The β2-adrenoceptor agonist formoterol improves structural and functional regenerative capacity of skeletal muscles from aged rat at the early stages of postinjury

Skeletal muscles from old rats fail to completely regenerate following injury. This study investigated whether pharmacological stimulation of β2-adrenoceptors in aged muscles following injury could improve their regenerative capacity, focusing on myofiber size recovery. Young and aged rats were treated with a subcutaneous injection of β2-adrenergic agonist formoterol (2 μg/kg/d) up to 10 and 21 days after soleus muscle injury. Formoterol-treated muscles from old rats evaluated at 10 and 21 days postinjury showed reduced inflammation and connective tissue but a similar number of regenerating myofibers of greater caliber when compared with their injured controls. Formoterol minimized the decrease in tetanic force and increased protein synthesis and mammalian target of rapamycin phosphorylation in old muscles at 10 days postinjury. Our results suggest that formoterol improves structural and functional regenerative capacity of regenerating skeletal muscles from aged rats by increasing protein synthesis via mammalian target of rapamycin activation. Furthermore, formoterol may have therapeutic benefits in recovery following muscle damage in senescent individuals.     

Selective downregulation of ubiquitin conjugation cascade mRNA occurs in the senescent rat soleus muscle

Aging-related alterations of the ubiquitin proteasome pathway (UPP) have been reported in locomotor skeletal muscle. Specifically, declines in proteasome activity have been observed in the soleus of senescent animals compared to the soleus of young controls. However, the influence of aging on the mRNA levels of key components within the ubiquitin conjugation cascade (UCC) remains unknown. We hypothesized that aged soleus muscle would exhibit downregulated expression of select UCC mRNA and decreased levels of ubiquitin-protein conjugates. To test this postulate, we harvested soleus muscles from 6 and 24-26 month old Fisher 344 rats. Aging resulted in a decline in mRNA expression of two key UCC components in soleus muscle; ubiquitin conjugating enzyme E2(14k) (E2(14k)) and muscle ring finger-1 (MuRF1). Surprisingly, no age-related differences existed in the total content of endogenous ubiquitin-protein conjugates in the soleus muscle. Nonetheless, a selective decrease in the level of ubiquitin-protein conjugates ( approximately 30kDa) was detected in the soleus of senescent animals. These results indicate that the soleus muscle displays a differential mRNA response of select UCC components to aging. Furthermore, the decline in E2(14k) and MuRF1 mRNA levels may contribute to altered substrate degradation by the UCC in the soleus muscle of senescent rats.     

Aging-associated oxidative stress modulates the acute inflammatory response in skeletal muscle after contusion injury

Inflammation is an integral component of the response of skeletal muscle to a contusion injury that can be modulated by acute oxidative stress. Less is known regarding the effect of aging-associated oxidative stress on the inflammatory response in injured skeletal muscle. The purpose of this project was to assess the level of oxidative stress in skeletal muscles of young, adult, and old rats and determine its effect on the acute inflammatory response to a contusion injury. Inherent oxidative stress in the muscle was determined by measuring the glutathione:glutathione disulfide ratio, and levels of GP91^phox. Elevated oxidative stress was observed in uninjured muscles of adult and old rats and was accompanied by increased levels of lipid peroxidation and neutrophil chemoattractant CINC-1. After injury, the acute inflammatory response (8 h, 3 d) was determined from markers of neutrophil (myeloperoxidase) and macrophage (CD68) content and by expression of NFκB, CINC-1 and TGF-β1. Compared to injured muscles of young rats, NFκB, myeloperoxidase activity (8 h), macrophage content (3 d), and TGF-β1 (8 h and 3 d) were significantly greater in injured muscles of old rats. We conclude that aging-associated oxidative stress in muscles of old rats exacerbated the inflammatory response to contusion injury and leads to increased TGF-β1-induced collagen content.     

Attenuation of age-related muscle wasting and weakness in rats after formoterol treatment: therapeutic implications for sarcopenia

We investigated the potential of the beta(2)-adrenoceptor agonist formoterol to increase mass and force-producing capacity of extensor digitorum longus (EDL) and soleus muscles from young, adult, and old rats. In addition, we examined the result of formoterol withdrawal. Young (3 month), adult (16 month), and old (27 month) F344 rats were treated with either formoterol (25 microg/kg/day, i.p.) or saline vehicle for 4 weeks. Another group of rats (for each age) was similarly treated with formoterol, followed by a withdrawal period of 4 weeks. Formoterol treatment increased EDL muscle mass and the force-producing capacity of both EDL and soleus muscles, without a concomitant increase in heart mass in adult and old rats. The hypertrophy and increased force-producing capacity of EDL muscles persisted 4 weeks after withdrawal of treatment. The findings have major implications for potential clinical trials utilizing beta(2)-agonists for sarcopenia.     

Response to trauma of protein, amino acid, and carbohydrate metabolism in injured and uninjured rat skeletal muscles

Soft tissue injury to one hindlimb produced trauma in rats without affecting their food intake or weight gain. Histologic examination showed damage to the soleus and gastrocnemius muscles but not to the extensor digitorum longus muscle. The protein content of the injured soleus muscle was lower than that of the contralateral soleus at one day after injury, and was reflected in vitro by a faster rate of protein degradation. The injured soleus also showed greater rates of protein synthesis, glucose uptake, glycolysis, oxidation of glucose, pyruvate, and leucine, and de novo synthesis of alanine. During three days after the injury, urinary nitrogen excretion increased progressively and was paralleled by a faster rate of protein degradation in uninjured muscles incubated with glucose, insulin, and amino acids. In these muscles, the inhibition of protein degradation by insulin diminished, while its stimulation of protein synthesis was unaffected. This insensitivity of proteolysis to insulin in trauma can explain the increased rate of this process. The oxidation of glucose and pyruvate were lower in the diaphragms of traumatized than of normal rats incubated with leucine, while glycolysis and uptake of 2-deoxyglucose did not differ. The degradation of leucine and isoleucine was greater in the diaphragms of traumatized animals and was associated with a faster de novo synthesis of alanine. For the uninjured soleus muscles of the traumatized rats, the slower rates of oxidation of glucose, glycolysis, and uptake of 2-deoxyglucose in the presence of insulin showed an insensitivity of glucose metabolism to this hormone. In contrast, no differences were seen in these various metabolic processes between the extensor digitorum longus muscles of traumatized and normal rats. These data suggest that the response of skeletal muscles to trauma may depend on their physiologic and biochemical characteristics.     

The influence of insulin-like growth factor-1 on protein turnover rates following exercise in young and mature animals

The purpose of this study was to determine the effect of an acute exercise bout on rates of skeletal muscle protein turnover in the presence and absence of a sub-maximal dose of insulin-like growth factor-1 (IGF-1) in young and mature animals. Mice (C57BI/6) were sacrificed 12 hours following a low intensity treadmill run (12 m/min @ 8% grade for either 1 or 2.5 hours). Protein synthesis, degradation and net protein degradation were measured in an in vitro isolated soleus muscle preparation in the absence or presence of IGF-1 in control and acutely exercised animals. Protein synthesis rates were lower in mature compared to young animals, but rates of protein degradation were unchanged. In the soleus from the 4 month control mice, protein synthesis increased in response to a submaximal dose of IGF-1 (20nM), but this response was abolished in the 12 month old tissue. Following an acute exercise bout (2.5 hours), protein synthesis increased in the soleus of both age groups, however net protein degradation increased in mature but not young animals. Protein synthesis rates of acutely exercised soleus of mature animals were increased in response to 20nM IGF-1 up to levels seen in the younger animals post-exercise. Results show that rates of protein synthesis decline from adolescence to maturity and protein synthesis is elevated in young and mature animals during the recovery from an exercise bout of long duration. It was also concluded that the ability of IGF-1 to stimulate protein synthesis was abolished in mature muscle, however this response was restored following low intensity aerobic exercise.     

Dynamics of postdenervation atrophy of young and old skeletal muscles: differential responses of fiber types and muscle types

We investigated the dynamics of muscle fiber atrophy in denervated fast and slow muscles of young and old rats. Hind limbs of 4-month-old and 24-month-old male rats were denervated, and soleus and tibialis anterior muscles were examined morphometrically 1 and 2 months after denervation. In all denervated muscles, type II muscle fibers underwent rapid atrophy, although muscle-specific differences in rate were observed. In both young and old denervated soleus muscles, the type I fibers underwent a pattern of atrophy closely paralleling that of the type II fibers, but in the tibialis anterior muscle, the mean cross-sectional area of the type I fibers actually increased during the first 2 months postdenervation. This study has shown that, among different muscles and between young and old rats, there is considerable variation in the response of the muscle fibers to denervation and that one cannot generalize from one muscle or one age to another.     

Age-related dystrophin-glycoprotein complex structure and function in the rat extensor digitorum longus and soleus muscle

This study tested the hypothesis that age-related changes in the dystrophin-glycoprotein complex (DGC) may precede age-associated alterations in muscle morphology and function. Compared to those in adult (6 month) rats, extensor digitorum longus (EDL) and soleus muscle mass was decreased in old (30 month) and very old (36 month) Fischer 344/NNiaHSD x Brown Norway/BiNia rats. The amount of dystrophin, beta-dystroglycan, and alpha-sarcoglycan increased with aging in the EDL and decreased with aging in the soleus. alpha-Dystroglycan levels were increased with aging in both muscles and displayed evidence of altered glycosylation. Immunostaining for the presence of antibody infiltration and dystrophin following increased muscle stretch suggested that the aging in the soleus was characterized by diminished membrane integrity. Together, these data suggest that aging is associated with alterations in EDL and soleus DGC protein content and localization. These results may implicate the DGC as playing a role in age-associated skeletal muscle remodeling.     

Decreased protein synthesis by microsomes isolated from senescent rat liver

Protein synthesis as indicated by the incorporation of ¹⁴C-leucine or a mixture of ¹⁴C-labelled amino acids into cold TCA-insoluble material by liver microsomal fractions of mature adult (12 months) and old (20–31 months) female rats was investigated. A decreased amino acid incorporation into protein occurred in the cell-free system from senescent rat liver. Also, cross-mixing experiments indicate that the cytosol(105,000g supernatant) from senescent animals is inhibitory to protein synthesis with microsomes from 12 month old rats. The combination of cytosol from 12 month old animals and microsomes from senescent animals functioned most often like the senescent system.     

Denervation stimulates apoptosis but not Id2 expression in hindlimb muscles of aged rats

Inhibitors of differentiation (Id) proteins are repressors of myogenic regulatory factors and have been implicated in apoptosis and muscle atrophy during aging. Indeed, we have previously found that Id levels are elevated in muscles from old rodents, possibly as a consequence of loss of alpha-motoneurons during senescence. To determine if Id2 proteins increase after denervation and if this is accompanied by increased apoptosis in aged as compared with adult animals, the gastrocnemius and soleus muscles were denervated in 1 limb of Fischer 344 x Brown Norway rats aged 9 months (adult, n = 12) and 33 months (aged, n = 9), while the contralateral limb served as the intra-animal control. After 14 days, the muscles in each limb were removed. The levels of Id1, Id2, and Id3 mRNA and protein were significantly greater in muscles of old as compared with young adult rats. Denervation, however, did not significantly increase Id1, Id2, and Id3 mRNA in soleus or gastrocnemius muscles from either young or old rats. Also Id2 protein levels were similar in denervated and control muscles from young adult and old rats. In young adult rats only, denervation induced an increase in Id1 and Id3 protein levels in both the soleus (Id1 113%; Id3 900%) and gastrocnemius (Id1 86%; Id3 80%). Denervation induced a significant increase in caspase 8 in both soleus and gastrocnemius muscles from young (101% and 147%, respectively) and old rats (167% and 190%, respectively). Bax protein levels, as estimated by western blots, increased by 726% and 1087% after denervation in the soleus and by 368% and 49% in the gastrocnemius muscles of young and old rats, respectively. The data suggest that the denervation-induced muscle loss was at least partly due to apoptosis as indicated by elevated caspase 8 and Bax levels in denervated muscles. While Id2 may have a role in aging-induced sarcopenia, Id2 does not appear to directly regulate apoptosis during denervation. The elevated Id expression in muscles from aged animals is therefore not a direct consequence of loss of alpha-motoneurons during senescence.     

Chronic resistance training activates autophagy and reduces apoptosis of muscle cells by modulating IGF-1 and its receptors,Akt/mTOR and Akt/FOXO3a signaling in aged rats

Resistance exercise training (RET) remains the most effective treatment for the loss of muscle mass and strength in elderly people. However, the underlying cellular and molecular mechanisms are not well understood. Recent evidence suggests that autophagic signaling is altered in aged skeletal muscles. This study aimed to investigate if RET affects IGF-1 and its receptors, the Akt/mTOR, and Akt/FOXO3a signaling pathways and regulates autophagy and apoptosis in the gastrocnemius muscles of 18–20 month old rats. The results showed that 9 weeks of RET prevented the loss of muscle mass and improved muscle strength, accompanied by reduced LC3-II/LC3-I ratio, reduced p62 protein levels, and increased levels of autophagy regulatory proteins, including Beclin 1, Atg5/12, Atg7, and the lysosomal enzyme cathepsin L. RET also reduced cytochrome c level in the cytosol but increased its level in mitochondrial fraction, and inhibited cleaved caspase 3 production and apoptosis. Furthermore, RET upregulated the expression of IGF-1 and its receptors but downregulated the phosphorylation of Akt and mTOR. In addition, RET upregulated the expression of total AMPK, phosphorylated AMPK, and FOXO3a. Taken together, these results suggest that the benefits of RET are associated with increased autophagy activity and reduced apoptosis of muscle cells by modulating IGF-1 and its receptors, the Akt/mTOR and Akt/FOXO3a signaling pathways in aged skeletal muscles.     

Effect of chronic renal failure on skeletal and diaphragmatic muscle contraction.

The changes in muscle mechanical properties caused by the myopathy of chronic uremia was examined in the soleus and the diaphragm muscles of rats in which chronic uremia was produced by subtotal nephrectomy. Using an in vitro muscle preparation in two groups of rats (moderately and severely uremic), we determined that uremia had a significant detrimental effect on both muscles with respect to the force-frequency relationships. In the diaphragm it decreased by 15% in the moderate group and by 43% in the severe group. In the soleus it decreased by 20% in both groups. Twitch characteristics behaved differently in the soleus and the diaphragm muscles in that 1/2 RT and TPT increased significantly (p < 0.05) in the soleus (severely uremic group) but not in the diaphragm. Fatigability was increased in both muscles in the moderately uremic rats and in the diaphragm in the severely uremic rats; however, the fatigability of the soleus in the severely uremic group was not different from that in the control group. Our findings suggest that the myopathic changes occurring in chronic uremia affects the function of the soleus and the diaphragm in different ways. Other findings in the severely uremic group indicate that additional factors such as marked electrolyte imbalances may also affect the excitation-contraction coupling in different ways.     

Intramuscular administration of PEGylated IGF-I improves skeletal muscle regeneration after myotoxic injury

ObjectiveMusculoskeletal injuries represent a major public health problem and drugs that can improve muscle repair and restore function are needed for patients with these conditions and other related muscular pathologies. Increasing insulin-like growth factor-I (IGF-I) levels in skeletal muscle improves regeneration after myotoxic injury and while administration of IGF-I has a potential for accelerating healing after trauma, optimizing its method of delivery and obviating potential side-effects currently associated with recombinant human (rh) IGF-I, remain a hurdle.DesignWe compared the treatment efficacy of rhIGF-I with a polyethylene glycol modified IGF-I (PEG-IGF-I) analog to improve functional repair of mouse tibialis anterior muscles after myotoxic injury, testing the hypothesis that PEG-IGF-I would exert greater beneficial effects on regenerating skeletal muscles than rhIGF-I due to improved pharmacokinetic properties. We also examined the relative efficacy of systemic versus local delivery of these IGF-I variants for improving functional muscle regeneration.ResultsLocal delivery of PEG-IGF-I, but not rhIGF-I, at 4 days post-injury significantly improved early functional recovery as evident by a 27% increase in normalized force compared with saline control (P < 0.05), whereas systemic application of either IGF-I variant was not effective. The improved function with intramuscular PEG-IGF-I administration was attributed to a greater and prolonged residence within the regenerating muscles, resulting in increased Akt activation and a 13% larger fiber cross-sectional area compared with rhIGF-I (P < 0.05).ConclusionsThese data support the hypothesis that PEG-IGF-I is more efficacious than rhIGF-I in hastening early fiber regeneration and improving muscle function after injury, highlighting its therapeutic potential for muscular pathologies.     

Elevated GLUT 1 level in crude muscle membranes from diabetic Zucker rats despite a normal GLUT 1 level in perineurial sheaths

Summary Recently, we demonstrated that approximately 60 % of GLUT 1 in a crude membrane fraction of rat skeletal muscle originates from perineurial sheaths. To study the in vivo regulation of GLUT 1 expression in different tissues in muscles, we measured the level of GLUT 1 in crude muscle membranes and in perineurial sheaths in diabetic (fa/fa) Zucker rats and lean controls, with and without metformin treatment. The GLUT 1 concentration in perineurial sheaths was identical in all four groups of rats, both when measured by quantitative immunofluorescence and by immunoblotting and densitometry. In a fraction of crude membranes of soleus muscles GLUT 1 expression was more than two-fold higher in (fa/fa) rats than in lean controls (p<0.005). Metformin treatment significantly elevated GLUT 1 in control rats (p<0.05) and tended to decrease GLUT 1 in diabetic rats (p<0.075). The expressions of GLUT 1 and GLUT 4 in crude muscle membranes were inversely correlated (p<0.01), and GLUT 1 expression correlated positively with fasting glucose (p<0.05). In conclusion, GLUT 1 expression in perineurial sheaths is unaffected by alterations in glucose homeostasis and by the genes responsible for obesity and diabetes in the Zucker rat. GLUT 1 expression in a crude membrane fraction of soleus muscle is increased in the diabetic animals, likely due to an increased expression in muscle cells proper. [Diabetologia (1994) 37: 443–448] Received: 17 June 1993 and in revised form: 19 November 1993     

A persistent increase in insulin-stimulated glucose uptake by both fast-twitch and slow-twitch skeletal muscles after a single exercise session by old rats

Exercise has been demonstrated to enhance subsequent insulin-stimulated glucose uptake (GU) by predominantly type II (fast-twitch) muscle of old rats, but previous research has not evaluated exercise effects on GU by type I (slow-twitch) muscle from old rats. Accordingly, we studied male Fischer 344/Brown Norway rats (24 months old) and determined GU (0, 100, 200, and 5,000 μU/ml insulin) of isolated soleus (predominantly type I) and epitrochlearis (predominantly type II) muscles after one exercise session. Epitrochlearis (100, 200, and 5,000 μU/ml insulin) and soleus (100 and 200 μU/ml insulin) GU were greater at 3-h postexercise vs. age-matched sedentary controls. Insulin receptor tyrosine phosphorylation (Tyr1162/1163) was unaltered by exercise in either muscle. Akt phosphorylation (pAkt) was greater for exercised vs. sedentary rats in the epitrochlearis (Ser473 and Thr308 with 100 and 200 μU/ml, respectively) and soleus (Ser473 with 200 μU/ml). AS160 phosphorylation (pAS160) was greater for exercised vs. sedentary rats in the epitrochlearis (Thr642 with 100 μU/ml), but not the soleus. Exercised vs. sedentary rats did not differ for total protein abundance of insulin receptor, Akt, AS160, or GLUT4 in either muscle. These results demonstrate that both predominantly type I and type II muscles from old rats are susceptible to exercise-induced improvement in insulin-mediated GU by mechanisms that are independent of enhanced insulin receptor tyrosine phosphorylation or altered abundance of important signaling proteins or GLUT4. Exercise-induced elevation in pAkt, and possibly pAS160, may contribute to this effect in the epitrochlearis of old rats, but other mechanisms are likely important for the soleus.     

Vascular smooth muscle cell outgrowth, proliferation, and apoptosis in young and old rats.

We attempted to identify the effects of aging on the response of vascular smooth muscle cells (VSMCs) to injury. Rat aortas were injured by ballooning, and cell outgrowth of the aortic explants, as well as cell proliferation and apoptosis induced by 7-ketocholesterol in the culture system were compared in young (6-7 weeks) and old (40-50 weeks) rats. Explant outgrowth in uninjured rats did not differ between young and old rats. However, 14 days after balloon injury, the number of explants with outgrowth increased by a factor of four (P<0.01) in young rats, while that of the old rats did not change. Cell proliferation in cultured VSMCs also did not differ between young and old uninjured rats, but, in the injured group, proliferation doubled in young rats (P<0.01), but did not change in the older group. The rate of unadhered cells in the presence of 7-ketocholesterol (30 microM) did not differ between young and old uninjured rats. In the injured rats, however, that of young rats was lower (P<0.01) while that of older rats was higher (P<0.01). The extent of DNA fragmentation (%) after the addition of 7-ketocholesterol (30 microM) did not differ between young and old uninjured rats. In the injured groups, DNA fragmentation was lower in the young rats (P<0.01), and higher in the old ones (P<0.01), when compared to their respective controls. These results indicate that the VSMC injury responses of cell outgrowth and proliferation were more pronounced in the young, and that 7-ketocholesterol-induced apoptosis was more extensive in old rats.     

Age-related differences in the adaptive potential of type I skeletal muscle fibers

This study was designed to investigate whether the relationship of fiber size and force is maintained with aging and inactivity. We hypothesized that fiber size and fiber force-generating capacity would decrease in parallel, thus resulting in no change in specific force with either age or inactivity. Thirty male Fischer 344/Brown Norway F1 hybrid rats, 6-23-month old (young adult), 24-33-month old (middle-aged) and 34-40 month old (old) were hindlimb unweighted for 14 days. Single permeabilized type I fibers from the soleus and gastrocnemius muscles were evaluated for size and contractile function. The diameter of the fibers from the soleus muscle declined with age and unweighting. In contrast, the fibers from the gastrocnemius showed no age-related atrophy. In both the soleus and gastrocnemius, there was a significant decrease in the force generation with age and unweighting. When comparing size and peak absolute force in the type I fibers from control young and middle-aged animals a positive relationship was observed whereas no significant relationship between size and peak absolute force was observed in the old animals. Following unweighting, fibers from young and middle aged rats showed a significant relationship between force and size. Fibers from old animals did not exhibit a relationship between size and force following unweighting. These results suggest aged skeletal muscle has an attenuated ability to adapt to inactivity by altering its size in response to inactivity.     

Influence of age on biochemical parameters of the rat liver following partial hepatectomy (author's transl)]

In the present paper measurements of DNA and protein content and lysosomal enzyme (beta-glucuronidase, beta-acetylglucosaminidase, cathepsin D) acitivities were performed in the rat liver following partial hepatectomy. The rats were divided into three age groups: 6 weeks, 10 months and 18 months. The regenerating liver of the 6 week and 10 month old animals disclosed significant higher concentrations of DNA than the controls. The 18 month old rats revealed no differences of the DNA content. In all age groups the protein content of the regenerating liver was significant diminished. There were age dependent differences of the activities of the three lysosomal enzymes. In comparison to the controls the beta-glucuronidase activity of the regenerating liver was significantly decreased in the 6 week and 18 month old animals, but significantly increased in the 10 month old rats. Refering to the protein content there were no differences of the activities of beta-acetylglucosaminidase and cathepsin D between the regenerating and control livers. Refering to the liver fresh weight the beta-acetylglucosaminidase activity of the regenerating liver was significant diminished in the 10 and 18 month old rats.