The action of the beta-agonist clenbuterol on protein metabolism in innervated and denervated phasic muscles.

1. Clenbuterol treatment in innervated and denervated phasic extensor digitorum longus, plantaris and gastrocnemius muscles from rats caused a significant increase in RNA and protein contents in all muscles except denervated extensor digitorum longus. 2. All muscles showed an increase in the fractional rate of protein synthesis (Ks) with clenbuterol, but the temporal response varied. 3. The data suggest that the effect of clenbuterol on protein metabolism in innervated muscles is muscle-type specific, and demonstrate the homology of response for denervated muscles.     

Inhibition and reversal of denervation-induced atrophy by theβ-agonist growth promoter,clenbuterol

Dietary administration of the growth promoter, clenbuterol, ameliorated denervation-induced atrophy in rat soleus muscles. In acutely denervated muscles the drug inhibited the appearance of atrophy, and in chronically denervated muscles the atrophy was almost fully reversed. Responses in slow twitch oxidative fibres were particularly marked.     

Histological evidences of reparative and regenerative effects of beta-adrenoceptor agonists, clenbuterol and isoproterenol, in denervated rat skeletal muscle

The aim of this study was to determine the contribution of beta-adrenoceptor activation in the reconstruction of the structural and functional organization of denervated skeletal muscle. beta-agonists, clenbuterol (1.2 mg/kg body weight) and isoproterenol (2 mg/kg body weight), administration (daily oral administration; maximum 7 days) to normal innervated rats as well as denervated animals caused muscle hypertrophy. An increase in mean fiber diameter confirmed this stimulated growth both in normal innervated and denervated rat gastrocnemius muscle. Examination of muscle nuclei from treated but normal innervated rat gastrocnemius exhibited features like large size, active nucleoplasm and an increase in their number per fiber cross section and per mm mean fiber length indicating towards an elevated biosynthetic activity in tissue in the presence of beta adrenoceptor agonists. Administration of drugs to normal innervated animals resulted in an emergence of central muscle nuclei. The hyperactive and enlarged muscle nuclei ultimately organized themselves into unusually elongated nuclear streaks. beta agonist treatment to denervated rats resulted in amelioration of atrophic state of tissue characterized by hypertrophy of muscle fibers thus lending to a restoration of structural organization of tissue. Bizarre shapes of nuclei in denervated muscle tend to recover to that characteristic to normal innervated muscle in presence of clenbuterol and isoproterenol hydrochloride. All observations were confirmed by administering butoxamine, a beta-adrenoceptor antagonist along with beta-agonists. The results suggests that both clenbuterol and isoproterenol hydrochloride are capable of mimicking normal innervation functions in skeletal muscle and thus play important role in the structural and functional reorganization of tissue. Amelioration of denervation atrophy in rat gastrocnemius in the presence of beta-agonists supports this.     

Analysis of the interaction between properdin and factor B, components of the alternative-pathway C3 convertase of complement.

Denervated (1-10 days) rat epitrochlearis muscles were isolated, and basal and insulin-stimulated protein and glucose metabolism were studied. Although basal rates of glycolysis and glucose transport were increased in 1-10-day-denervated muscles, basal glycogen-synthesis rates were unaltered and glycogen concentrations were decreased. Basal rates of protein degradation and synthesis were increased in 1-10-day-denervated muscles. The increase in degradation was greater than that in synthesis, resulting in muscle atrophy. Increased rates of proteolysis and glycolysis were accompanied by elevated release rates of leucine, alanine, glutamate, pyruvate and lactate from 3-10-day-denervated muscles. ATP and phosphocreatine were decreased in 3-10-day-denervated muscles. Insulin resistance of glycogen synthesis occurred in 1-10-day denervated muscles. Insulin-stimulated glycolysis and glucose transport were inhibited by day 3 of denervation, and recovered by day 10. Inhibition of insulin-stimulated protein synthesis was observed only in 3-day-denervated muscles, whereas regulation by insulin of net proteolysis was unaffected in 1-10-day-denervated muscles. Thus the results demonstrate enhanced glycolysis, proteolysis and protein synthesis, and decreased energy stores, in denervated muscle. They further suggest a defect in insulin's action on protein synthesis in denervated muscles as well as on glucose metabolism. However, the lack of concurrent changes in all insulin-sensitive pathways and the absence of insulin-resistance for proteolysis suggest multiple and specific cellular defects in insulin's action in denervated muscle.     

The adaptive response of MyoD family proteins in overloaded, regenerating and denervated rat muscles.

Using Western blot analysis, we investigated whether the amount of myogenic regulatory factors differs in slow-type and fast-type muscles. In addition, we examined the adaptive response of myogenic regulatory factor protein in the overloaded rat muscles by the ablation of synergists, in the regenerating muscles following bupivacaine injection and in the denervated muscle. The amount of myogenin protein in the slow-type muscle was markedly greater. In contrast, the proteins MyoD and Myf-5 were selectively accumulated in the fast-type muscles. A gradual down-regulation of MyoD and Myf-5 proteins was detected in the denervated fast-type muscles, but not in the myogenin protein content. A rapid down-regulation of myogenic regulatory factor protein was observed both of the mechanically overloaded and in the regenerating muscles. These results indicate that the fast-type-specific gene expression in muscle is modulated by MyoD and Myf-5 proteins and suggest that myogenin protein plays an important role in the reconstruction of damaged neuromuscular connections.     

Role of different proteolytic systems in the degradation of muscle proteins during denervation atrophy

In order to clarify the cellular mechanisms of denervation atrophy of skeletal muscle, we have studied protein turnover in denervated and control rat soleus muscles in vitro under different conditions. By 24 h after cutting the sciatic nerve, overall protein breakdown was greater in the denervated soleus than in the contralateral control muscle, and by 3 days, net proteolysis had increased about 3-fold. Since protein synthesis increased slightly following denervation, the rise in proteolysis must be responsible for the muscle atrophy and the differential loss of contractile proteins. Like overall proteolysis, the breakdown of actin (as shown by 3-methyl-histidine production by the muscles) increased each day after denervation and by 3 days was 2.5 times faster than in controls. Treatments that block the lysosomal and Ca2(+)-dependent proteolytic systems did not reduce the increase in overall protein degradation and actin breakdown in the denervated muscles (maintained in complete medium at resting length). However, the content of the lysosomal protease, cathepsin B, increased about 2-fold by 3 days after denervation. Furthermore, conditions that activate intralysosomal proteolysis (incubation without insulin or amino acids) stimulated proteolysis 2-3-fold more in the denervated muscles than in controls. Also, incubation conditions that activate the Ca2(+)-dependent pathway (incubation with Ca2+ ionophores or allowing muscles to shorten) were 2-3 times more effective in enhancing overall proteolysis in the denervated muscle. None of these treatments affected 3-methylhistidine production. Thus, multiple proteolytic systems increase in parallel in the denervated muscle, but a nonlysosomal process (independent of Ca2+) appears mainly responsible for the rapid loss of cell proteins, especially of myofibrillar components.     

Normal distribution of presenilin-1 and nicastrin in skeletal muscle and the differential responses of these proteins after denervation

Presenilin-1 and nicastrin, two components of gamma-secretase associated with Alzheimer's disease plaques, are present in the synapses of the brain and in various peripheral organs, including skeletal muscle. In the present study, we examined the expression pattern of presenilin-1 and nicastrin in normal and denervated hindlimb muscles of the rat. Using immunohistochemical approaches, we found that presenilin-1 and AChRalpha was co-localized at the neuromuscular junction in the normal skeletal muscles of rats. The immunoreactivities of both presenilin-1 and nicastrin were also observed at the sarcolemma of muscle fibers. We discovered that presenilin-1 mRNA and its protein are upregulated after denervation of the soleus and tibialis anterior muscles. Furthermore, clear co-localization between presenilin-1 and DAPI, but not nicastrin, was noted in several myonuclei in the denervated muscles. We recognized a few fibers possessing both ubiquitin and presenilin-1 protein in the cytosol. The amount of presenilin-1 in the nucleus and membrane fraction was more abundantly expressed in the denervated muscle fibers. In contrast, no significant difference in the nicastrin protein level was observed between normal and denervated muscle fibers. These data suggest that enhanced presenilin-1 protein may play a role in the degeneration and regeneration of skeletal muscle.     

IGF and myostatin pathways are respectively induced during the earlier and the later stages of skeletal muscle hypertrophy induced by clenbuterol,a β2‐adrenergic agonist

Clenbuterol, a β₂‐adrenergic agonist, increases the hypertrophy of skeletal muscle. Insulin‐like growth factor (IGF) is reported to work as a potent positive regulator in the clenbuterol‐induced hypertrophy of skeletal muscles. However, the precise regulatory mechanism for the hypertrophy of skeletal muscle induced by clenbuterol is unknown. Myostatin, a member of the TGFβ super family, is a negative regulator of muscle growth. The aim of the present study is to elucidate the function of myostatin and IGF in the hypertrophy of rat masseter muscle induced by clenbuterol. To investigate the function of myostatin and IGF in regulatory mechanism for the clenbuterol‐induced hypertrophy of skeletal muscles, we analysed the expression of myostatin and phosphorylation levels of myostatin and IGF signaling components in the masseter muscle of rat to which clenbuterol was orally administered for 21 days. Hypertrophy of the rat masseter muscle was induced between 3 and 14 days of oral administration of clenbuterol and was terminated at 21 days. The expression of myostatin and the phosphorylation of smad2/3 were elevated at 21 days. The phosphorylation of IGF receptor 1 (IGFR1) and akt1 was elevated at 3 and 7 days. These results suggest that myostatin functions as a negative regulator in the later stages in the hypertrophy of rat masseter muscle induced by clenbuterol, whereas IGF works as a positive regulator in the earlier stages. Copyright © 2012 John Wiley & Sons, Ltd.     

Beta 2-agonist fenoterol has greater effects on contractile function of rat skeletal muscles than clenbuterol

Potential treatments for skeletal muscle wasting and weakness ideally possess both anabolic and ergogenic properties. Although the beta(2)-adrenoceptor agonist clenbuterol has well-characterized effects on skeletal muscle, less is known about the therapeutic potential of the related beta(2)-adrenoceptor agonist fenoterol. We administered an equimolar dose of either clenbuterol or fenoterol to rats for 4 wk to compare their effects on skeletal muscle and tested the hypothesis that fenoterol would produce more powerful anabolic and ergogenic effects. Clenbuterol treatment increased fiber cross-sectional area (CSA) by 6% and maximal isometric force (P(o)) by 20% in extensor digitorum longus (EDL) muscles, whereas fiber CSA in soleus muscles decreased by 3% and P(o) was unchanged, compared with untreated controls. In the EDL muscles, fenoterol treatment increased fiber CSA by 20% and increased P(o) by 12% above values achieved after clenbuterol treatment. Soleus muscles of fenoterol-treated rats exhibited a 13% increase in fiber CSA and a 17% increase in P(o) above that of clenbuterol-treated rats. These data indicate that fenoterol has greater effects on the functional properties of rat skeletal muscles than clenbuterol.     

Protease Inhibitors Reduce Effects of Denervation on Muscle End-Plate Acetylcholinesterase

The effects of certain protease inhibitors on end-plate acetylcholinesterase (AChE) activity, as well as on wet weight and total protein, were studied in vivo in intact and denervated anterior gracilis muscles from the rat. A combination of leupeptin, pepstatin, and aprotinin, administered intraarterially, partly prevented the early (24 h) denervation-induced decrease in muscle weight and protein content. In turn, leupeptin and aprotinin, either alone or in combination, markedly reduced the decay of AChE activity in the denervated muscles, whereas pepstatin alone was ineffective. Such effects were additive in that the inhibitors in combination were more effective than when they were used separately. Additional experiments indicated that none of the inhibitors, at the concentrations used, affected AChE activity directly, nor did they have a significant effect during processing of the muscle samples. These findings indicate that the initial decay of AChE activity with denervation was effectively reduced by the inhibitors, probably through inactivation of proteolytic enzymes which, otherwise, would be increase in denervated muscle.     

Atrophy, but not necrosis, in rabbit skeletal muscle denervated for periods up to one year

Our understanding of the effects of long-term denervation on skeletal muscle is heavily influenced by an extensive literature based on the rat. We have studied physiological and morphological changes in an alternative model, the rabbit. In adult rabbits, tibialis anterior muscles were denervated unilaterally by selective section of motor branches of the common peroneal nerve and examined after 10, 36, or 51 wk. Denervation reduced muscle mass and cross-sectional area by 50–60% and tetanic force by 75%, with no apparent reduction in specific force (force per cross-sectional area of muscle fibers). The loss of mass was associated with atrophy of fast fibers and an increase in fibrous and adipose connective tissue; the diameter of slow fibers was preserved. Within fibers, electron microscopy revealed signs of ultrastructural disorganization of sarcomeres and tubular systems. This, rather than the observed transformation of fiber type from IIx to IIa, was probably responsible for the slow contractile speed of the muscles. The muscle groups denervated for 10, 36, or 51 wk showed no significant differences. At no stage was there any evidence of necrosis or regeneration, and the total number of fibers remained constant. These changes are in marked contrast to the necrotic degeneration and progressive decline in mass and force that have previously been found in long-term denervated rat muscles. The rabbit may be a better choice for a model of the effects of denervation in humans, at least up to 1 yr after lesion. force; shortening velocity; electron microscopy; histochemistry     

Thyroid hormone induces a nerve-independent precocious expression of fast myosin heavy chain mRNA in rat hindlimb skeletal muscle

The appearance of the mRNA for the adult fast IIB myosin heavy chain (MHC) was examined during postnatal development of rats using an S1 nuclease assay. In normal rats, a large increase in the adult MHC mRNA began at 6-7 days after birth, whereas daily injections of newborn rats with 3 micrograms of triiodothyronine (T3) resulted in a precocious increase of the mRNA as early as 3 days after birth. Injection of a range of doses of T3 demonstrated that a large effect was obtained between 30 and 300 ng of T3/day/rat. Fast myosin protein was also precociously induced over the same range of T3 doses. This effect was also seen in denervated muscles, and muscles responded similarly to the different doses of T3 whether they were denervated or not. These results suggest that either thyroid hormone or some circulating factors induced by thyroid hormone are limiting factors in controlling the neonatal-to-adult fast MHC transition and that these factors may act directly on muscle tissue.     

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.     

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.     

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.     

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.