Differential morphofunctional characteristics and gene expression in fast and slow muscle of rats with monocrotaline-induced heart failure

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

Remodeling of calcium handling in skeletal muscle through PGC-1?: impact on force, fatigability, and fiber type

Regular endurance exercise remodels skeletal muscle, largely through the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). PGC-1α promotes fiber type switching and resistance to fatigue. Intracellular calcium levels might play a role in both adaptive phenomena, yet a role for PGC-1α in the adaptation of calcium handling in skeletal muscle remains unknown. Using mice with transgenic overexpression of PGC-1α, we now investigated the effect of PGC-1α on calcium handling in skeletal muscle. We demonstrate that PGC-1α induces a quantitative reduction in calcium release from the sarcoplasmic reticulum by diminishing the expression of calcium-releasing molecules. Concomitantly, maximal muscle force is reduced in vivo and ex vivo. In addition, PGC-1α overexpression delays calcium clearance from the myoplasm by interfering with multiple mechanisms involved in calcium removal, leading to higher myoplasmic calcium levels following contraction. During prolonged muscle activity, the delayed calcium clearance might facilitate force production in mice overexpressing PGC-1α. Our results reveal a novel role of PGC-1α in altering the contractile properties of skeletal muscle by modulating calcium handling. Importantly, our findings indicate PGC-1α to be both down- as well as upstream of calcium signaling in this tissue. Overall, our findings suggest that in the adaptation to chronic exercise, PGC-1α reduces maximal force, increases resistance to fatigue, and drives fiber type switching partly through remodeling of calcium transients, in addition to promoting slow-type myofibrillar protein expression and adequate energy supply.

Phenotypic conversion leads to structural and functional changes of smooth muscle sarcolemma

Continuous changes in the length of smooth muscles require a highly organized sarcolemmal structure. Yet, smooth muscle cells also adapt rapidly to altered environmental cues. Their sarcolemmal plasticity must lead to profound changes which affect transmembrane signal transduction as well as contractility. We have established porcine vascular and human visceral smooth muscle cultures of epithelioid and spindle-shaped morphology and determined their plasma membrane properties. Epithelioid cells from both sources contain a higher ratio of cholesterol to glycerophospholipids, and express a less diverse range of lipid-associated annexins. These findings point to a reduction in efficiency of membrane segregation in epithelioid cells. Moreover, compared to spindle-shaped cells, cholesterol is more readily extracted from epithelioid cells with methyl-beta-cyclodextrin and its synthesis is more susceptible to inhibition with lovastatin. The inability of epithelioid cells to process vasoactive metabolites, such as angiotensin or nucleotides further indicates that contractile properties are impaired. Phenotypic plasticity extends beyond the loss of smooth muscle cell marker genes. The plasma membrane has undergone profound functional changes which are incompatible with cyclic foreshortening, but might be important in the development of vascular disease.

Myostatin dysfunction is associated with reduction in overload induced hypertrophy of soleus muscle in mice

Acknowledgements This project was also supported by Marie Curie International Reintegration Grant 249156 (A. Lionikas) and the grants VP1-3.1-SMM-01-V-02-003 (A. Kilikevicius) and MIP-067/2012 (T. Venckunas) from the Research Council of Lithuania as well as the grant from the Ministry of Higher Education of Saudi Arabia (Y. Alhind). We wish also to thank Mrs Indre Libnickiene for her excellent technical assistance provided during the project

Interindividual variation of isolated muscle performance and fibre-type composition in the toad Bufo viridus

Interindividual analyses of physiological performance represent one of the most powerful tools for identifying functional positive and negative linkages between various performance traits. In this study we investigated functional linkages in the whole-gastrocnemius performance of juvenile Bufo viridis by examining interindividual variation in in vitro muscle performance and muscle fibre-type composition. We used the work-loop technique to investigate the maximum in vitro power output and fatigue resistance of the gastrocnemius muscle during repeated sets of three cycles at the cycle frequency of 5 Hz, simulating an intermittent style of locomotion. We found several significant correlations between different measures of in vitro muscle performance, including a negative correlation between maximum net power output and fatigue resistance of power, indicating functional trade-offs between these performance traits. We also investigated the extent of individual variation in the proportions of different fibre types, and tested for correlations between individual variation in muscle fibre-type composition and the previously measured isolated muscle performance. Fast glycolytic fibres represented 84.0+/-3.4% of the muscle, while the combined slow oxidative and fast oxidative-glycolytic fibres represented 16+/-3.4%. We found no significant correlations between measures of in vitro muscle performance and the proportion of different fibre types in the gastrocnemius muscle. However, despite this lack of correlation between whole-muscle performance and muscle fibre-type composition data, we suggest the functional linkages detected between different measures of in vitro muscular performance have important ecological and evolutionary consequences.

Reduced inspiratory muscle endurance following successful weaning from prolonged mechanical ventilation

Study objectives: Respiratory muscle weakness and decreased endurance have been demonstrated following mechanical ventilation. However, its relationship to the duration of mechanical ventilation is not known. The aim of this study was to assess respiratory muscle endurance and its relationship to the duration of mechanical ventilation. Design: Prospective study. Setting: Tertiary teaching hospital ICU. Patients: Twenty subjects were recruited for the study who had received mechanical ventilation for a 48 h and had been discharged from the ICU. Measurements: FEV1 FVC, and maximal inspiratory pressure (Pimax) at functional residual capacity were recorded. The Pimax attained following resisted inspiration at 30% of the initial Pimax for 2 min was recorded, and the fatigue resistance index (FRI) [Pimax final/Pimax initial] was calculated. The duration of ICU length of stay (ICULOS), duration of mechanical ventilation (MVD), duration of weaning (WD), and Charlson comorbidities score (CCS) were also recorded. Relationships between fatigue and other parameters were analyzed using the Spearman correlations (p). Results: Subjects were admitted to the ICU for a mean duration of 7.7 days (SD, 3.7 days) and required mechanical ventilation for a mean duration of 4.6 days (SD, 2.5 days). The mean FRI was 0.88 (SD, 0.13), indicating a 12% fall in Pimax, and was negatively correlated with MVD (r = -0.65; p = 0.007). No correlations were found between the FRI and FEV1, FVC, ICULOS, WD, or CCS. Conclusions: Patients who had received mechanical ventilation for > 48 h have reduced inspiratory muscle endurance that worsens with the duration of mechanical ventilation and is present following successful weaning. These data suggest that patients needing prolonged mechanical ventilation are at risk of respiratory muscle fatigue and may benefit from respiratory muscle training.

Physiological role of carnosine in contracting muscle

High-intensity exercise leads to reductions in muscle substrates (ATP, PCr, and glycogen) and a subsequent accumulation of metabolites (ADP, Pi, H+, and M2+) with a possible increase in free radical production. These factors independently and collectively have deleterious effects on muscle, with significant repercussions on high-intensity performance or training sessions. The effect of carnosine on overcoming muscle fatigue appears to be related to its ability to buffer the increased H+ concentration following high-intensity work. Carnosine, however, has other roles such as an antioxidant, a metal chelator, a Ca2+ and enzyme regulator, an inhibitor of protein glycosylation and protein-protein cross-linking. To date, only 1 study has investigated the effects of carnosine supplementation (not in pure form) on exercise performance in human subjects and found no improvement in repetitive high-intensity work. Much data has come from in vitro work on animal skeletal muscle fibers or other components of muscle contractile mechanisms. Thus further research needs to be carried out on humans to provide additional understanding on the effects of carnosine in vivo.

Myostatin dysfunction is associated with reduction in overload induced hypertrophy of soleus muscle in mice

Acknowledgements This project was also supported by Marie Curie International Reintegration Grant 249156 (A. Lionikas) and the grants VP1-3.1-SMM-01-V-02-003 (A. Kilikevicius) and MIP-067/2012 (T. Venckunas) from the Research Council of Lithuania as well as the grant from the Ministry of Higher Education of Saudi Arabia (Y. Alhind). We wish also to thank Mrs Indre Libnickiene for her excellent technical assistance provided during the project

Myostatin dysfunction is associated with reduction in overload induced hypertrophy of soleus muscle in mice

Acknowledgements This project was also supported by Marie Curie International Reintegration Grant 249156 (A. Lionikas) and the grants VP1-3.1-SMM-01-V-02-003 (A. Kilikevicius) and MIP-067/2012 (T. Venckunas) from the Research Council of Lithuania as well as the grant from the Ministry of Higher Education of Saudi Arabia (Y. Alhind). We wish also to thank Mrs Indre Libnickiene for her excellent technical assistance provided during the project

Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.

Biomechanical effects of immobilization and rehabilitation on the skeletal muscle of trained and sedentary rats

Because of the scarcity of information about the comparison of training to sedentarism beforehand immobilization and rehabilitation through muscle mechanical properties, the present work investigates this theme. Seventy rats were divided into 7 groups: 1-control (C); 2-trained (T); 3-sedentary (S); 4-trained and immobilized (TI); 5-sedentary and immobilized (SI); 6-trained, immobilized and rehabilitated (TIR); 7-sedentary, immobilized and rehabilitated (SIR). Interventions: Swimming training; Sedentarism (reduced size cages); Cast immobilization (pelvic limb) and water rehabilitation. Load at the limit of proportionality (LLP), maximum limit load (MLL) and stiffness (St) were the mechanical properties determined after a mechanical test of traction of the gastrocnemius. The training improved all mechanical properties when compared to sedentarism. After immobilization, LLP and MLL were reduced in TI and SI. However, there was no difference in St between C and TI. Additionally, TI showed improved MLL when compared to SI. The comparison of TI and TIR showed significant melioration in all properties after remobilization. SIR showed an improvement only in MLL when compared to SI. Significant melioration in LLP and St was observed in TIR compared to SIR. We demonstrated that the training before immobilization and rehabilitation had a positive effect on the muscle mechanical behavior compared to sedentarism. This analysis is of fundamental importance because it helps characterize the muscle tissue under different functional demands.

Long-term metabolic and skeletal muscle adaptations to short-sprint training: Implications for sprint training and tapering

The adaptations of muscle to sprint training can be separated into metabolic and morphological changes. Enzyme adaptations represent a major metabolic adaptation to sprint training, with the enzymes of all three energy systems showing signs of adaptation to training and some evidence of a return to baseline levels with detraining. Myokinase and creatine phosphokinase have shown small increases as a result of short-sprint training in some studies and elite sprinters appear better able to rapidly breakdown phosphocreatine (PCr) than the sub-elite. No changes in these enzyme levels have been reported as a result of detraining. Similarly, glycolytic enzyme activity (notably lactate dehydrogenase, phosphofructokinase and glycogen phosphorylase) has been shown to increase after training consisting of either long (> 10-second) or short (< 10-second) sprints. Evidence suggests that these enzymes return to pre-training levels after somewhere between 7 weeks and 6 months of detraining. Mitochondrial enzyme activity also increases after sprint training, particularly when long sprints or short recovery between short sprints are used as the training stimulus. Morphological adaptations to sprint training include changes in muscle fibre type, sarcoplasmic reticulum, and fibre cross-sectional area. An appropriate sprint training programme could be expected to induce a shift toward type Ha muscle, increase muscle cross-sectional area and increase the sarcoplasmic reticulum volume to aid release of Ca2+. Training volume and/or frequency of sprint training in excess of what is optimal for an individual, however, will induce a shift toward slower muscle contractile characteristics. In contrast, detraining appears to shift the contractile characteristics towards type IIb, although muscle atrophy is also likely to occur. Muscle conduction velocity appears to be a potential non-invasive method of monitoring contractile changes in response to sprint training and detraining. In summary, adaptation to sprint training is clearly dependent on the duration of sprinting, recovery between repetitions, total volume and frequency of training bouts. These variables have profound effects on the metabolic, structural and performance adaptations from a sprint-training programme and these changes take a considerable period of time to return to baseline after a period of detraining. However, the complexity of the interaction between the aforementioned variables and training adaptation combined with individual differences is clearly disruptive to the transfer of knowledge and advice from laboratory to coach to athlete.

Locomotion at-1.0 degrees C: burst swimming performance of five species of Antarctic fish

We investigated the burst swimming performance of five species of Antarctic fish at -1.0degreesC. The species studied belonged to the suborder, Notothenioidei, and from the families, Nototheniidae and Bathydraconidae. Swimming performance of the fish was assessed over the initial 300 ms of a startle response using surgically attached miniature accelerometers. Escape responses in all fish consisted of a C-type fast start; consisting of an initial pronounced bending of the body into a C-shape, followed by one or more complete tail-beats and an un-powered glide. We found significant differences in the swimming performance of the five species of fish examined, with average maximum swimming velocities (U-max) ranging from 0.91 to 1.39 m s(-1) and maximum accelerations (A(max)) ranging from 10.6 to 15.6 m s(-2). The cryopelagic species, Pagothenia borchgrevinki, produced the fastest escape response, reaching a U-max and A(max) of 1.39 m s(-1) and 15.6 m s(-2), respectively. We also compared the body shapes of each fish species with their measures of maximum burst performance. The dragonfish, Gymnodraco acuticeps, from the family Bathdraconidae, did not conform to the pattern observed for the other four fish species belonging to the family Nototheniidae. However, we found a negative relationship between buoyancy of the fish species and burst swimming performance. (C) 2002 Elsevier Science Ltd. All rights reserved.

Mdx myotubes have normal excitability but show reduced contraction-relaxation dynamics.

The pathogenesis of Duchenne muscular dystrophy (DMD), characterised by lack of the cytoskeletal protein dystrophin, is not completely understood. An early event in the degenerative process of DMD muscle could be a rise in cytosolic calcium concentration. In order to investigate whether this leads to alterations of contractile behaviour, we studied the excitability and contractile properties of cultured myotubes from control (C57BL/10) and mdx mice, an animal model for DMD. The myotubes were stimulated electrically and their motion was recorded photometrically. No significant differences were found between control and mdx myotubes with respect to the following parameters: chronaxy and rheobase (0.33 +/- 0.03 ms and 23 +/- 4 V vs. 0.39 +/- 0.07 ms and 22 +/- 2 V for C57 and mdx myotubes, respectively), tetanisation frequency (a similar distribution pattern was found between 5 and 30 Hz), fatigue during tetanus (found in 35% of both types of myotubes) and post-tetanic contracture. In contrast, contraction and relaxation times were longer (P < 0.005) in mdx (36 +/- 2 and 142 +/- 13 ms, respectively) than in control myotubes (26 +/- 1 and 85 +/- 9 ms, respectively). Together with our earlier findings, these results suggest a decreased capacity for calcium removal in mdx cells leading, in particular, to alterations of muscle relaxation.

Double gene deletion reveals the lack of cooperation between PPARalpha and PPARbeta in skeletal muscle.

The peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of most of the pathways linked to lipid metabolism. PPARalpha and PPARbeta isotypes are known to regulate muscle fatty acid oxidation and a reciprocal compensation of their function has been proposed. Herein, we investigated muscle contractile and metabolic phenotypes in PPARalpha-/-, PPARbeta-/-, and double PPARalpha-/- beta-/- mice. Heart and soleus muscle analyses show that the deletion of PPARalpha induces a decrease of the HAD activity (beta-oxidation) while soleus contractile phenotype remains unchanged. A PPARbeta deletion alone has no effect. However, these mild phenotypes are not due to a reciprocal compensation of PPARbeta and PPARalpha functions since double gene deletion PPARalpha-PPARbeta mostly reproduces the null PPARalpha-mediated reduced beta-oxidation, in addition to a shift from fast to slow fibers. In conclusion, PPARbeta is not required for maintaining skeletal muscle metabolic activity and does not compensate the lack of PPARalpha in PPARalpha null mice.