patients with the Botox injection and nerve amputation of normal side for the mandibular marginal ramus paralysis, 93 patients had a good therapeutic result. The follow-up for 3 months to 10 years in all the patients revealed that 75 patients were satisfied with their treatment results and 13 patients were almost satisfied according to the criteria for the facial symmetrical checking and the House-Brackmann grading system. The technique had advantages of a complete survival of the related small muscles, a hidden incision, less trauma for the Botox injection, and the denerved muscles for surrounding nerve inserting. CONCLUSION: The synthetical technique combining static and dynamic states for treating facial paralysis is a good therapeutic method and the therapies for facial paralysis should vary with the different conditions of the paralysis. We advocate an individuation treatment for facial paralysis.

Adolescent↗

The relationship between myenteric neuronal denervation, smooth muscle thickening and epithelial cell proliferation in the rat colon.

The effects of myenteric neuronal denervation on smooth muscle thickening and epithelial cell proliferation were studied in the descending colon of rats treated by serosal application of 2 mM benzalkonium chloride (BAC) for 30 min. Control animals were treated with saline (0.9% NaCl). The animals were divided into six groups of 13 animals each and killed 10, 45 and 120 days after BAC treatment. A significant reduction in neuron number was observed in the myenteric plexus of animals treated with BAC, as well as smooth muscle thickening and an increase in crypt cell population, crypt cell production per crypt and a decrease in cell cycle time. These findings permit us to conclude that a relationship may exist between the increase of epithelial cell proliferation, smooth muscle thickening and myenteric neuron denervation in the descending colon caused by BAC, the latter probably playing an important role in the integration of the other two.

Animals↗

TTX-sensitive and TTX-insensitive sodium channel mRNA transcripts are independently regulated in adult skeletal muscle after denervation.

The expression of mRNA encoding the TTX-sensitive (SkM1) and TTX-insensitive (SkM2) voltage-dependent sodium channels in adult skeletal muscle is independently regulated. In normal skeletal muscle, only the SkM1 message is expressed and the level varies with muscle fiber type. After surgical denervation, the steady-state SkM1 mRNA level declines transiently, but returns to control levels within 5 days. Expression of SkM2 transcripts is markedly activated, reaching a peak 3 days after axotomy and then declining to a maintained level at approximately 30% of peak. Chemical denervation with botulinum toxin results in higher levels of SkM2 mRNA, which by 7 days posttreatment are 7-fold greater than levels in paired axotomized muscles. SkM2 expression subsequently declines as functional reinnervation appears. Quantal acetylcholine release appears to play a major role in suppression of SkM2 expression in adult innervated or reinnervated muscle, whereas nonquantal factors in toxin-treated, but not axotomized, muscle may sustain high level SkM2 mRNA expression.

Animals↗

[Differences in the thermostability of aminoacyl-mRNA-synthetases from intact and denervated rabbit muscles].

The stability of alanyl-tRNA synthetase and valyl-tRNA synthetase from the intact and denervated for 30 days rabbit skeletal muscles towards the inactivating effect of heat (42 degrees for 10 min) has been studied. The activity of the enzymes was measured in the supernatant fraction obtained by centrifugation of muscle homogenates at 105, 000 g for 1 h. The stability of both alanyl- and valyl-tRNA synthetases from the denervated m. soleus and m. gastrocnemius was shown to be considerably decreased as compared with the enzymes from the intact muscles.

Alanine-tRNA Ligase↗

Extrasynaptic accumulations of acetylcholinesterase in the rat sternocleidomastoid muscle after neonatal denervation. Light and electron microscopic localization and molecular forms.

Denervated neonatal rat sternocleidomastoid muscle has decreased levels of total AChE when compared to control muscle. Denervated versus control values of total muscle AChE present a three-phase curve in function of time after denervation. There is a rapid initial fall 0-3 days after denervation, an increase during about 2 weeks, then again a decrease in total AChE. Thus, there is a transitory net accumulation of AChE after the initial fall of activity in denervated developing muscle. Extrasynaptic areas of high AChE activity develop between 1 and 2 weeks after denervation and remain visible up to 1 month after denervation before vanishing. An electron microscope study shows that these accumulations are internal to the muscle fiber, close to a limited number of muscle nuclei and associated to the sarcoplasmic reticulum and nuclear envelope, but not to the T-tubule system. As found in adult rat muscle, the initial fall in AChE affects first the 16 S AChE form, and soon after, the 4 S and 10 S AChE forms. A main difference with adult muscle is the sudden increase and predominance over other forms of 10 S AChE 2 weeks after denervation at birth. Later, the decrease in AChE affects 16 S and 4 S AChE before 10 S AChE. The regions rich in extrasynaptic sites of AChE accumulation possess a very high proportion of 10 S AChE. Thus, the mechanisms of biosynthesis, intracellular transport and/or secretion of AChE may be very different in young, developing muscle compared to adult muscle.

Acetylcholinesterase↗

Fiber splitting in tonic and phasic skeletal muscles following denervation, chloroquine and triton WR-1339 treatments.

Fiber splitting was studied in the tonic, anterior (ALD) and phasic, posterior (PLD) latissimus dorsi muscles using three experimental models, denervation, chloroquine administration and Triton WR-1339 administration. Fiber splitting was observed in the PLD in all three models, but in the ALD only after chloroquine administration. A basement membrane was always present in the split fibers, and acid phosphatase was localized between the split fibers in the PLD. However, no evidence of lysosomal involvement in the fiber splitting process was observed.

Acid Phosphatase↗

Dihydropyridine receptor and ryanodine receptor gene expression in long-term denervated rat muscles.

Following disruption of the nerve supply, extensor digitorum longus (EDL) and soleus (SOL) muscles in rats are known to exhibit alterations in excitation-contraction coupling. After total RNA isolation from the denervated and the contralateral control muscles performed at 25 and 50 days following denervation, RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac isoforms of both the DHPR alpha 1-subunit and the RyR. Longterm denervation increased the expression of the mRNA for skeletal DHPR and skeletal RyR in SOL muscle, but it also significantly increased the expression of the mRNA for the cardiac isoform of the DHPR alpha 1 subunit in EDL muscle.

Animals↗

Reinnervation of developing rat muscle by non-axotomized motoneurons.

To study the ability of developing motoneurons to reinnervate their denervated muscle, axotomized motoneurons in rat neonates and pups were retrogradely labeled with two fluorescent tracers. Fluorogold (FG), a long-lasting fluorescent dye, was injected into intercostal muscle T8 to retrogradely label the motoneurons that innervated it. Two days later intercostal nerves T7-T9 were cut. The intercostal muscle denervated at birth was reinnervated within 10-20 days, as evidenced by nerve-evoked muscle contraction. Three weeks following axotomy, tetramethylrhodamine isothiocyanate (TRITC) was injected into the same muscle to label the motoneurons that reinnervated it. The motoneurons double-labeled with FG and TRITC were, therefore, axotomized motoneurons that regenerated to reinnervate T8. In neonates, axotomy resulted in a significant reduction in the number of FG-labeled motoneurons, which suggests that axon transection at early postnatal days causes a massive motoneuron death. The percentage of double-labeled motoneurons was significantly smaller than that in non-axotomized rats. TRITC-labeled motoneurons constituted the majority of stained motoneurons; these were located in different nuclei than the intercostal motoneurons. These findings suggest that muscle reinnervation is, at least in part, by motoneurons which originally did not innervate intercostal muscle T8. Unlike axotomy at birth, axotomy performed 2-3 weeks after birth did not result in a significant motoneuron loss. The number of stained motoneurons labeled with both FG and TRITC was significantly smaller, however, than in non-axotomized spinal cords. Our data indicate that in pups only a small percentage of axotomized motoneurons reinnervated the denervated muscle.

Animals↗