Aerobic exercise training upregulates skeletal muscle calpain and ubiquitin-proteasome systems in healthy mice

Cunha TF, Moreira JB, Paixao NA, Campos JC, Monteiro AW, Bacurau AV, Bueno CR Jr., Ferreira JC, Brum PC. Aerobic exercise training upregulates skeletal muscle calpain and ubiquitin-proteasome systems in healthy mice. J Appl Physiol 112: 1839-1846, 2012. First published March 29, 2012; doi:10.1152/japplphysiol.00346.2011.-Aerobic exercise training (AET) is an important mechanical stimulus that modulates skeletal muscle protein turnover, leading to structural rearrangement. Since the ubiquitin-proteasome system (UPS) and calpain system are major proteolytic pathways involved in protein turnover, we aimed to investigate the effects of intensity-controlled AET on the skeletal muscle UPS and calpain system and their association to training-induced structural adaptations. Long-lasting effects of AET were studied in C57BL/6J mice after 2 or 8 wk of AET. Plantaris cross-sectional area (CSA) and capillarization were assessed by myosin ATPase staining. mRNA and protein expression levels of main components of the UPS and calpain system were evaluated in plantaris by real-time PCR and Western immunoblotting, respectively. No proteolytic system activation was observed after 2 wk of AET. Eight weeks of AET resulted in improved running capacity, plantaris capillarization, and CSA. Muscle RING finger-1 mRNA expression was increased in 8-wk-trained mice. Accordingly, elevated 26S proteasome activity was observed in the 8-wk-trained group, without accumulation of ubiquitinated or carbonylated proteins. In addition, calpain abundance was increased by 8 wk of AET, whereas no difference was observed in its endogenous inhibitor calpastatin. Taken together, our findings indicate that skeletal muscle enhancements, as evidenced by increased running capacity, plantaris capillarization, and CSA, occurred in spite of the upregulated UPS and calpain system, suggesting that overactivation of skeletal muscle proteolytic systems is not restricted to atrophying states. Our data provide evidence for the contribution of the UPS and calpain system to metabolic turnover of myofibrillar proteins and skeletal muscle adaptations to AET.

Capillary growth, ultrastructure remodelling and exercise training in skeletal muscle of essential hypertensive patients

AIM The aim was to elucidate whether essential hypertension is associated with altered capillary morphology and density and to what extent exercise training can normalize these parameters. METHODS To investigate angiogenesis and capillary morphology in essential hypertension, muscle biopsies were obtained from m. vastus lateralis in subjects with essential hypertension (n = 10) and normotensive controls (n = 11) before and after 8 weeks of aerobic exercise training. Morphometry was performed after transmission electron microscopy, and protein levels of several angioregulatory factors were determined. RESULTS At baseline, capillary density and capillary-to-fibre ratio were not different between the two groups. However, the hypertensive subjects had 9% lower capillary area (12.7 ± 0.4 vs. 13.9 ± 0.2 μm(2)) and tended to have thicker capillary basement membranes (399 ± 16 vs. 358 ± 13 nm; P = 0.094) than controls. Protein expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 and thrombospondin-1 were similar in normotensive and hypertensive subjects, but tissue inhibitor of matrix metalloproteinase was 69% lower in the hypertensive group. After training, angiogenesis was evident by 15% increased capillary-to-fibre ratio in the hypertensive subjects only. Capillary area and capillary lumen area were increased by 7 and 15% in the hypertensive patients, whereas capillary basement membrane thickness was decreased by 17% (P < 0.05). VEGF expression after training was increased in both groups, whereas VEGF receptor-2 was decreased by 25% in the hypertensive patients(P < 0.05). CONCLUSION Essential hypertension is associated with decreased lumen area and a tendency for increased basement membrane thickening in capillaries of skeletal muscle. Exercise training may improve the diffusion conditions in essential hypertension by altering capillary structure and capillary number.

Effect of exercise training on skeletal muscle cytokine expression in the elderly

Aging is associated with increased circulating pro-inflammatory and lower anti-inflammatory cytokines. Exercise training, in addition to improving muscle function, reduces these circulating pro-inflammatory cytokines. Yet, few studies have evaluated changes in the expression of cytokines within skeletal muscle after exercise training. The aim of the current study was to examine the expression of cytokines both at rest and following a bout of isokinetic exercise performed before and after 12 weeks of resistance exercise training in young (n = 8, 20.3 ± 0.8 yr) and elderly men (n = 8, 66.9 ± 1.6 yr). Protein expression of various cytokines was determined in muscle homogenates. The expression of MCP-1, IL-8 and IL-6 (which are traditionally classified as ‘pro-inflammatory’) increased substantially after acute exercise. By contrast, the expression of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 increased only slightly (or not at all) after acute exercise. These responses were not significantly different between young and elderly men, either before or after 12 weeks of exercise training. However, compared with the young men, the expression of pro-inflammatory cytokines 2 h post exercise tended to be greater in the elderly men prior to training. Training attenuated this difference. These data suggest that the inflammatory response to unaccustomed exercise increases with age. Furthermore, regular exercise training may help to normalize this inflammatory response, which could have important implications for muscle regeneration and adaptation in the elderly.

Exercise, diet and skeletal muscle gene expression

Skeletal muscle, as a consequence of its mass and great capacity for altered metabolism, has a major impact on whole-body metabolic homeostasis and is capable of remarkable adaptation in response to various physiological stimuli, including exercise and dietary intervention. Exercise-induced increases in skeletal muscle mRNA levels of a number of genes have been reported, due to transcriptional activation and/or increased mRNA stability. The cellular adaptations to exercise training appear to be due to the cumulative effects of transient increases in gene transcription after repeated exercise bouts. The relative importance of transcriptional (mRNA synthesis) and translational (mRNA stability or translational efficiency) mechanisms for the training-induced increases in skeletal muscle protein abundance remains to be fully elucidated. Dietary manipulation, and the associated alterations in nutrient availability and hormone levels, can also modify skeletal muscle gene expression, although fewer studies have been reported. A major challenge is to understand how exercise and diet exert their effects on gene and protein expression in skeletal muscle. In relation to exercise, potential stimuli include stretch and muscle tension, the pattern of motor nerve activity and the resultant calcium transients, the energy charge of the cell and substrate availability, oxygen tension and circulating hormones. These are detected by various cellular signaling mechanisms, acting on a range of downstream targets and a wide range of putative transcription factors. A key goal in the years ahead is to identify how alterations at the level of gene expression are coupled to the changes in skeletal muscle phenotype. It is clear that gene expression, although representing a specific site of regulation, is only one step in a complex cascade from the initial stimulus to the final phenotypic adaptation and integrated physiological response.

Lack of beta(2)-AR improves exercise capacity and skeletal muscle oxidative phenotype in mice

beta(2)-adrenergic receptor (beta(2)-AR) agonists have been used as ergogenics by athletes involved in training for strength and power in order to increase the muscle mass. Even though anabolic effects of beta(2)-AR activation are highly recognized, less is known about the impact of beta(2)-AR in endurance capacity. We presently used mice lacking beta(2)-AR [beta(2)-knockout (beta(2) KO)] to investigate the role of beta(2)-AR on exercise capacity and skeletal muscle metabolism and phenotype. beta(2) KO mice and their wild-type controls (WT) were studied. Exercise tolerance, skeletal muscle fiber typing, capillary-to-fiber ratio, citrate synthase activity and glycogen content were evaluated. When compared with WT, beta 2KO mice displayed increased exercise capacity (61%) associated with higher percentage of oxidative fibers (21% and 129% of increase in soleus and plantaris muscles, respectively) and capillarity (31% and 20% of increase in soleus and plantaris muscles, respectively). In addition, beta 2KO mice presented increased skeletal muscle citrate synthase activity (10%) and succinate dehydrogenase staining. Likewise, glycogen content (53%) and periodic acid-Schiff staining (glycogen staining) were also increased in beta 2KO skeletal muscle. Altogether, these data provide evidence that disruption of beta(2)AR improves oxidative metabolism in skeletal muscle of beta 2KO mice and this is associated with increased exercise capacity.

Exercise training prevents hyperinsulinemia, muscular glycogen loss and muscle atrophy induced by dexamethasone treatment

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Short-term intensified cycle training alters acute and chronic responses of PGC1α and cytochrome C oxidase IV to exercise in human skeletal muscle

Reduced activation of exercise responsive signalling pathways have been reported in response to acute exercise after training; however little is known about the adaptive responses of the mitochondria. Accordingly, we investigated changes in mitochondrial gene expression and protein abundance in response to the same acute exercise before and after 10-d of intensive cycle training.

Nine untrained, healthy participants (mean±SD; VO2peak 44.1±17.6 ml/kg/min) performed a 60 min bout of cycling exercise at 164±18 W (72% of pre-training VO2peak). Muscle biopsies were obtained from the vastus lateralis muscle at rest, immediately and 3 h after exercise. The participants then underwent 10-d of cycle training which included four high-intensity interval training sessions (6×5 min; 90–100% VO2peak) and six prolonged moderate-intensity sessions (45–90 min; 75% VO2peak). Participants repeated the pre-training exercise trial at the same absolute work load (64% of pre-training VO2peak). Muscle PGC1-α mRNA expression was attenuated as it increased by 11- and 4- fold (P<0.001) after exercise pre- and post-training, respectively. PGC1-α protein expression increased 1.5 fold (P<0.05) in response to exercise pre-training with no further increases after the post-training exercise bout. RIP140 protein abundance was responsive to acute exercise only (P<0.01). COXIV mRNA (1.6 fold; P<0.01) and COXIV protein expression (1.5 fold; P<0.05) were increased by training but COXIV protein expression was decreased (20%; P<0.01) by acute exercise pre- and post-training.

These findings demonstrate that short-term intensified training promotes increased mitochondrial gene expression and protein abundance. Furthermore, acute indicators of exercise-induced mitochondrial adaptation appear to be blunted in response to exercise at the same absolute intensity following short-term training.

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

Bacurau AV, Jardim MA, Ferreira JC, Bechara LR, Bueno CR Jr, Alba-Loureiro TC, Negrao CE, Casarini DE, Curi R, Ramires PR, Moriscot AS, Brum PC. Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure: role of exercise training. J Appl Physiol 106: 1631-1640, 2009. First published January 29, 2009; doi:10.1152/japplphysiol.91067.2008.-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.

Exercise and skeletal muscle gene expression

1. Skeletal muscle is a complex and heterogenous tissue capable of remarkable adaptation in response to exercise training. The role of gene transcription, as an initial target to control protein synthesis, is poorly understood.
2. Mature myofibres contain several hundred nuclei, all of which maintain transcriptional competency, although the localized responsiveness of nuclei is not well known. Myofibres are capable of hypertrophy. These processes require the activation and myogenic differentiation of mononuclear satellite cells that fuse with the enlarging or repairing myofibre.
3. A single bout of exercise in human subjects is capable of activating the expression of many diverse groups of genes.
4. The impact of repeated exercise bouts, typical of exercise training, on gene expression has yet to receive systematic investigation.
5. The molecular programme elicited by resistance exercise and endurance exercise differs markedly. Muscular hypertrophy following resistance exercise is dependent on the activation of satellite cells and their subsequent myogenic maturation. Endurance exercise requires the simultaneous activation of mitochondrial and nuclear genes to enable mitochondrial biogenesis.
6. Future analysis of the regulation of genes by exercise may combine high-throughput technologies, such as gene-chips, enabling the rapid detection and analysis of changes in the expression of many thousands of genes.

Early signaling responses to divergent exercise stimuli in skeletal muscle from well-trained humans

Skeletal muscle from strength- and endurance-trained individuals represents diverse adaptive states. In this regard, AMPK-PGC-1α signaling mediates several adaptations to endurance training, while up-regulation of the Akt-TSC2-mTOR pathway may underlie increased protein synthesis after resistance exercise. We determined the effect of prior training history on signaling responses in seven strength-trained and six endurance-trained males who undertook 1 h cycling at 70% VO2peak or eight sets of five maximal repetitions of isokinetic leg extensions. Muscle biopsies were taken at rest, immediately and 3 h postexercise. AMPK phosphorylation increased after cycling in strength-trained (54%; P<0.05) but not endurance-trained subjects. Conversely, AMPK was elevated after resistance exercise in endurance- (114%; P<0.05), but not strengthtrained subjects. Akt phosphorylation increased in endurance- (50%; P<0.05), but not strengthtrained subjects after cycling but was unchanged in either group after resistance exercise. TSC2 phosphorylation was decreased (47%; P<0.05) in endurance-trained subjects following resistance exercise, but cycling had little effect on the phosphorylation state of this protein in either group. p70S6K phosphorylation increased in endurance- (118%; P<0.05), but not strength-trained subjects after resistance exercise, but was similar to rest in both groups after cycling. Similarly, phosphorylation of S6 protein, a substrate for p70 S6K, was increased immediately following resistance exercise in endurance- (129%; P<0.05), but not strength-trained subjects. In conclusion, a degree of “response plasticity” is conserved at opposite ends of the endurancehypertrophic adaptation continuum. Moreover, prior training attenuates the exercise specific signaling responses involved in single mode adaptations to training.

The effect of exercise on the skeletal muscle phospholipidome of rats fed a high-fat diet

The aim of this study was to examine the effect of endurance training on skeletal muscle phospholipid molecular species from high-fat fed rats. Twelve female Sprague-Dawley rats were fed a high-fat diet (78.1% energy). The rats were randomly divided into two groups, a sedentary control group and a trained group (125 min of treadmill running at 8 m/min, 4 days/wk for 4 weeks). Forty-eight hours after their last training bout phospholipids were extracted from the red and white vastus lateralis and analyzed by electrospray-ionization mass spectrometry. Exercise training was associated with significant alterations in the relative abundance of a number of phospholipid molecular species. These changes were more prominent in red vastus lateralis than white vastus lateralis. The largest observed change was an increase of similar to 30% in the abundance of 1-palmitoyl-2-linoleoyl phosphatidylcholine ions in oxidative fibers. Reductions in the relative abundance of a number of phospholipids containing long-chain n-3 polyunsaturated fatty acids were also observed. These data suggest a possible reduction in phospholipid remodeling in the trained animals. This results in a decrease in the phospholipid n-3 to n-6 ratio that may in turn influence endurance capacity.

Effect of Cluster Multi-Diode Light Emitting Diode Therapy (LEDT) on Exercise-Induced Skeletal Muscle Fatigue and Skeletal Muscle Recovery in Humans

Background and Objectives: There are some indications that low-level laser therapy (LLLT) may delay the development of skeletal muscle fatigue during high-intensity exercise. There have also been claims that LED cluster probes may be effective for this application however there are differences between LED and laser sources like spot size, spectral width, power output, etc. In this study we wanted to test if light emitting diode therapy (LEDT) can alter muscle performance, fatigue development and biochemical markers for skeletal muscle recovery in an experimental model of biceps humeri muscle contractions. Study Design/Materials and Methods: Ten male professional volleyball players (23.6 [SD +/- 5.6] years old) entered a randomized double-blinded placebo-controlled crossover trial. Active cluster LEDT (69 LEDs with wavelengths 660/850 nm, 10/30 mW, 30 seconds total irradiation time, 41.7J of total energy irradiated) or an identical placebo LEDT was delivered under double-blinded conditions to the middle of biceps humeri muscle immediately before exercise. All subjects performed voluntary biceps humeri contractions with a workload of 75% of their maximal voluntary contraction force (MVC) until exhaustion. Results: Active LEDT increased the number of biceps humeri contractions by 12.9% (38.60 [SD +/- 9.03] vs. 34.20 [SD +/- 8.68], P = 0.021) and extended the elapsed time to perform contractions by 11.6% (P = 0.036) versus placebo. In addition, post-exercise levels of biochemical markers decreased significantly with active LEDT: Blood Lactate (P = 0.042), Creatine Kinase (P = 0.035), and C-Reative Protein levels (P = 0.030), when compared to placebo LEDT. Conclusion: We conclude that this particular procedure and dose of LEDT immediately before exhaustive biceps humeri contractions, causes a slight delay in the development of skeletal muscle fatigue, decreases post-exercise blood lactate levels and inhibits the release of Creatine Kinase and C-Reative Protein. Lasers Surg. Med. 41:572-577, 2009. (C) 2009 Wiley-Liss, Inc.

Exercise and skeletal muscle glucose transporter 4 expression: molecular mechanisms

1.      Skeletal muscle is a highly plastic tissue that has a remarkable ability to adapt to external demands, such as exercise. Many of these adaptations can be explained by changes in skeletal muscle gene expression. A single bout of exercise is sufficient to induce the expression of some metabolic genes. We have focused our attention on the regulation of glucose transporter isoform 4 (GLUT-4) expression in human skeletal muscle.

2.      Glucose transporter isoform 4 gene expression is increased immediately following a single bout of exercise, and the GLUT-4 enhancer factor (GEF) and myocyte enhancer factor 2 (MEF2) transcription factors are required for this response. Glucose transporter isoform enhancer factor and MEF2 DNA binding activities are increased following exercise, and the molecular mechanisms regulating MEF2 in exercising human skeletal muscle have also been examined.

3.      These studies find possible roles for histone deacetylase 5 (HDAC5), adenosine monophosphate–activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) and p38 mitogen-activated protein kinase (MAPK) in regulating MEF2 through a series of complex interactions potentially involving MEF2 repression, coactivation and phosphorylation.

4.      Given that MEF2 is a transcription factor required for many exercise responsive genes, it is possible that these mechanisms are responsible for regulating the expression of a variety of metabolic genes during exercise. These mechanisms could also provide targets for the treatment and management of metabolic disease states, such as obesity and type 2 diabetes, which are characterized by mitochondrial dysfunction and insulin resistance in skeletal muscle.

Exercise and Sports Science Australia position statement on exercise training and chronic heart failure

Chronic heart failure (CHF) is a complex syndrome characterised by progressive decline in left ventricular function, low exercise tolerance and raised mortality and morbidity. Regular exercise participation has been shown to be a safe and effective treatment modality in the majority of CHF patients, partially reversing some of the maladaptations evident in myocardial and skeletal muscle function, and resulting in improvements in physical fitness and quality of life, and perhaps reduced mortality. The volume and intensity of exercise that is recommended depends on the syndrome severity, however in most patients it should consist of a combination of low-to-moderate intensity aerobic (endurance) exercise on most days of the week and individually prescribed low-to-moderate intensity resistance (strength) training at least twice per week. Additionally, all patients should be closely monitored prior to and during exercise for contraindications by an appropriately trained health professional. The purpose of this statement is to inform and guide exercise practitioners and health professionals in the safe and effective prescription and supervision of exercise for patients with CHF.