Anselmo S. Moriscot

LEUCINE ATTENUATES SKELETAL MUSCLE WASTING VIA INHIBITION OF UBIQUITIN LIGASES

The aim of this study was to assess the effect of leucine supplementation on elements of the ubiquitin–proteasome system (UPS) in rat skeletal muscle during immobilization. This effect was evaluated by submitting the animals to a leucine supplementation protocol during hindlimb immobilization, after which different parameters were determined, including: muscle mass; cross‐sectional area (CSA); gene expression of E3 ligases/deubiquitinating enzymes; content of ubiquitinated proteins; and rate of protein synthesis. Our results show that leucine supplementation attenuates soleus muscle mass loss driven by immobilization. In addition, the marked decrease in the CSA in soleus muscle type I fibers, but not type II fibers, induced by immobilization was minimized by leucine feeding. Interestingly, leucine supplementation severely minimized the early transient increase in E3 ligase [muscle ring finger 1 (MuRF1) and muscle atrophy F‐box (MAFbx)/atrogin‐1] gene expression observed during immobilization. The reduced peak of E3 ligase gene expression was paralleled by a decreased content of ubiquitinated proteins during leucine feeding. The protein synthesis rate decreased by immobilization and was not affected by leucine supplementation. Our results strongly suggest that leucine supplementation attenuates muscle wasting induced by immobilization via minimizing gene expression of E3 ligases, which consequently could downregulate UPS‐driven protein degradation. It is notable that leucine supplementation does not restore decreased protein synthesis driven by immobilization. Muscle Nerve, 2010

Exercise Training Prevents Oxidative Stress and Ubiquitin-Proteasome System Overactivity and Reverse Skeletal Muscle Atrophy in Heart Failure

Background Heart failure (HF) is known to lead to skeletal muscle atrophy and dysfunction. However, intracellular mechanisms underlying HF-induced myopathy are not fully understood. We hypothesized that HF would increase oxidative stress and ubiquitin-proteasome system (UPS) activation in skeletal muscle of sympathetic hyperactivity mouse model. We also tested the hypothesis that aerobic exercise training (AET) would reestablish UPS activation in mice and human HF. Methods/Principal Findings Time-course evaluation of plantaris muscle cross-sectional area, lipid hydroperoxidation, protein carbonylation and chymotrypsin-like proteasome activity was performed in a mouse model of sympathetic hyperactivity-induced HF. At the 7th month of age, HF mice displayed skeletal muscle atrophy, increased oxidative stress and UPS overactivation. Moderate-intensity AET restored lipid hydroperoxides and carbonylated protein levels paralleled by reduced E3 ligases mRNA levels, and reestablished chymotrypsin-like proteasome activity and plantaris trophicity. In human HF (patients randomized to sedentary or moderate-intensity AET protocol), skeletal muscle chymotrypsin-like proteasome activity was also increased and AET restored it to healthy control subjects’ levels. Conclusions Collectively, our data provide evidence that AET effectively counteracts redox imbalance and UPS overactivation, preventing skeletal myopathy and exercise intolerance in sympathetic hyperactivity-induced HF in mice. Of particular interest, AET attenuates skeletal muscle proteasome activity paralleled by improved aerobic capacity in HF patients, which is not achieved by drug treatment itself. Altogether these findings strengthen the clinical relevance of AET in the treatment of HF.

Effect of bench press exercise intensity on muscle soreness and inflammatory mediators

Abstract This study compared four different intensities of a bench press exercise for muscle soreness, creatine kinase activity, interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), and prostaglandin E2 (PGE2) concentrations in the blood. Thirty-five male Brazilian Army soldiers were randomly assigned to one of five groups: 50% one-repetition maximum (1-RM), 75% 1-RM, 90% 1-RM, 110% 1-RM, and a control group that did not perform the exercise. The total volume (sets × repetitions × load) of the exercise was matched among the exercise groups. Muscle soreness and plasma creatine kinase activity increased markedly (P < 0.05) after exercise, with no significant differences among the groups. Serum PGE2 concentration also increased markedly (P < 0.05) after exercise, with a significantly (P < 0.05) greater increase in the 110% 1-RM group compared with the other groups. A weak but significant (P < 0.05) correlation was found between peak muscle soreness and peak PGE2 concentration, but no significant correlation was evident between peak muscle soreness and peak creatine kinase activity, or peak creatine kinase activity and peak PGE2 concentration. All groups showed no changes in IL-1β, IL-6 or TNF-α. Our results suggest that the intensity of bench press exercise does not affect the magnitude of muscle soreness and blood markers of muscle damage and inflammation.

Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure : role of exercise training

Sympathetic hyperactivity (SH) is a hallmark of heart failure (HF), and several lines of evidence suggest that SH contributes to HF-induced skeletal myopathy. However, little is known about the influence of SH on skeletal muscle morphology and metabolism in a setting of developing HF, taking into consideration muscles with different fiber compositions. The contribution of SH on exercise tolerance and skeletal muscle morphology and biochemistry was investigated in 3- and 7-mo-old mice lacking both alpha(2A)- and alpha(2C)-adrenergic receptor subtypes (alpha(2A)/alpha(2C)ARKO mice) that present SH with evidence of HF by 7 mo. To verify whether exercise training (ET) would prevent skeletal muscle myopathy in advanced-stage HF, alpha(2A)/alpha(2C)ARKO mice were exercised from 5 to 7 mo of age. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF and preserved exercise tolerance and muscular norepinephrine with no changes in soleus morphology. In contrast, plantaris muscle of alpha(2A)/alpha(2C)ARKO mice displayed hypertrophy and fiber type shift (IIA --> IIX) paralleled by capillary rarefaction, increased hexokinase activity, and oxidative stress. At 7 mo, alpha(2A)/alpha(2C)ARKO mice displayed exercise intolerance and increased muscular norepinephrine, muscular atrophy, capillary rarefaction, and increased oxidative stress. ET reestablished alpha(2A)/alpha(2C)ARKO mouse exercise tolerance to 7-mo-old wild-type levels and prevented muscular atrophy and capillary rarefaction associated with reduced oxidative stress. Collectively, these data provide direct evidence that SH is a major factor contributing to skeletal muscle morphological changes in a setting of developing HF. ET prevented skeletal muscle myopathy in alpha(2A)/alpha(2C)ARKO mice, which highlights its importance as a therapeutic tool for HF.

Resistance Training with Vascular Occlusion in Inclusion Body Myositis: A Case Study

UNLABELLED Inclusion body myositis (IBM) is a rare idiopathic inflammatory myopathy that produces remarkable muscle weakness. Resistance training with vascular occlusion has been shown to improve muscle strength and cross-sectional area in other muscle wasting conditions. PURPOSE We evaluated the efficacy of a moderate-intensity resistance training program combined with vascular occlusion by examining functional capacity, muscle morphology, and changes in the expression of genes related to muscle protein synthesis and proteolysis in a patient with IBM. METHODS A 65-yr-old man with IBM resistant to all proposed treatments underwent resistance training with vascular occlusion for 12 wk. Leg press one-repetition maximum; thigh cross-sectional area; balance, mobility, and muscle function; quality of life; and blood markers of inflammation and muscle damage were assessed at baseline and after the 12-wk program. The messenger RNA (mRNA) expression levels of mechanogrowth factor, mammalian target of rapamycin, atrogin-1, and muscle RING finger-1 were also quantified. RESULTS After the 12-wk training program, the patients leg press one-repetition maximum, balance and mobility function, and thigh cross-sectional area increased 15.9%, 60%, and 4.7%, respectively. All Short Form-36 Health Survey Questionnaire subscales demonstrated improvements as well, varying from 18% to 600%. mRNA expression of mechanogrowth factor increased 3.97-fold, whereas that of atrogin-1 decreased 0.62-fold. Muscle RING finger-1 and mammalian target of rapamycin mRNA levels were only slightly altered, 1.18- and 1.28-fold, respectively. Importantly, the exercise did not induce disease flare. CONCLUSIONS We describe a novel, and likely the first, nonpharmacological therapeutic tool that might be able to counteract the muscle atrophy and the declining strength that usually occur in IBM.

MuRF1 is a muscle fiber-type II associated factor and together with MuRF2 regulates type-II fiber trophicity and maintenance.

MuRF1 is a member of the RBCC (RING, B-box, coiled-coil) superfamily that has been proposed to act as an atrogin during muscle wasting. Here, we show that MuRF1 is preferentially induced in type-II muscle fibers after denervation. Fourteen days after denervation, MuRF1 protein was further elevated but remained preferentially expressed in type-II muscle fibers. Consistent with a fiber-type dependent function of MuRF1, the tibialis anterior muscle (rich in type-II muscle fibers) was considerably more protected in MuRF1-KO mice from muscle wasting when compared to soleus muscle with mixed fiber-types. We also determined fiber-type distributions in MuRF1/MuRF2 double-deficient KO (dKO) mice, because MuRF2 is a close homolog of MuRF1. MuRF1/MuRF2 dKO mice showed a profound loss of type-II fibers in soleus muscle. As a potential mechanism we identified the interaction of MuRF1/MuRF2 with myozenin-1, a calcineurin/NFAT regulator and a factor required for maintenance of type-II muscle fibers. MuRF1/MuRF2 dKO mice had lost myozenin-1 expression in tibialis anterior muscle, implicating MuRF1/MuRF2 as regulators of the calcineurin/NFAT pathway. In summary, our data suggest that expression of MuRF1 is required for remodeling of type-II fibers under pathophysiological stress states, whereas MuRF1 and MuRF2 together are required for maintenance of type-II fibers, possibly via the regulation of myozenin-1.

Insulin resistance of pregnancy involves estrogen-induced repression of muscle GLUT4.

Pregnancy is accompanied by hyperestrogenism, however, the role of estrogens in the gestational-induced insulin resistance is unknown. Skeletal muscle plays a fundamental role in this resistance, where GLUT4 regulates glucose uptake. We investigated: (1) effects of oophorectomy and estradiol (E2) on insulin sensitivity and GLUT4 expression. E2 ( approximately 200nM) for 7 days decreased sensitivity, reducing approximately 30% GLUT4 mRNA and protein (P<0.05) and plasma membrane expression in muscle; (2) the expression of ERalpha and ERbeta in L6 myotubes, showing that both coexpress in the same nucleus; (3) effects of E2 on GLUT4 in L6, showing a time- and dose-dependent response. High concentration (100nM) for 6 days reduced approximately 25% GLUT4 mRNA and protein (P<0.05). Concluding, E2 regulates GLUT4 in muscle, and at high concentrations, such as in pregnancy, reduces GLUT4 expression and, in vivo, decreases insulin sensitivity. Thus, hyperestrogenism may be involved in the pregnancy-induced insulin resistance and/or gestational diabetes.

The Thyroid Hormone Receptor β‐Specific Agonist GC‐1 Selectively Affects the Bone Development of Hypothyroid Rats

We investigated the effects of GC‐1, a TRβ‐selective thyromimetic, on bone development of hypothyroid rats. Whereas T3 reverted the IGF‐I deficiency and the skeletal defects caused by hypothyroidism, GC‐1 had no effect on serum IGF‐I or on IGF‐I protein expression in the epiphyseal growth plate of the femur, but induced selective effects on bone development. Our findings indicate that T3 exerts some essential effects on bone development that are mediated by TRβ1.

Thyroid hormone receptor-β-selective agonist GC-24 spares skeletal muscle type I to II fiber shift

Triiodothyronine (T3) is known to play a key role in the function of several tissues/organs via the thyroid hormone receptor isoforms alpha (TRα) and beta (TRβ). We have investigated the effects of GC-24, a novel synthetic TRβ-selective compound, on skeletal muscle fiber-type determination, cross-sectional area, and gene expression in rat skeletal muscles. For fiber typing, cross sections of soleus and extensor digitorum longus (EDL) muscles were stained for myosin ATPase activity at various pHs. Serum T3, T4, and cholesterol levels were also determined. Analysis of highly T3-responsive genes, viz., myosin heavy chain IIa (MHCIIa) and sarcoendoplasmic reticulum adenosine triphosphatase (SERCA1), was performed by quantitative real-time polymerase chain reaction. Equimolar doses of T3 and GC-24 had a similar cholesterol-lowering effect. T3, but not GC-24, decreased fiber type I and increased fiber type II abundance in soleus and EDL muscles. Conversely, in EDL, both T3 and GC-24 decreased the mean cross-sectional area of type I fibers. MHCIIa gene expression was reduced (approximately 50%) by T3 and unchanged by GC-24. SERCA1 gene expression was strongly induced by T3 (approximately 20-fold) and mildly induced by GC-24 (approximately two-fold). These results show that GC-24 does not significantly alter the composition of skeletal muscle fiber type and further strengthens the putative use of GC compounds as therapeutic agents.

A TRβ-selective agonist confers resistance to diet-induced obesity

Thyroid hormone receptor beta (TRbeta also listed as THRB on the MGI Database)-selective agonists activate brown adipose tissue (BAT) thermogenesis, while only minimally affecting cardiac activity or lean body mass. Here, we tested the hypothesis that daily administration of the TRbeta agonist GC-24 prevents the metabolic alterations associated with a hypercaloric diet. Rats were placed on a high-fat diet and after a month exhibited increased body weight (BW) and adiposity, fasting hyperglycemia and glucose intolerance, increased plasma levels of triglycerides, cholesterol, nonesterified fatty acids and interleukin-6. While GC-24 administration to these animals did not affect food ingestion or modified the progression of BW gain, it did increase energy expenditure, eliminating the increase in adiposity without causing cardiac hypertrophy. Fasting hyperglycemia remained unchanged, but treatment with GC-24 improved glucose tolerance by increasing insulin sensitivity, and also normalized plasma triglyceride levels. Plasma cholesterol levels were only partially normalized and liver cholesterol content remained high in the GC-24-treated animals. Gene expression in liver, skeletal muscle, and white adipose tissue was only minimally affected by treatment with GC-24, with the main target being BAT. In conclusion, during high-fat feeding treatment with the TRbeta-selective agonist, GC-24 only partially improves metabolic control probably as a result of accelerating the resting metabolic rate.