Myofibrillar protein synthesis in myostatin-deficient mice

Either increased protein synthesis or prolonged protein half-life is necessary to support the excessive muscle growth and maintenance of enlarged muscles in myostatin-deficient mice. This issue was addressed by determining in vivo rates of myofibrillar protein synthesis in mice with constitutive myostatin deficiency (Mstn(DeltaE3/DeltaE3)) or normal myostatin expression (Mstn(+/+)) by measuring tracer incorporation after a systemic flooding dose of l-[ring-(2)H(5)]phenylalanine. At 5-6 wk of age, Mstn(DeltaE3/DeltaE3) mice had increased muscle mass (40%), fractional rates of myofibrillar synthesis (14%), and protein synthesis per whole muscle (60%) relative to Mstn(+/+) mice. With maturation, fractional rates of synthesis declined >50% in parallel with decreased DNA and RNA [total, 28S rRNA, and poly(A) RNA] concentrations in muscle. At 6 mo of age, Mstn(DeltaE3/DeltaE3) mice had even greater increases in muscle mass (90%) and myofibrillar synthesis per muscle (85%) relative to Mstn(+/+) mice, but the fractional rate of synthesis was normal. Estimated myofibrillar protein half-life was not affected by myostatin deficiency. Muscle DNA concentrations were reduced in both young and mature Mstn(DeltaE3/DeltaE3) mice, whereas RNA concentrations were normal, so the ratio of RNA to DNA was approximately 30% greater than normal in Mstn(DeltaE3/DeltaE3) mice. Thus the increased protein synthesis and RNA content per muscle in myostatin-deficient mice cannot be explained entirely by an increased number of myonuclei.     

Altered expression of genes regulating skeletal muscle mass in the portacaval anastomosis rat

We examined the temporal relationship between portacaval anastomosis (PCA), weight gain, changes in skeletal muscle mass and molecular markers of protein synthesis, protein breakdown, and satellite cell proliferation and differentiation. Male Sprague-Dawley rats with end to side PCA (n=24) were compared with sham-operated pair-fed rats (n=24). Whole body weight, lean body mass, and forelimb grip strength were determined at weekly intervals. The skeletal muscle expression of the ubiquitin proteasome system, myostatin, its receptor (the activin 2B receptor) and its signal, cyclin-dependent kinase inhibitor (CDKI) p21, insulin-like growth factor (IGF)-I and its receptor (IGF-I receptor-alpha), and markers of satellite cell proliferation and differentiation were quantified. PCA rats did not gain body weight and had lower lean body mass, forelimb grip strength, and gastrocnemius muscle weight. The skeletal muscle expression of the mRNA of ubiquitin proteasome components was higher in PCA rats in the first 2 wk followed by a lower expression in the subsequent 2 wk (P<0.01). The mRNA and protein of myostatin, activin 2B receptor, and CDKI p21 were higher, whereas IGF-I and its receptor as well as markers of satellite cell function (proliferating nuclear cell antigen, myoD, myf5, and myogenin) were lower at weeks 3 and 4 following PCA (P < 0.05). We conclude that PCA resulted in uninhibited proteolysis in the initial 2 wk. This was followed by an adaptive response in the later 2 wk consisting of an increased expression of myostatin that may have contributed to reduced muscle protein synthesis, impaired satellite cell function, and lower skeletal muscle mass.     

Myofibrillar protein degradation in the chicken. 3-Methylhistidine release in vivo and in vitro in normal and genetically muscular-dystrophic chickens

Myofibrillar protein degradation was measured in 4-week-old normal (line 412) and genetically muscular-dystrophic (line 413) New Hampshire chickens by monitoring the rates of 3-methylhistidine excretion in vivo and in vitro. A method of perfusing breast and wing muscles was developed and the rate of 3-methylhistidine release in vitro was measured between 30 and 90min of perfusion. During this perfusion period, 3-methylhistidine release from the muscle preparation was linear, indicating that changes in 3-methylhistidine concentration of the perfusate were the result of myofibrillar protein degradation. Furthermore, the viability of the perfused muscle was maintained during this interval. After 60min of perfusion, ATP, ADP and creatine phosphate concentrations in pectoral muscle were similar to muscle freeze-clamped in vivo. Rates of glucose uptake and lactate production were constant during the perfusion. In dystrophic-muscle preparations, the rate of 3-methylhistidine release in vitro (nmol/h per g of dried muscle) was elevated 2-fold when compared with that in normal muscle. From these data the fractional degradation rates of myofibrillar protein in normal and dystrophic pectoral muscle were calculated to be 12 and 24% respectively. Daily 3-methylhistidine excretion (nmol/day per g body wt.) in vivo was elevated 1.35-fold in dystrophic chickens. Additional studies revealed that the anti-dystrophic drugs diphenylhydantoin and methylsergide, which improve righting ability of dystrophic chickens, did not alter 3-methylhistidine release in vitro. This result implies that changes in myofibrillar protein turnover are not the primary lesion in avian muscular dystrophy. From tissue amino acid analysis, the myofibrillar 3-methylhistidine content per g dry weight of muscle was similar in normal and dystrophic pectoral muscle. More than 96% of the 3-methylhistidine present in pectoral muscle was associated with the myofibrillar fraction. Dystrophic myofibrillar protein contained significantly less 3-methylhistidine (nmol/g of myofibrillar protein) than protein from normal muscle. This observation supports the hypothesis that there may be a block in the biochemical maturation and development of dystrophic muscle after hatching. Free 3-methylhistidine (nmol/g wet wt.) was elevated in dystrophic muscle, whereas blood 3-methylhistidine concentrations were similar in both lines. In summary, the increased myofibrillar protein catabolism demonstrated in dystrophic pectoral muscle correlates with the increased lysosomal cathepsin activity in this tissue as reported by others.     

Acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA without increasing proteolysis in skeletal muscle

Acute alcohol intoxication decreases muscle protein synthesis, but there is a paucity of data on the ability of alcohol to regulate muscle protein degradation. Furthermore, various types of atrophic stimuli appear to regulate ubiquitin-proteasome-dependent proteolysis by increasing the muscle-specific E3 ligases atrogin-1 and MuRF1 (i.e., "atrogenes"). Therefore, the present study was designed to test the hypothesis that acute alcohol intoxication increases atrogene expression leading to an elevated rate of muscle protein breakdown. In male rats, the intraperitoneal injection of alcohol dose- and time-dependently increased atrogin-1 and MuRF1 mRNA in gastrocnemius, the latter of which was most pronounced. A comparable change was absent in the soleus and heart. The ability of in vivo-administered ethanol to increase atrogene expression was independent of the route of alcohol administration (intraperitoneal vs. oral), as well as of nutritional status (fed vs. fasted) and gender (male vs. female). The increase in atrogin-1 and MuRF1 was independent of alcohol metabolism, and the overproduction of endogenous glucocorticoids and could not be prevented by maintaining the circulating concentration of insulin-like growth factor-I. Despite marked changes in atrogene expression, acute alcohol in vivo did not alter the release of either 3-methylhistidine (MH) or tyrosine from the isolated perfused hindlimb, suggesting that the rate of muscle proteolysis remains unchanged. Moreover, alcohol did not increase the directly determined rate of protein degradation in isolated epitrochlearis muscles or cultured myocytes. Finally, no increase in atrogene expression or 3-MH release was detected in muscle from rats fed an alcohol-containing diet. Our results indicate that although acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA preferentially in fast-twitch skeletal muscle, this change was not associated with increased rates of muscle proteolysis. Therefore, the loss of muscle mass/protein in response to chronic alcohol abuse appears to result primarily from a decrement in muscle protein synthesis, not an increase in degradation.     

Muscle growth after postdevelopmental myostatin gene knockout

Constitutive myostatin gene knockout in mice causes excessive muscle growth during development. To examine the effect of knocking out the myostatin gene after muscle has matured, we generated mice in which myostatin exon 3 was flanked by loxP sequences (Mstn[f/f]) and crossed them with mice bearing a tamoxifen-inducible, ubiquitously expressed Cre recombinase transgene. At 4 mo of age, Mstn[f/f]/Cre+ mice that had not received tamoxifen had a 50-90% reduction in myostatin expression due to basal Cre activity but were not hypermuscular relative to Mstn[w/w]/Cre+ mice (homozygous for wild-type myostatin gene). Three months after tamoxifen treatment (initiated at 4 mo of age), muscle mass had not changed from the pretreatment level in Mstn[w/w]/Cre+ control mice. Tamoxifen administration to 4-mo-old Mstn[f/f]/Cre+ mice reduced myostatin mRNA expression to less than 1% of normal, which increased muscle mass approximately 25% over the following 3 mo in both male and female mice (P<0.005 vs. control). Fiber hypertrophy appeared to be sufficient to explain the increase in muscle mass. The pattern of expression of genes encoding the various myosin heavy-chain isoforms was unaffected by postdevelopmental myostatin knockout. We conclude that, even after developmental muscle growth has ceased, knockout of the myostatin gene induces a significant increase in muscle mass.     

The p97/VCP ATPase is critical in muscle atrophy and the accelerated degradation of muscle proteins

The p97/VCP ATPase complex facilitates the extraction and degradation of ubiquitinated proteins from larger structures. We therefore studied if p97 participates to the rapid degradation of myofibrillar proteins during muscle atrophy. Electroporation of a dominant negative p97 (DNp97), but not the WT, into mouse muscle reduced fibre atrophy caused by denervation and food deprivation. DNp97 (acting as a substrate-trap) became associated with specific myofibrillar proteins and its cofactors, Ufd1 and p47, and caused accumulation of ubiquitinated components of thin and thick filaments, which suggests a role for p97 in extracting ubiquitinated proteins from myofibrils. DNp97 expression in myotubes reduced overall proteolysis by proteasomes and lysosomes and blocked the accelerated proteolysis induced by FoxO3, which is essential for atrophy. Expression of p97, Ufd1 and p47 increases following denervation, at times when myofibrils are rapidly degraded. Surprisingly, p97 inhibition, though toxic to most cells, caused rapid growth of myotubes (without enhancing protein synthesis) and hypertrophy of adult muscles. Thus, p97 restrains post-natal muscle growth, and during atrophy, is essential for the accelerated degradation of most muscle proteins.     

Effects of serum deprivation on myofibrillar proteolysis in chick myotube cultures

We examined the effects of serum deprivation on myofibrillar proteolysis in chick myotubes. Myotubes were incubated with serum-free medium for 24 hours. N(tau)-methylhistidine release, as an index of myofibrillar proteolysis, as well as protease activities such as calpain, proteasome, and cathepsins (B+L and D) activities were increased by serum deprivation. These results indicate that serum deprivation induces calpain, proteasome, and cathepsins activities, resulting in an increase in myofibrillar proteolysis in chick myotubes.     

Time course of the effect of catabolic doses of corticosterone on protein turnover in rat skeletal muscle and liver.

The time course of the response of protein synthesis in muscle and liver to catabolic doses of corticosterone (10 mg/day per 100 g body wt.) was studied in vivo in growing rats over a 12-day period. The rate of protein synthesis in muscle and liver and the rate of actomyosin synthesis in muscle were measured by the phenylalanine-flooding technique, and 3-methylhistidine (N tau-methylhistidine) synthesis was measured by injection of labelled histidine. 3-Methylhistidine concentrations in tissue free pools and urinary excretion were also measured to compare directly with the rate of muscle protein degradation determined as the difference between synthesis and growth each day during the treatment. The overall rate of protein synthesis in muscle fell gradually over the first 4 days, reaching a rate after 5 days that was 36% of the initial rate, and this lower rate was then maintained for the following week. This decrease in the overall rate was accompanied with changes in the relative rate of synthesis in muscle proteins, since during the first 4 days there was a disproportionate decrease in the rate of actomyosin synthesis, and specifically 3-methylhistidine synthesis. In the latter case the synthesis rate was decreased to only 4% of its initial rate after 4 days. These changes in protein synthesis in muscle were accompanied by a transient increase in the rate of protein degradation, which was more than doubled on days 2 and 3 of treatment but which returned to the original rate on day 5, and a similar pattern of response was indicated by urinary 3-methylhistidine excretion, which also exhibited a transient increase. Thus in this case 3-methylhistidine excretion and measured rates of protein degradation in muscle do correlate. The transient effects of the glucocorticoids on degradation compared with the sustained effect on synthesis suggest that these two responses are achieved by different mechanisms. The hepatic size and protein mass were increased by the treatment, and protein synthesis was well maintained until after 12 days, when the rate was suppressed. Although the fractional synthesis rate was transiently increased for 24 h, it is argued that the enlarged liver most likely reflects a decrease in protein degradation resulting from the increased amino acid supply to the liver. This would result from the cessation of muscle growth while dietary supply was maintained.     

Myostatin expression in age and denervation-induced skeletal muscle atrophy

Myostatin is hypothesized to regulate skeletal muscle mass and to be a potential target for therapeutic intervention in sarcopenia. To clarify whether myostatin is invariably associated with sarcopenia, this study examined the levels of expression of myostatin mRNA and protein in Sprague Dawley rats during aging- and denervation-induced sarcopenia. The level of myostatin mRNA in the gastrocnemius decreased progressively with age being 9, 34 and 56% lower at 6, 12 and 27 months, respectively, compared with mRNA levels at 1.5 months. In contrast, two low molecular mass isoforms of myostatin protein identified by Western blotting increased progressively with age. With denervation, myostatin mRNA was 31% higher on day 1 but by 14 days after sciatic neurectomy when the muscle had atrophied 50%, myostatin expression decreased 34% relative to the sham operated limb. Western analysis of the denervated gastrocnemius showed that myostatin protein levels varied in parallel with mRNA. These disparate patterns of expression of myostatin during age- and denervation-induced atrophy suggest that the regulation of myostatin is complex and variable depending on whether the atrophy is slowly or rapidly progressive.     

Protein metabolism in rat gastrocnemius muscle after stimulated chronic concentric exercise.

Previous results by use of a model of resistance exercise consisting of nonvoluntary electrical contraction of rat skeletal muscle have shown that significant gastrocnemius muscle enlargement was produced after 16 wk of chronic concentric resistance training with progressively increased weights but not after the same training program without weights (J. Appl. Physiol. 65: 950-954, 1988). In the present study we examined whether this differential effect on muscle mass between high- and low-resistance exercise is mediated through differential actions on muscle protein synthesis rates. In addition, we determined whether accumulation of specific mRNA quantities had a primary role in the protein synthesis response to this type of exercise. The data revealed that as little as 8 min of total contractile duration increased gastrocnemius protein synthesis rates by nearly 50%. Contrary to our hypothesis, post-exercise protein synthesis rates do not appear to be differentially regulated by the resistance imposed on the muscle during exercise but rather by the number of repetitions performed during the acute bout. This observation, the failure of high-frequency chronic training to produce gastrocnemius enlargement, and the relatively minor effects on mRNA levels collectively suggest that translational and posttranslational mechanisms, including protein degradation, may be the principal processes by which gastrocnemius protein expression is regulated in this model of stimulated concentric exercise.     

Skeletal and cardiac myopathy in HIV-1 transgenic rats

The mechanism by which human immunodeficiency virus (HIV)-1 infection in humans leads to the erosion of lean body mass is poorly defined. Therefore, the purpose of the present study was to determine whether transgenic (Tg) rats that constitutively overexpress HIV-1 viral proteins exhibit muscle wasting and to elucidate putative mechanisms. Over 7 mo, Tg rats gained less body weight than pair-fed controls exclusively as a result of a proportional reduction in lean, not fat, mass. Fast- and slow-twitch muscle atrophy in Tg rats did not result from a reduction in the in vivo-determined rate of protein synthesis. In contrast, urinary excretion of 3-methylhistidine, as well as the content of atrogin-1 and the 14-kDa actin fragment, was elevated in gastrocnemius of Tg rats, suggesting increased muscle proteolysis. Similarly, Tg rats had reduced cardiac mass, which was independent of a change in protein synthesis. This decreased cardiac mass was associated with a reduction in stroke volume, but cardiac output was maintained by a compensatory increase in heart rate. The HIV-induced muscle atrophy was associated with increased whole body energy expenditure, which was not due to an elevated body temperature or secondary bacterial infection. Furthermore, the atrophic response could not be attributed to the development of insulin resistance, decreased levels of circulating amino acids, or increased tissue cytokines. However, skeletal muscle and, to a lesser extent, circulating insulin-like growth factor I was reduced in Tg rats. Although hepatic injury was implicated by increased plasma levels of aspartate and alanine aminotransferases, hepatic protein synthesis was not different between control and Tg rats. Hence, HIV-1 Tg rats develop atrophy of cardiac and skeletal muscle, the latter of which results primarily from an increased protein degradation and may be related to the marked reduction in muscle insulin-like growth factor I.     

Classification of Chitosanases by Hydrolytic Specificity toward N 1,N 4-Diacetylchitohexaose

The immediate response of protein degradation to food intake and the factors for its regulation in rat skeletal muscle were examined. The concentration of N ^τ-methylhistidine (MeHis) in serum and the rates of MeHis release from isolated soleus and extensor digitorum longus muscles were reduced in the period from 3 to 6h after refeeding, indicating that the rate of myofibrillar protein degradation in the rat decreased immediately after refeeding. Changes in the serum concentration of insulin and corticosterone were not synchronized with those in the myofibrillar protein degradation. When rats were fed on a protein-free diet, no reduction of serum MeHis concentration or of the rate of MeHis release from isolated muscles after refeeding was apparent. Furthermore, there was a tendency toward suppressing myofibrillar protein degradation with a higher protein content of the diet. These results suggest that the suppression of myofibrillar protein degradation by food intake was regulated by dietary proteins.     

Lysine suppresses protein degradation through autophagic–lysosomal system in C2C12 myotubes

Muscle mass is determined between protein synthesis and protein degradation. Reduction of muscle mass leads to bedridden condition and attenuation of resistance to diseases. Moreover, bedridden condition leads to additional muscle loss due to disuse muscle atrophy. In our previous study (Sato et al. 2013), we showed that administered lysine (Lys), one of essential amino acid, suppressed protein degradation in skeletal muscle. In this study, we investigated that the mechanism of the suppressive effects of Lys on skeletal muscle proteolysis in C2C12 cell line. C2C12 myotubes were incubated in the serum-free medium containing 10 mM Lys or 20 mM Lys, and myofibrillar protein degradation was determined by the rates of 3-methylhistidine (MeHis) release from the cells. The mammalian target of rapamycin (mTOR) activity from the phosphorylation levels of p70-ribosormal protein S6 kinase 1 and eIF4E-binding protein 1 and the autophagic–lysosomal system activity from the ratio of LC3-II/I in C2C12 myotubes stimulated by 10 mM Lys for 0–3 h were measured. The rates of MeHis release were markedly reduced by addition of Lys. The autophagic–lysosomal system activity was inhibited upon 30 min of Lys supplementation. The activity of mTOR was significantly increased upon 30 min of Lys supplementation. The suppressive effect of Lys on the proteolysis by the autophagic–lysosomal system was maintained partially when mTOR activity was inhibited by 100 nM rapamycin, suggesting that some regulator other than mTOR signaling, for example, Akt, might also suppress the autophagic–lysosomal system. From these results, we suggested that Lys suppressed the activity of the autophagic–lysosomal system in part through activation of mTOR and reduced myofibrillar protein degradation in C2C12 myotubes.     

Alloxan-induced luminol luminescence as a tool for investigating mechanisms of radical-mediated diabetogenicity.

It is not clear whether the muscle wasting commonly observed in hyperthyroidism is due to alteration in the rate of protein synthesis or degradation. The effect of experimental hyperthyroidism on skeletal-muscle proteolysis in the rat was studied by measuring alanine and tyrosine release from isolated skeletal muscles in vitro and 3-methyl-histidine excretion in vivo. Alanine release from the isolated epitrochlaris-muscle preparation was increased as soon as 24h after a 25 microgram dose of L-tri-iodothyronine in vivo. Conversely, alanine release from muscles of hypothyroid rats was decreased, but restored by L-tri-iodothyronine supplementation before death. Furthermore, 3-methylhistidine excretion was increased in hyperthyroid rats throughout an 18-day treatment period. The increased amino acid release from isolated muscles and the increased 3-methylhistidine excretion in vivo strongly suggests that hyperthyroidism increases skeletal-muscle proteolysis. Furthermore, the thyroid-hormone concentration may be an important factor in regulating muscle proteolysis.     

Effect of corticosterone treatment on muscle protein turnover in adrenalectomized rats and diabetic rats maintained on insulin

The effect of corticosterone on protein turnover in skeletal muscle was investigated in growing rats. Protein synthesis was measured in vivo by the constant infusion of [(14)C]tyrosine. The extent to which any effect of corticosterone is modulated by the hyperinsulinaemia induced by steroid treatment was examined by giving the hormone not only to adrenalectomized rats but also to streptozotocin-induced diabetic rats maintained throughout the treatment period on two dosages of insulin by an implanted osmotic minipump. Approximate rates of protein degradation were also estimated in some cases as the difference between synthesis and net change in muscle protein mass. Measurements were also made of free 3-methylhistidine concentration in muscle and plasma. At 10mg of corticosterone/100g body wt. per day, growth stopped and muscle wasting occurred, whereas at 5 mg of corticosterone/100g body wt. per day no net loss of protein occurred. However, this low dose did induce muscle wasting when insulin concentration was regulated by a dose of 1.2 units/day. Protein synthesis was markedly depressed in all treated groups, the depression in the insulin-maintained rats being marginally more than in the hyperinsulinaemic adrenalectomized rats. The oxidative soleus muscle appeared to be less susceptible to the effect of the corticosterone than was the more glycolytic plantaris or gastrocnemius muscle. Any effect of the corticosterone on protein degradation was much less than its effects on protein synthesis. Where increases in the degradation rates appeared to occur in the rats treated with 10mg of corticosterone/100g body wt. per day, the increases were less than 20%. The free intracellular 3-methylhistidine concentrations were doubled in all groups treated with 5 mg of corticosterone/100g body wt. per day and increased 5-fold in the adrenalectomized rats treated with 10mg of corticosterone/100g body wt. per day, with no change in plasma concentration in any of the groups. It is therefore concluded that: (a) the suppression of protein synthesis is the main effect of glucocorticoids in muscle; (b) marked increases in insulin afford only minor protection against this effect; (c) stimulation of protein degradation may occur, but to a much lesser extent.     

Mechanisms of protein degradation in mantle muscle and proposed gill remodeling in starved Sepia officinalis

Cephalopods have relatively high rates of protein synthesis compared to rates of protein degradation, along with minimal carbohydrate and lipid reserves. During food deprivation on board protein is catabolized as a metabolic fuel. The aim of the current study was to assess whether biochemical indices of protein synthesis and proteolytic mechanisms were altered in cuttlefish, Sepia officinalis, starved for 7 days. In mantle muscle, food deprivation is associated with a decrease in protein synthesis, as indicated by a decrease in the total RNA level and dephosphorylation of key signaling molecules, such as the eukaryote binding protein, 4E-BP1 (regulator of translation) and Akt. The ubiquitination-proteasome system (UPS) is activated as shown by an increase in the levels of proteasome β-subunit mRNA, polyubiquitinated protein, and polyubiquitin mRNA. As well, cathepsin activity levels are increased, suggesting increased proteolysis through the lysosomal pathway. Together, these mechanisms could supply amino acids as metabolic fuels. In gill, the situation is quite different. It appears that during the first stages of starvation, both protein synthesis and protein degradation are enhanced in gill. This is based upon increased phosphorylation of 4E-BP1 and enhanced levels of UPS indicators, especially 20S proteasome activity and polyubiquitin mRNA. It is proposed that an increased protein turnover is related to gill remodeling perhaps to retain essential hemolymph-borne compounds.