Satellite cells on isolated myofibers from normal and denervated adult rat muscle.

Satellite cells (SCs) in normal adult muscle are quiescent. They can enter the mitotic program when stimulated with growth factors such as basic FGF. Short-term denervation stimulates SC to enter the mitotic cycle in vivo, whereas long-term denervation depletes the SC pool. The molecular basis for the neural influence on SCs has not been established. We studied the phenotype and the proliferative capacity of SCs from muscle that had been denervated before being cultured in vitro. The expression of PCNA, myogenin, and muscle (M)-cadherin in SCs of normal and denervated muscle fibers was examined at the single-cell level by immunolabeling in a culture system of isolated rat muscle fibers with attached SCs. Immediately after plating (Day 0), neither PCNA nor myogenin was present on normal muscle fibers, but we detected an average of 0.5 M-cadherin(+) SCs per muscle fiber. The number of these M-cadherin(+) cells (which are negative for PCNA and myogenin) increased over the time course examined. A larger fraction of cells negative for M-cadherin underwent mitosis and expressed PCNA, followed by myogenin. The kinetics of SCs from muscle fibers denervated for 4 days before culturing were similar to those of normal controls. Denervation from 1 to 32 weeks before plating, however, suppressed PCNA and myogenin expression almost completely. The fraction of M-cadherin(+) (PCNA(-)/myogenin(-)) SCs was decreased after 1 week of denervation, increased above normal after denervation for 4 or 8 weeks, and decreased again after denervation for 16 or 32 weeks. We suggest that the M-cadherin(+) cells are nondividing SCs because they co-express neither PCNA or myogenin, whereas the cells positive for PCNA or myogenin (and negative for M-cadherin) have entered the mitotic cycle. SCs from denervated muscle were different from normal controls when denervated for 1 week or longer. The effect of denervation on the phenotypic modulation of SCs includes resistance to recruitment into the mitotic cycle under the conditions studied here and a robust extension of the nonproliferative compartment. These characteristics of SCs deprived of neural influence may account for the failure of denervated muscle to fully regenerate. (J Histochem Cytochem 47:1375-1383, 1999)     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.     

Muscle mechanical changes following denervation and their modification by use of an inhibitor of RNA synthesis.

Mechanical properties of soleus muscles from adult Wistar rats were studied “in vitro”. Contraction time, time for half relaxation, duration of the active state, fusion frequency and tetanus-twitch ratio were measured. In a first group of experiments normal innervated muscles were compared with muscles denervated for ten days. Significative differences in their contractile properties were found. In a second group of rats Actinomycin D was injected intravenously eight days after denervation and soleus contractile parameters compared 48 hours later with those obtained in the same muscle of denervated untreated rats. Treated muscles did not show significative differences with the untreated ones. However, when Actinomycin D was injected at the time of denervation differences in the duration of the active state were detected. It is suggested in the present paper that mechanical changes following denervation may be considered an induced phenomenon, as was demonstrated in other denervatory changes.     

Aging of skeletal muscle does not affect the response of satellite cells to denervation

Satellite cells (SCs) are the main source of new fibers in regenerating skeletal muscles and the key contributor to extra nuclei in growing fibers during postnatal development. Aging results in depletion of the SC population and in the reduction of its proliferative activity. Although it has been previously determined that under conditions of massive fiber death in vivo the regenerative potential of SCs is not impaired in old muscle, no studies have yet tested whether advanced age is a factor that may restrain the response of SCs to muscle denervation. The present study is designed to answer this question, comparing the changes of SC numbers in tibialis anterior (TA) muscles from young (4 months) and old (24 months) WI/HicksCar rats after 2 months of denervation. Immunostaining with antibodies against M-cadherin and NCAM was used to detect and count the SCs. The results demonstrate that the percentages of both M-cadherin- and NCAM-positive SCs (SC/Fibers x 100) in control TA muscles from young rats (5.6 +/- 0.5% and 1.4 +/- 0.2%, respectively) are larger than those in old rats (2.3 +/- 0.3% and 0.5 +/- 0.1%, respectively). At the same time, in 2-month denervated TA muscles the percentages of M-cadherin and NCAM positive SC are increased and reach a level that is comparable between young (16.2 +/- 0.9% and 7.5 +/- 0.5%, respectively) and old (15.9 +/- 0.7% and 10.1 +/- 0.5%, respectively) rats. Based on these data, we suggest that aging does not repress the capacity of SC to become activated and grow in the response to muscle denervation.     

Nerve activity-independent regulation of skeletal muscle atrophy: role of MyoD and myogenin in satellite cells and myonuclei

Electrical activity is thought to be the primary neural stimulus regulating muscle mass, expression of myogenic regulatory factor genes, and cellular activity within skeletal muscle. However, the relative contribution of neural influences that are activity-dependent and -independent in modulating these characteristics is unclear. Comparisons of denervation (no neural influence) and spinal cord isolation (SI, neural influence with minimal activity) after 3, 14, and 28 days of treatment were used to demonstrate whether there are neural influences on muscle that are activity independent. Furthermore, the effects of these manipulations were compared for a fast ankle extensor (medial gastrocnemius) and a fast ankle flexor (tibialis anterior). The mass of both muscles plateaued at approximately 60% of control 2 wk after SI, whereas both muscles progressively atrophied to <25% of initial mass at this same time point after denervation. A rapid increase in myogenin and, to a lesser extent, MyoD mRNAs and proteins was observed in denervated and SI muscles: at the later time points, these myogenic regulatory factors remained elevated in denervated, but not in SI, muscles. This widespread neural activity-independent influence on MyoD and myogenin expression was observed in myonuclei and satellite cells and was not specific for fast or slow fiber phenotypes. Mitotic activity of satellite and connective tissue cells also was consistently lower in SI than in denervated muscles. These results demonstrate a neural effect independent of electrical activity that 1) helps preserve muscle mass, 2) regulates muscle-specific genes, and 3) potentially spares the satellite cell pool in inactive muscles.     

In vivo expression patterns of MyoD, p21, and Rb proteins in myonuclei and satellite cells of denervated rat skeletal muscle

MyoD, a myogenic regulatory factor, is rapidly expressed in adult skeletal muscles in response to denervation. However, the function(s) of MyoD expressed in denervated muscle has not been adequately elucidated. In vitro, it directly transactivates cyclin-dependent kinase inhibitor p21 (p21) and retinoblastoma protein (Rb), a downstream target of p21. These factors then act to regulate cell cycle withdrawal and antiapoptotic cell death. Using immunohistochemical approaches, we characterized cell types expressing MyoD, p21, and Rb and the relationship among these factors in the myonucleus of denervated muscles. In addition, we quantitatively examined the time course changes and expression patterns among distinct myofiber types of MyoD, p21, and Rb during denervation. Denervation induced MyoD expression in myonuclei and satellite cell nuclei, whereas p21 and Rb were found only in myonuclei. Furthermore, coexpression of MyoD, p21, and Rb was induced in the myonucleus, and quantitative analysis of these factors determined that there was no difference among the three myofiber types. These observations suggest that MyoD may function in myonuclei in response to denervation to protect against denervation-induced apoptosis via perhaps the activation of p21 and Rb, and function of MyoD expressed in satellite cell nuclei may be negatively regulated. The present study provides a molecular basis to further understand the function of MyoD expressed in the myonuclei and satellite cell nuclei of denervated skeletal muscle. denervation; protein expression; apoptotic cell death; immunohistochemistry     

1,2-Diacylglycerol and ceramide levels in insulin-resistant tissues of the rat in vivo

Phorbol esters have been reported to decrease sensitivity or responsiveness to insulin in cells in vitro. Since phorbol esters are analogues of endogenously produced 1,2-diacylglycerol, the present study investigated whether 1,2-diacylglycerol concentration is elevated in insulin-resistant tissues of the rat in vivo. Studies were done on 11-12-week-old genetically obese Zucker rats, which are insulin-resistant. Lean Zucker rats served as controls. Levels of 1,2-diacylglycerol in obese rats were increased 82% in liver, 136% in calf muscles, 72% in soleus muscle, a slow-twitch muscle, and 40% in plantaris muscle, a fast-twitch muscle. Ceramide levels in the same tissues were increased 26, 52, 69, and 13%, respectively. Studies were also done on normal, non-obese Sprague-Dawley rats 3 h, 1, 3, 8, and 15 days after interrupting the nerve supply to hindlimb muscles. We have previously shown that 3-17 days after denervation, soleus muscles are completely unresponsive to insulin and do not increase glucose uptake in response to insulin stimulation in vivo, whereas plantaris muscles show a normal glucose uptake when stimulated by insulin; however, the insulin-induced increment in glucose uptake is reduced 68% because it is superimposed on already elevated basal glucose uptake (Turinsky, J. (1987) Am. J. Physiol. 252, R531-R537). In the present study, the denervated soleus muscles exhibited a sustained increase of 23-56% in 1,2-diacylglycerol concentration between 3 h and 15 days after interruption of nerve supply. The denervated soleus muscles also showed 34 and 42% increases in ceramide concentration at 3 and 8 days after denervation, respectively. In contrast, no increases in 1,2-diacylglycerol concentration were observed in plantaris muscles at shorter intervals than 15 days after denervation. Ceramide concentrations in plantaris muscles were increased 43 and 75% at 8 and 15 days after denervation, respectively. These observations demonstrate that tissue insulin resistance is frequently associated with a long term increase in tissue 1,2-diacylglycerol concentration. This suggests the possibility that augmented 1,2-diacylglycerol levels contribute to the development of some types of tissue insulin resistance.     

The effects of denervation on protein turnover of the soleus and extensor digitorum longus muscles of adult mice.

1. Changes in protein turnover of the soleus and EDL muscles of adult mice have been studied 1, 7 and 80 days after denervation. 2. Increased rates of protein degradation 7 and 80 days post-denervation correlated with the atrophy and loss of protein from these muscles. 3. Rates of protein synthesis in the EDL decreased 24 hr after nerve section. However, these synthetic rates increased again to become higher in the 7 day denervated muscles compared with their controls. These latter anabolic changes are inconsistent with the concept of a denervated muscle being inactive. 4. These findings have been compared with a similar study on muscles of growing rats. Any passive stretching of the denervated muscles by continued bone growth appears unlikely to be a crucial factor explaining the increased rates of protein synthesis 7 days after denervation.     

Unlike effects of denervation on the rate of entry of inorganic phosphate into rat slow and fast muscles.

The increased inorganic phosphate flow, characteristic of denervated gastrocnemius muscle is shown to be present in additional denervated fast muscles, i.e. the plantaris, tibialis anterior and extensor digitorum longus muscles. The response of the soleus, a slow muscle, to denervation is biphasic. After an initial decrease of the phosphate flow at the end of the first postoperative day, there is a secondary rise which has the same general characteristics as the rise observed in fast muscles i.e. an exponential or hyperbolic increase to an asymptotic value reached after thirty days. The denervated fast and slow muscles are not converging to an intermediate metabolic pattern. The changes in phosphate flow induced by denervation are reversible in the soleus as well as in the gastrocnemius muscles.     

Changes in isometric contractile properties of extensor digitorum longus and soleus muscles of C57BL/6J mice following denervation

In this study, conducted on mice of the C57BL/6J+/+ strain, we investigated the differential effects of denervation on the isometric contractile properties of the extensor digitorum longus (EDL) and soleus (SOL) muscles. The contractile properties were studied at 1, 28, 84, and 210 days following unilateral section of the sciatic nerve at 12 weeks of age. When isometric tetanus tension was expressed relative to wet weight, the denervated SOL showed an earlier and more pronounced loss in tension generating capacity than the EDL. Both the denervated SOL and EDL showed potentiation of the twitch tension at 28 days postdenervation. The time to peak twitch tension (TTP) and the time to half-relaxation (1/2RT) were prolonged by 28 days postdenervation in both muscles. This trend continued to the oldest age-groups studied in the EDL, but reached an apparent plateau in the SOL at 84 days postdenervation. In response to fatigue, the denervated SOL showed a marked decrease in resistance to fatigue at 1 day but a relatively normal response thereafter, whereas the denervated EDL showed an increase in resistance to fatigue at and beyond the 28-day period. In spite of the fact that the total contraction time of both muscles increased following denervation, the predominantly oxidative SOL remained a slower contracting muscle than the more glycolytic EDL.     

Expression of ARPP-16/19 in rat denervated skeletal muscle

It is known that denervation of rat skeletal muscle causes atrophy and this is often adopted as a model for human muscle atrophy. To understand the molecular changes that occur, it is important to identify the profiles of differential gene expression. In the present study, we investigated differentially expressed genes in denervated muscle using DNA microarrays with printed genes preferentially expressed in skeletal muscle. We found that several genes are differentially expressed. Of these genes, ARPP-16/19 (cAMP-regulated phosphoprotein 16/19) is selectively enhanced after denervation. The expression of ARPP-16/19 in denervated muscles starts to increase from two days after denervation surgery. On the other hand, the expression of ARPP-16/19 does not change in hind-limb suspended muscles, such as EDL and soleus muscles. These results suggest that the increase in ARPP-16/19 mRNA expression is regulated by unknown factor(s) secreted from nerves, and not by electrical muscle activity.     

Innervation is necessary for the development of fast contraction kinetics of singing muscles in a katydid

The twitch duration of mesothoracic wing muscles of the male katydid Neoconocephalus robustus (Insecta; Orthoptera; Tettigoniidae) decreases rapidly within the first 5 days of adulthood, to about half of its value in newly molted adults. To determine if this change is dependent upon neural input, male mesothoracic first tergocoxal muscles were unilaterally denervated on the second day of adulthood. The contraction kinetics of the denervated and contralateral innervated muscles were tested four days later. The development of rapid contraction kinetics was slowed or stopped in the denervated muscles, while the contralateral innervated muscles did become faster. Mesothoracic wing muscles of females do not develop faster contraction kinetics. When the female mesothoracic first tergocoxal muscle is denervated, there is no difference in twitch duration after 4 days between the innervated and contralateral denervated muscles. Therefore, denervation in newly molted adult male katydids interrupts a developmental program for the acquisition of adult contraction kinetics.     

Denervation provokes greater reductions in insulin-stimulated glucose transport in muscle than severe diabetes

We have examined the independent and combined effects of insulin insufficiency (streptozotocin (STZ)-induced diabetes, 85 mg/kg i.p.) and reduced muscle activity (denervation) (7 days) on basal, insulin-stimulated and contraction-stimulated glucose transport in rat muscles (soleus, red and white gastrocnemius). There were four treatments: control, denervated, diabetic, and denervated + diabetic muscles. Contraction-stimulated glucose transport was lowered (~ 50%) (p < 0.05) to the same extent in all experimental groups. In contrast, there was a much smaller reduction insulin-stimulated glucose transport in muscles from diabetic animals (18-24% reduction, p < 0.05) than in denervated muscles (40-60% reduction, p < 0.05) and in denervated + diabetic muscles (40-60% reduction, p < 0.05). GLUT-4 mRNA reduction was greatest in denervated + diabetic muscles (~ -75%, p < 0.05). GLUT-4 protein was decreased (p < 0.05) to a similar extent in all three experimental conditions (~ -30-40%). In conclusion, (1) muscle inactivity (denervation) and STZ-induced diabetes had similar effects on reducing contraction-stimulated glucose transport, but (2) muscle inactivity (denervation), rather than severe diabetes, produced a 2-fold greater impairment in skeletal muscle insulin-stimulated glucose transport.     

Effects of actinomycin D on fibrillation activity in denervated skeletal muscles of the rat

The effects of actinomycin D on fibrillation activity, acetylcholine sensitivity and resting membrane potential of denervated muscles of the rat was studied. Actinomycin D (0.7 mg/kg I.V.) administered 1 day after denervation delays the appearance of fibrillation for approximately 3 days. If this drug is given 5–7 days after denervation, it is also capable of blocking the already established fibrillation but fails to suppress extrajunctional cholinergic receptors and to reverse the fall in resting potential. The mechanical responses of denervated muscles are unaffected by actinomycin D. These results suggest that in fibrillation a genetic induction of newly formed RNA and protein is involved. It is also suggested that these molecules probably have a more rapid turnover than those required for the formation of extrasynaptic receptors in denervated muscle.     

AMP-activated protein kinase activation prevents denervation-induced decline in gastrocnemius GLUT-4.

This study was designed to determine whether the reductions in GLUT-4 seen in 3-day-denervated muscles can be prevented through chemical activation of 5'-AMP-activated protein kinase (AMPK). Muscle AMPK can be chemically activated in rats using subcutaneous injections with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). In this study, the tibial nerve was sectioned on one side; the other was sham operated but without nerve section. Acute injections of AICAR resulted in significantly increased AMPK activity in denervated gastrocnemius but not soleus muscles. Acetyl-CoA carboxylase activity, a reporter of AMPK activation, declined in both gastrocnemius and soleus in both denervated and contralateral muscles. Three days after denervation, GLUT-4 levels were significantly decreased by approximately 40% in gastrocnemius muscles and by approximately 30% in soleus muscles. When rats were injected with AICAR (1 mg/g body wt) for 3 days, the decline in GLUT-4 levels was prevented in denervated gastrocnemius muscles but not in denervated soleus muscles. The extent of denervation-induced muscle atrophy was similar in AICAR-treated vs. saline-treated rats. These studies provide evidence that some effects of denervation may be prevented by chemical activation of the appropriate signaling pathways.