EXPRESSION OF GENES RELATED TO MYOSTATIN SIGNALING DURING RAT SKELETAL MUSCLE LONGITUDINAL GROWTH

In this study we investigated the gene expression of proteins related to myostatin (MSTN) signaling during skeletal muscle longitudinal growth. To promote muscle growth, Wistar male rats were submitted to a stretching protocol for different durations (12, 24, 48, and 96 hours). Following this protocol, soleus weight and length and sarcomere number were determined. In addition, expression levels of the genes that encode MSTN, follistatin isoforms 288 and 315 (FLST288 and FLST315), follistatin-like 3 protein (FLST-L3), growth and differentiation factor-associated protein-1 (GASP-1), activin IIB receptor (ActIIB), and SMAD-7 were determined by real-time polymerase chain reaction. Prolonged stretching increased soleus weight, length, and sarcomere number. In addition, MSTN gene expression was increased at 12-24 hours, followed by a decrease at 96 hours when compared with baseline values. FLST isoforms, FLST-L3, and GASP-1 mRNA levels increased significantly over all time-points. ActIIB gene expression decreased quickly at 12-24 hours. SMAD-7 mRNA levels showed a late increase at 48 hours, which peaked at 96 hours. The gene expression pattern of inhibitory proteins related to MSTN signaling suggests a strong downregulation of this pathway in response to prolonged stretching. Muscle Nerve 40: 992-999, 2009

Tubular system volume changes in twitch fibres from toad and rat skeletal muscle assessed by confocal microscopy

The volume of the extracellular compartment (tubular system) within intact muscle fibres from cane toad and rat was measured under various conditions using confocal microscopy. Under physiological conditions at rest, the fractional volume of the tubular system (t-sys(Vol)) was 1.38 +/- 0.09% (n = 17),1.41 +/- 0.09% (n = 12) and 0.83 +/- 0.07% (n = 12) of the total fibre volume in the twitch fibres from toad iliofibularis muscle, rat extensor digitorum longus muscle and rat soleus muscle, respectively. In toad muscle fibres, the t-sys(Vol) decreased by 30% when the tubular system was fully depolarized and decreased by 15% when membrane cholesterol was depleted from the tubular system with methyl-beta-cyclodextrin but did not change as the sarcomere length was changed from 1.93 to 3.30 mum. There was also an increase by 30% and a decrease by 25% in t-sys(Vol) when toad fibres were equilibrated in solutions that were 2.5-fold hypertonic and 50% hypotonic, respectively. When the changes in total fibre volume were taken into consideration, the t-sys(Vol) expressed as a percentage of the isotonic fibre volume did actually decrease as tonicity increased, revealing that the tubular system in intact fibres cannot be compressed below 0.9% of the isotonic fibre volume. The results can be explained in terms of forces acting at the level of the tubular wall. These observations have important physiological implications showing that the tubular system is a dynamic membrane structure capable of changing its volume in response to the membrane potential, cholesterol depletion and osmotic stress but not when the sarcomere length is changed in resting muscle.

Osmotic properties of the sealed tubular system of toad and rat skeletal muscle

A method was developed that allows conversion of changes in maximum Ca2+-dependent fluorescence of a fixed amount of fluo-3 into volume changes of the fluo-3-containing solution. This method was then applied to investigate by confocal microscopy the osmotic properties of the sealed tubular (t-) system of toad and rat mechanically skinned fibers in which a certain amount Of fluo-3 was trapped. When the osmolality of the myoplasmic environment was altered by simple dilution or addition of sucrose within the range 190-638 mosmol kg(-1), the sealed t-system of toad fibers behaved almost like an ideal osmometer, changing its volume inverse proportionally to osmolality However, increasing the osmolality above 638 to 2,550 mosmol kg(-1) caused hardly any change in t-system volume. In myoplasmic solutions made hypotonic to 128 mosmol kg(-1), a loss of Ca2+ from the sealed t-system of toad fibers Occurred, presumably through either stretch-activated cationic channels or store-operated Ca2+ channels. In contrast to the behavior of the t-system in toad fibers, the volume of the sealed t-system of rat fibers changed little (by

Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo

Pires-Oliveira M, Maragno AL, Parreiras-E-Silva LT, Chiavegatti T, Gomes MD, Godinho RO. Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo. J Appl Physiol 108: 266-273, 2010. First published November 19, 2009; doi:10.1152/japplphysiol.00490.2009.-Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.

Use of alprostadil, a stable prostaglandin E(1) analogue, for the attenuation of rat skeletal muscle ischemia and reperfusion injury

Aim. Some stable prostaglandin analogues such as alprostadil have been used to attenuate the deleterious effects of ischemia and reperfusion injury. The aim of this paper was to test if alprostadil can decrease the ischemia- reperfusion injury in rat skeletal muscle using muscular enzymes as markers, such as aspartate aminotransferase (AST), creatine kinase (CPK), lactate dehydrogenase (LDH); degeneration products of cell membrane-malondialdehyde (MDA) and muscle glycogen storage. Methods. Thirty male Wistar rats were used in a model of hind limb ischemia achieved by infrarenal aortic cross-clamping. The animals were randomized into three equal groups (N=10) submitted to 5 hours of ischemia followed by one hour of reperfusion. The first group (control) received continuous intravenous infusion of saline solution and the second group (preischemia, GPI) received continuous intravenous infusion of alprostadil throughout the experiment starting 20 minutes before the aortic cross-clamping. The third group, prereperfusion (GPR), received alprostadil only during the reperfusion period, with intravenous infusion being started 10 min before the clamp release. Results. There was no difference in CPK, LDH, AST or tissue glycogen values between groups. However, a significant elevation in MDA was observed in the GPI and GPR groups compared to the control group, with no difference between the GPI and GPR. Conclusion. Under conditions of partial skeletal muscle ischemia, alprostadil did not reduce the release of muscular enzymes, the consumption of tissue glycogen or the effects of ischemia and reperfusion on the cell membrane, characterized by lipid peroxidation.

Regenerated rat skeletal muscle after periodic contusions

In the present study we evaluated the morphological aspect and changes in the area and incidence of muscle fiber types of long-term regenerated rat tibialis anterior (TA) muscle previously submitted to periodic contusions. Animals received eight consecutive traumas: one trauma per week, for eight weeks, and were evaluated one (N = 8) and four (N = 9) months after the last contusion. Serial cross-sections were evaluated by toluidine blue staining, acid phosphatase and myosin ATPase reactions. The weight of injured muscles was decreased compared to the contralateral intact one (one month: 0.77 ± 0.15 vs 0.91 ± 0.09 g, P = 0.03; four months: 0.79 ± 0.14 vs 1.02 ± 0.07 g, P = 0.0007, respectively) and showed abundant presence of split fibers and fibers with centralized nuclei, mainly in the deep portion. Damaged muscles presented a higher incidence of undifferentiated fibers when compared to the intact one (one month: 3.4 ± 2.1 vs 0.5 ± 0.3%, P = 0.006; four months: 2.3 ± 1.6 vs 0.3 ± 0.3%, P = 0.007, respectively). Injured TA evaluated one month later showed a decreased area of muscle fibers when compared to the intact one (P = 0.003). Thus, we conclude that: a) muscle fibers were damaged mainly in the deep portion, probably because they were compressed against the tibia; b) periodic contusions in the TA muscle did not change the percentage of type I and II muscle fibers; c) periodically injured TA muscles took four months to reach a muscle fiber area similar to that of the intact muscle.

Histomorphometric analysis of the response of rat skeletal muscle to swimming, immobilization and rehabilitation

The objective of the present study was to determine to what extent, if any, swimming training applied before immobilization in a cast interferes with the rehabilitation process in rat muscles. Female Wistar rats, mean weight 260.52 ± 16.26 g, were divided into 4 groups of 6 rats each: control, 6 weeks under baseline conditions; trained, swimming training for 6 weeks; trained-immobilized, swimming training for 6 weeks and then immobilized for 1 week; trained-immobilized-rehabilitated, swimming training for 6 weeks, immobilized for 1 week and then remobilized with swimming for 2 weeks. The animals were then sacrificed and the soleus and tibialis anterior muscles were dissected, frozen in liquid nitrogen and processed histochemically (H&E and mATPase). Data were analyzed statistically by the mixed effects linear model (P < 0.05). Cytoarchitectural changes such as degenerative characteristics in the immobilized group and regenerative characteristics such as centralized nucleus, fiber size variation and cell fragmentation in the groups submitted to swimming were more significant in the soleus muscle. The diameters of the lesser soleus type 1 and type 2A fibers were significantly reduced in the trained-immobilized group compared to the trained group (P < 0.001). In the tibialis anterior, there was an increase in the number of type 2B fibers and a reduction in type 2A fibers when trained-immobilized rats were compared to trained rats (P < 0.001). In trained-immobilized-rehabilitated rats, there was a reduction in type 2B fibers and an increase in type 2A fibers compared to trained-immobilized rats (P < 0.009). We concluded that swimming training did not minimize the deleterious effects of immobilization on the muscles studied and that remobilization did not favor tissue re-adaptation.

The acute effects of strength, endurance and concurrent exercises on the Akt/mTOR/p70S6K1 and AMPK signaling pathway responses in rat skeletal muscle

The activation of competing intracellular pathways has been proposed to explain the reduced training adaptations after concurrent strength and endurance exercises (CE). The present study investigated the acute effects of CE, strength exercises (SE), and endurance exercises (EE) on phosphorylated/total ratios of selected AMPK and Akt/mTOR/p70S6K1 pathway proteins in rats. Six animals per exercise group were killed immediately (0 h) and 2 h after each exercise mode. In addition, 6 animals in a non-exercised condition (NE) were killed on the same day and under the same conditions. The levels of AMPK, phospho-Thr172AMPK (p-AMPK), Akt, phospho-Ser473Akt (p-Akt), p70S6K1, phospho-Thr389-p70S6K1(p-p70S6K1), mTOR, phospho-Ser2448mTOR (p-mTOR), and phospho-Thr1462-TSC2 (p-TSC2) expression were evaluated by immunoblotting in total plantaris muscle extracts. The only significant difference detected was an increase (i.e., 87%) in Akt phosphorylated/total ratio in the CE group 2 h after exercise compared to the NE group (P = 0.002). There were no changes in AMPK, TSC2, mTOR, or p70S6K1 ratios when the exercise modes were compared to the NE condition (P ≥ 0.05). In conclusion, our data suggest that low-intensity and low-volume CE might not blunt the training-induced adaptations, since it did not activate competing intracellular pathways in an acute bout of strength and endurance exercises in rat skeletal muscle.

Glucose dynamics in rat skeletal muscle : recovery from osmotic stress

The purpose of this study was to examine cell glucose kinetics in rat skeletal muscle during iso-osmotic recovery from hyper- and hypo-osmotic stress. Rat EDL muscles were incubated for sixty minutes in either HYPO (190 mmol/kg), ISO (290 mmol/kg), or HYPER (400 mmol/kg) media (Sigma medium-199, 8 mM glucose) according to an established in vitro whole muscle model. In addition to sixty minute baseline measures in aniso-osmotic conditions, (HYPO-0 n=8; ISO- 0, n=S; HYPER-0, n=8), muscles were subjected to either one minute (HYPO-1 n=8; ISO-1, n=8; HYPER-1, n=8) or five minutes (HYPO-5 n=8; ISO-5, n=8; HYPER-5, n=8) of iso-osmotic recovery media and analyzed for metabolite content and glycogen synthase percent activation. To determine glucose uptake during iso-osmotic recovery, muscles (n=6 per group) were incubated for sixty minutes in either hypo-, iso-, or hyper-osmotic media immediately followed by five minutes of iso-osmotic media containing 3H-glucose and 14 C-mannitol. Increased relative water content/decreased [glucose] (observed in HYPO-0) and decreased water content/increased [glucose] (observed in HYPER-0) returned to ISO levels within 5 minutes of recovery. Glycogen synthase percent activation increased significantly in HYPO-5 over iso-osmotic controls. Glucose uptake measurements revealed no significant differences between groups. It was determined that [glucose] and muscle water content rapidly recovered from osmotic stress demonstrating skeletal muscle's resilience to osmotic stress.

Protein Turnover in Rat Skeletal Muscle During In Vitro Hypo-Osmotic Stress

Hypo-osmolality influences tissue metabolism, but research on protein turnover in skeletal muscle is limited. The purpose of this investigation was to examine the effects of hypo-osmotic stress on protein turnover in rat skeletal muscle. We hypothesized increased protein synthesis and reduced degradation following hypo-osmotic exposure. EDL muscles (n=8/group) were incubated in iso-osmotic (290 Osm/kg) or hypo-osmotic (190 Osm/kg) modified medium 199 (95% O2, 5% CO2, pH 7.4, 30±2 °C) for 60 min, followed by 75 min incubations with L-U[14C]phenylalanine or cycloheximide to determine protein synthesis and degradation. Immunoblotting was performed to assess signalling pathways involved. Phenylalanine uptake and incorporation were increased by 199% and 169% respectively in HYPO from ISO (p < 0.05). This was supported by elevated phosphorylation of mTOR Ser2448 (+12.5%) and increased Thr389 phosphorylation on p70s6 kinase (+23.6%) (p < 0.05). Hypo-osmotic stress increased protein synthesis and potentially amino acid uptake. Future studies should examine the upstream mechanisms involved.

Changes in mitochondrial PLIN3 and PLIN5 protein content in rat skeletal muscle following acute contraction and endurance training

Surrounding lipid droplets in skeletal muscle are the perilipin (PLIN2-5) family of proteins, regulating lipid droplet metabolism. During exercise lipid droplets provide fatty acids to the mitochondria for oxidation while increasing their proximity to each other. Whether PLIN3 and PLIN5 associate with mitochondria following contraction has not been examined. To determine whether contraction altered mitochondrial PLIN3 and PLIN5 content, sedentary and endurance trained rats underwent acute contraction. The main outcomes are; 1) mitochondrial PLIN3 content is unaltered while mitochondrial PLIN5 content is increased following an acute contraction 2) mitochondrial PLIN3 content is higher in endurance trained rats when compared to sedentary and mitochondrial PLIN5 content is similar in both conditions 3) only PLIN5 mitochondrial content is increased similarly in both groups following acute contraction. This work highlights the dynamics of these two PLIN proteins, which may have roles not only on the lipid droplet but also on the mitochondria.

Chronic low frequency/low volume resistance training reduces pro-inflammatory cytokine protein levels and TLR4 mRNA in rat skeletal muscle

Skeletal muscle is the source of pro- and anti-inflammatory cytokines, and recently, it has been recognized as an important source of interleukin 6 (IL-6), a cytokine that exerts inhibitory effects on several pro-inflammatory cytokines. Although dynamic chronic resistance training has been shown to produce the known ""repeated bout effect"", which abolishes the acute muscle damage, performing of high-intensity resistance training has been regarded highly advisable, at least from the hypertrophy perspective. On the other hand, a more therapeutic, ""non-damaging"" resistance training program, mainly composed of concentric forces, low frequency/low volume of training, and the same exercise, could theoretically benefit the muscle when the main issue is to avoid muscle inflammation (as in the treatment of several ""low-grade"" inflammatory diseases) because the acute effect of each resistance exercise session could be diminished/avoided, at the same time that the muscle is still being overloaded in a concentric manner. However, the benefits of such ""less demanding"" resistance training schedule on the muscle inflammatory profile have never been investigated. Therefore, we assessed the protein expression of IL-6, TNF-alpha, IL-10, IL-10/TNF-alpha ratio, and HSP70 levels and mRNA expression of SCF(beta-TrCP), IL-15, and TLR-4 in the skeletal muscle of rats submitted to resistance training. Briefly, animals were randomly assigned to either a control group (S, n = 8) or a resistance-trained group (T, n = 7). Trained rats were exercised over a duration of 12 weeks (two times per day, two times per week). Detection of IL-6, TNF-alpha, IL-10, and HSP70 protein expression was carried out by western blotting and SCF(beta-TrCP) (SKP Cullin F-Box Protein Ligases), a class of enzymes involved in the ubiquitination of protein substrates to proteasomal degradation, IL-15, and TLR-4 by RT-PCR. Our results show a decreased expression of TNF-alpha and TLR4 mRNA (40 and 60%, respectively; p < 0.05) in the plantar muscle from trained, when compared with control rats. In conclusion, exercise training induced decreased TNF-alpha and TLR-4 expressions, resulting in a modified IL-10/TNF-alpha ratio in the skeletal muscle. These data show that, in healthy rats, 12-week resistance training, predominantly composed of concentric stimuli and low frequency/low volume schedule, down regulates skeletal muscle production of cytokines involved in the onset, maintenance, and regulation of inXammation.

Chronic exercise decreases cytokine production in healthy rat skeletal muscle

Skeletal muscle is the source of pro- and anti-inflammatory cytokines, and recently, it has been recognized as an important source of interleukin-6 (IL-6). Acute physical exercise is known to induce a pro-inflammatory cytokine profile in the plasma. However, the effect of chronic physical exercise in the production of pro- and anti-inflammatory cytokines by the skeletal muscle has never been examined. We assessed IL-6, TNF-alpha, IL-1 beta and IL-10 levels in the skeletal muscle of rats submitted to endurance training. Animals were randomly assigned to either a Sedentary group (S, n = 7) or an endurance exercise trained group (T, n = 8). Trained rats ran on a treadmill for 5 days week(-1) for 8 weeks (60% VO(2max)). Detection of IL-6, TNF-alpha, IL-1 beta and IL-10 protein expression was carried out by ELISA. We found decreased expression of IL-1 beta, IL-6, TNF-alpha and IL-10 (28%, 27%. 32% and 37%, respectively, p < 0.05) in the extensor digital longus (EDL) from T, when compared with S. In the soleus, IL-1 beta, TNF-alpha and IL-10 protein levels were similarly decreased (34%, 42% and 50%, respectively, p < 0.05) in T in relation to S, while IL-6 expression was not affected by the training protocol. In conclusion, exercise training induced decreased cytokine protein expression in the skeletal muscle. These data show that in healthy rats, 8-week moderate-intensity aerobic training down regulates skeletal muscle production of cytokines involved in the onset, maintenance and regulation of inflammation, and that the response is heterogeneous according to fibre composition. Copyright (C) 2009 John Wiley & Sons, Ltd.

Heart failure alters matrix metalloproteinase gene expression and activity in rat skeletal muscle

Heart failure is associated with a skeletal muscle myopathy with cellular and extracellular alterations. The hypothesis of this investigation is that extracellular changes may be associated with enhanced mRNA expression and activity of matrix metalloproteinases (MMP). We examined MMP mRNA expression and MMP activity in Soleus (SOL), extensor digitorum longus (EDL), and diaphragm (DIA) muscles of young Wistar rat with monocrotaline-induced heart failure. Rats injected with saline served as age-matched controls. MMP2 and MMP9 mRNA contents were determined by RT-PCR and MMP activity by electrophoresis in gelatin-containing polyacrylamide gels in the presence of SDS under non-reducing conditions. Heart failure increased MMP9 mRNA expression and activity in SOL, EDL and DIA and MMP2 mRNA expression in DIA. These results suggest that MMP changes may contribute to the skeletal muscle myopathy during heart failure.

High-Intensity Resistance Training with Insufficient Recovery Time Between Bouts Induce Atrophy and Alterations in Myosin Heavy Chain Content in Rat Skeletal Muscle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)