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Bidirectional selection for either high or low responsiveness to endurance running has created divergent rat phenotypes of high-response trainers (HRT) and low-response trainers (LRT). We conducted proteome profiling of HRT and LRT gastrocnemius of 10 female rats (body weight 279 ± 35 g; n = 5 LRT and n = 5 HRT) from generation 8 of selection. Differential analysis of soluble proteins from gastrocnemius was conducted by label-free quantitation. Genetic association studies were conducted in 384 Russian international-level athletes (age 23.8 ± 3.4 yr; 202 men and 182 women) stratified to endurance or power disciplines. Proteomic analysis encompassed 1,024 proteins, 76 of which exhibited statistically significant ( P < 0.05, false discovery rate <1%) differences between HRT and LRT muscle. There was significant enrichment of enzymes involved in glycolysis/gluconeogenesis in LRT muscle but no enrichment of gene ontology phrases in HRT muscle. Striated muscle-specific serine/threonine-protein kinase-beta (SPEG-β) exhibited the greatest difference in abundance and was 2.64-fold greater ( P = 0.0014) in HRT muscle. Coimmunoprecipitation identified 24 potential binding partners of SPEG-β in HRT muscle. The frequency of the G variant of the rs7564856 polymorphism that increases SPEG gene expression was significantly greater (32.9 vs. 23.8%; OR = 1.6, P = 0.009) in international-level endurance athletes ( n = 258) compared with power athletes ( n = 126) and was significantly associated (β = 8.345, P = 0.0048) with a greater proportion of slow-twitch fibers in vastus lateralis of female endurance athletes. Coimmunoprecipitation of SPEG-β in HRT muscle discovered putative interacting proteins that link with previously reported differences in transforming growth factor-β signaling in exercised muscle.

We report automated and time-efficient (2 h per sample) profiling of muscle using ultra-performance LC coupled directly with high-definition MS (HDMS(E)). Soluble proteins extracted from rat gastrocnemius (n = 10) were digested with trypsin and analyzed in duplicate using a 90 min RPLC gradient. Protein identification and label-free quantitation were performed from HDMS(E) spectra analyzed using Progenesis QI for Proteomics software. In total 1514 proteins were identified. Of these, 811 had at least three unique peptides and were subsequently used to assess the dynamic range and precision of LC-HDMS(E) label-free profiling. Proteins analyzed by LC-HDMS(E) encompass the entire complement of glycolytic, β-oxidation, and tricarboxylic acid enzymes. In addition, numerous components of the electron transport chain and protein kinases involved in skeletal muscle regulation were detected. The dynamic range of protein abundances spanned four orders of magnitude. The correlation between technical replicates of the ten biological samples was R(2) = 0.9961 ± 0.0036 (95% CI = 0.9940 - 0.9992) and the technical CV averaged 7.3 ± 6.7% (95% CI = 6.87 - 7.79%). This represents the most sophisticated label-free profiling of skeletal muscle to date., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Loss of muscle mass and function occurs in various diseases. Myostatin blocking can attenuate muscle loss, but downstream signaling is not well known. Therefore, to elucidate associated signaling pathways, we used the soluble activin receptor IIb (sActRIIB-Fc) to block myostatin and activins in mice. Within 2 wk, the treatment rapidly increased muscle size as expected but decreased capillary density per area. sActRIIB-Fc increased muscle protein synthesis 1-2 days after the treatment correlating with enhanced mTORC1 signaling (phosphorylated rpS6 and S6K1, r = 0.8). Concurrently, increased REDD1 and eIF2Bε protein contents and phosphorylation of 4E-BP1 and AMPK was observed. In contrast, proangiogenic MAPK signaling and VEGF-A protein decreased. Hippo signaling has been characterized recently as a regulator of organ size and an important regulator of myogenesis in vitro. The phosphorylation of YAP (Yes-associated protein), a readout of activated Hippo signaling, increased after short- and longer-term myostatin and activin blocking and in exercised muscle. Moreover, dystrophic mdx mice had elevated phosphorylated and especially total YAP protein content. These results show that the blocking of myostatin and activins induce rapid skeletal muscle growth. This is associated with increased protein synthesis and mTORC1 signaling but decreased capillary density and proangiogenic signaling. It is also shown for the first time that Hippo signaling is activated in skeletal muscle after myostatin blocking and exercise and also in dystrophic muscle. This suggests that Hippo signaling may have a role in skeletal muscle in various circumstances.

Eccentric exercise induced by electrostimulation increases mRNA expression of titin-complex proteins in rodent skeletal muscle. In this study, mRNA expression of titin, muscle LIM protein (MLP), cardiac ankyrin repeat protein (CARP), ankyrin repeat domain protein 2 (Ankrd2), diabetes-related ankyrin repeat protein (DARP), and calcium-activated proteinases, calpains, were investigated in human skeletal muscle after fatiguing jumping exercise. Fatiguing jumping exercise did not change mRNA expression of titin, DARP, calpain 1, or calpain 3. MLP, Ankrd2 and calpain 2 mRNA levels were increased 2 days postexercise. CARP mRNA level was already elevated 30 min and remained elevated 2 days postexercise. Increased mRNA expression of MLP, CARP, and Ankrd2, observed for the first time in human skeletal muscle, may be part of the signaling activated by physical exercise. The rapid increase in the level of CARP mRNA nominates CARP as one of the first genes to respond to exercise. The increase in the mRNA level of calpain 2 suggests its involvement in myofiber remodeling after strenuous jumping exercise.

In striated muscle, a sarcomeric noncontractile protein, titin, is proposed to form the backbone of the stress- and strain-sensing structures. We investigated the effects of diabetes, physical training, and their combination on the gene expression of proteins of putative titin stretch-sensing complexes in skeletal and cardiac muscle. Mice were divided into control (C), training (T), streptozotocin-induced diabetic (D), and diabetic training (DT) groups. Training groups performed for 1, 3, or 5 wk of endurance training on a motor-driven treadmill. Muscle samples from T and DT groups together with respective controls were collected 24 h after the last training session. Gene expression of calf muscles (soleus, gastrocnemius, and plantaris) and cardiac muscle were analyzed using microarray and quantitative PCR. Diabetes induced changes in mRNA expression of the proteins of titin stretch-sensing complexes in Z-disc (MLP, myostatin), I-band (CARP, Ankrd2), and M-line (titin kinase signaling). Training alleviated diabetes-induced changes in most affected mRNA levels in skeletal muscle but only one change in cardiac muscle. In conclusion, we showed diabetes-induced changes in mRNA levels of several fiber-type-biased proteins (MLP, myostatin, Ankrd2) in skeletal muscle. These results are consistent with previous observations of diabetes-induced atrophy leading to slower fiber type composition. The ability of exercise to alleviate diabetes-induced changes may indicate slower transition of fiber type.

To investigate the role of placental glucose delivery in fetal growth, two glucose transporters (Glut3 and Glut4) were determined from term placentae. This was accomplished by immunoblotting from crude placental membrane samples from cases of fetal intrauterine growth retardation (IUGR, n = 6), macrosomia (n = 6), maternal diabetes mellitus (n = 4) and normal term (n = 8). Glut3 and Glut4 were detected in only very low numbers in all patient groups and there were no changes in their placental density, which suggests that the expression of these transporters is not involved in disorders of fetal growth. However, birth weight corresponded to placental weight, indicating that the total amount of Glut3 and Glut4 is reduced in IUGR and increased in macrosomia.

The in vivo glucose uptake and the levels of two glucose transporter proteins (GLUT1 and GLUT4) were measured in heart and in various types of skeletal muscle from streptozotocin-diabetic rats. Diabetes (12-16 weeks) reduced the in vivo glucose uptake (glucose metabolic index, GMI), and the levels of GLUT1 and GLUT4 in heart by 75%, 60% and 70%, respectively. In diaphragm consisting of approximately equal amounts of type I (slow-contracting oxidative), IIa (fast-contracting oxidative) and IIb (fast-contracting glycolytic) fibers, GMI and GLUT4 levels were reduced by 60% and 40%, respectively, with no change in GLUT1 levels. In muscle consisting mainly of type I fibers (e.g., m. soleus), GMI and GLUT4 levels were reduced by 60% and 30%, respectively, whereas GLUT1 levels were unaltered. In mixed-type muscle consisting of type IIa and IIb fibers (e.g., m. plantaris and red part of m. gastrocnemius), GMI and GLUT1 levels were unchanged, whereas GLUT4 levels were decreased by 45%. In contrast, GMI was increased by 100% in type IIb fibers (e.g., the white part of m. gastrocnemius), probably reflecting the 4-fold increase in blood glucose levels, whereas GLUT4 levels were lowered by 55% with no change in GLUT1 levels. These data demonstrate a marked difference in the response of in vivo glucose uptake to long-term hypoinsulinemia between oxidative (type I) and glycolytic (type IIb) fibers. Furthermore, in contrast to the GLUT4, GLUT1 levels are regulated differentially in heart and skeletal muscle in response to streptozotocin-induced diabetes.

The effect of 10 weeks of running training and termination of training on the regional distribution of cardiac glucose uptake and protein synthesis were studied in isolated perfused hearts in male rats. The left ventricular glucose uptake in hearts from sedentary rats was 1.87 +/- 0.14 mumol/min per g protein (mean +/- SE), being about 30% higher in the subendocardial than in the subepicardial layer (p less than 0.05). The gradient of left ventricular glucose uptake was similar to the controls in the rats retired from training, but was absent in the trained animals. The altered transmural glucose uptake probably reflects differences in the adaptive response of various myocardial muscle layers to a long-term intermittent increase in cardiac work load. Phenylalanine incorporation was evenly distributed through the left ventricle in all the groups, but was lowered in the left and right ventricles of the trained rats. Phenylalanine incorporation returned to the control level 5 weeks after the cessation of training.

Dystrophin associated protein α-syntrophin is known to interact with voltage-gated sodium ion channel (NaCh). Dystrophin is known to be sensitive to eccentric muscle actions. For this reason, the function of the NaChs might also be affected. Molecular adaptations of dystrophin, α-syntrophin and NaChs were investigated after fatiguing stretch-shortening cycle (SSC) exercise, which consisted of unilateral jumps on a sledge apparatus. Muscle biopsies were taken from the vastus lateralis muscle of eight healthy subjects immediately after (IA) and 2 days after (2D) the exercise to analyze mRNA levels and immunohistochemical staining patterns. SSC exercise resulted in decreased isometric maximal voluntary contraction (IA: −31±9%, 2D: −14±16%) and a delayed increase of plasma creatine kinase activity (2D: +178±211%). Despite muscle soreness ( P<0.001), no morphological damage was observed and no changes were found in the mRNA concentrations. However, the relative changes of the mRNA concentrations of α-syntrophin and NaChs were highly correlated ( r=0.93, P<0.001) 2D after SSC exercise. This consistent pattern of mRNA regulation may imply a functional relationship between these two proteins. In addition, the current experiment emphasises high inter-individual variation in molecular responses to heavy exercise. [ABSTRACT FROM AUTHOR]