[Preparation of Human Skeletal Muscle Samples for Proteomic Analysis with Isobaric iTRAQ Labels].

In the past decade, mass spectrometry studies of skeletal muscles have become common. In this tissue, the abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows improving assessment of the changes in the protein content, while analyzing up to 10 samples in a single run. Here we present the results of a comparative study of various methods for the fractionation of skeletal muscle peptides labeled with an isobaric label iTRAQ. Samples from m. vastus lateralis of eight young males were collected with a needle biopsy. After digestion into peptides and labeling, the preparations were carried out according to three different protocols: (1) peptide purification, HPLC-MS/MS; (2) peptide purification, isoelectric focusing, HPLC-MS/MS; (3) high pH reverse-phase LC fractionation, HPLC-MS/MS. Fractionation of labeled peptides by high pH reverse-phase LC was the optimal strategy for increasing the depth of the proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including the nucleic acids binding the proteins, chaperones, receptors, and transcription factors.

[Effect of Combined Aerobic and Strength Exercise on Regulation of Mitochondrial Biogenesis, Protein Synthesis and Degradation in Human Skeletal Muscle.]

We tested the hypothesis that strength exercise after intermittent aerobice exercise might activate signalling pathways related to mitochondrial biogenesis (phosphorylation level of AMPK, p38; expression of PGC-1a, NT-PGC-la, TFAM, VEGFA), to protein synthesis (phosphorylation level of p70S6Kl(Thr389) eEF2(Thr56) expression of IGF-lEa, IGF-lEc (MGF), REDDl) and to proteolysis (phosphorylation level of FOXOl(Ser256) and expression of MURFl, MAFbx, Myostatin) in trained skeletal muscle. Nine amateur endurance-trained athletes performed 70-Min bicycle intermittent exercise with both legs (E), followed by one-leg strength exercise (ES: 4 bouts of knee extensions at 75% MVC till exhaustion). Gene expression and protein level were evaluated in samples from m. vastus lateralis before, 40 min, 5 and 22 h after the aerobic exercise. The phosphorylation level of the ACC(Ser79/222)(an endogenous marker of AMPK activity) and expression of PGC-la-related TFAM - marker of mitochondrial biogenesis were increased after E exercise and did not changed after ES. Expression of PGC-lα and truncated isoform NT- PGC-lα was increased in both legs as well. Insulin concentration in blood was dramatically, 7.5-fold diminished after intermittent aerobic exercise. Phosphorylation of FOXO(Ser256) - regulator of ubiquitin-related proteolysis - was decreased after both E and ES exercise, it means it was activated in both cases, while expression of E3-ubiquitin ligase MURFl was increased only after E exercise. Both aerobic and combined exercise did not affect regulation of protein synthesis: neither expression of IGF-lEa and IGF-Ec (MGF) mRNA isoforms nor phosphorylation levels of markers of protein synthesis p70S6Kl(Thr389) and eEF2(Thr56) were changed. Thus effects of aerobic exercise in trained muscles are noticeably suppressed by performing strength exercise immediately after endurance one. In particular, the activity of signalling cascades and expression of genes regulating mitochondrial biogenesis are lessened, but protein synthesis regulation is not affected. And at last strength exercise suppresses induced by aerobic exercise expression of MURF1 gene - marker of ubiquitin proteasome system. It means that strength exercise just after intermittent aerobic exercise might have a negative effect on aerobic performance if used chronically.

[Determination of Aerobic-anaerobic Transition in Working Muscle Using EMG and Near Infrared-Spectroscopy Data].

A method to evaluate aerobic-anaerobic transition (AAT) during exercise is suggested. The subjects performed two tests with incremental increase of load: bicycle exercise and one leg knee extensions. In both tests the relation of deoxygenated hemoglobin (HHb) to EMG-activity of m. vastus lateralis during test has characteristic peak corresponding to AAT. The statistically significant correlation (r = 0.78, p < 0.05) between the load corresponding to AAT and anaerobic threshold (blood [La] = 4 mmol) was found during bicycle test. The method is applicable to determination of AAT during exercise of small muscle group.

[The Effect of Single Aerobic Training on the Regulation of Mitochondrial Biogenesis in Skeletal Muscles of Trained Men: a Time-Course Study].

Adaptation of skeletal muscles to physical training depends on intensity and duration of exercise sessions. The purpose of this study was to investigate the effect of the duration of moderately intensive single aerobic exercise session (60% V(O2max)) on the activation of signalling kinases which regulate PGC-1α gene expression and on the expression of regulatory genes of mitochondrial biogenesis and muscle catabolism. Nine athletes (V(O2max)) 59 mL/min/kg) performed 30-, 60-, and 90-min cycling sessions. An exercise-induced increase in PGC-1α gene expression was proved to occur without activation of AM PK, p38 MAPK and CAMKII. It was found that 60- and 90-min sessions result in comparable increases of PGC-lα gene expression, while VEGFA gene expression increased only after 90-min session. Even 90-min exercise did not induce the activation of FOXO1-E3 ubiquitin ligase pathway and did not result in an increase of expression of exercise-induced catabolic genes.

[THE INDICATORS OF GROWTH AND MYOGENESIS IN THE GLYCOLYTIC AND OXIDATIVE MUSCLES OF THE RAT AFTER INDIRECT ELECTROSTIMULATION].

A comparative analysis of the signaling pathways activity and gene expression in the red (RG) and white (WG) parts of the gastrocnemius muscle of rat after a series of short (1 s) tetanic contractions induced by motor nerve stimulation at a frequency of 100 Hz and with an amplitude that provides activation of all motor units of the muscle. WG compared to RG demonstrated a marked increase in the phosphorylation level of ERK1/2, although the increase in the phosphorylation of AMPK was not different in two muscles 2 h after the stimulation. Along with that, content of MyoD and myogenin mRNA in WG increased much higher than in RG, whereas the effect of stimulation on IGF-1, MaFbx and MuRF genes expression was weak and comparable in WG and RG. There was an increase of myostatin mRNA in RG. Thus, glycolytic muscle fibers of WG exhibit more pronounced regulatory shifts of hypertrophic character than oxidative muscle fibers of RG.

[Regional differences in the level of ERK1/2 phosphorylation and expression of the myogenic regulatory factors following electrostimulation with different mechanic and metabolic action on the gastrocnemius muscle].

Effect of high-frequency electrical stimulation of the sciatic nerve on ERK1/2 kinase phosphorylation and mRNA expression in MyoD (myogenic regulation factor) and myogenin in the red (RGM) and white (WGM) parts of the medial head in rat's m. gastrocnemius was studied. Two stimulation regimes were equalized both lengthwise and in total effort but differed in duration and number of contractions and, therefore, in mechanic and metabolic effects on the muscle. It was shown that growth of the number of phosphorylated ERK1/2 was particularly high in WCM due to application of the protocol for multiple short-time contractions. Whatever the stimulation regime, MyoD mRNA expression in RGM and WGM increases to the same extent, whereas myogenin mRNA expression does not change. Consequently, the regime with the predominantly mechanic effect is favorable to activation of the ERK signaling pathway in glycolytic myofibers.

Stress-associated cardiovascular reaction masks heart rate dependence on physical load in mice.

When tested on the treadmill mice do not display a graded increase of heart rate (HR), but rather a sharp shift of cardiovascular indices to high levels at the onset of locomotion. We hypothesized that under test conditions cardiovascular reaction to physical load in mice is masked with stress-associated HR increase. To test this hypothesis we monitored mean arterial pressure (MAP) and heart rate in C57BL/6 mice after exposure to stressful stimuli, during spontaneous locomotion in the open-field test, treadmill running or running in a wheel installed in the home cage. Mice were treated with ß1-adrenoblocker atenolol (2mg/kg ip, A), cholinolytic ipratropium bromide (2mg/kg ip, I), combination of blockers (A+I), anxiolytic diazepam (5mg/kg ip, D) or saline (control trials, SAL). MAP and HR in mice increased sharply after handling, despite 3weeks of habituation to the procedure. Under stressful conditions of open field test cardiovascular parameters in mice were elevated and did not depend on movement speed. HR values did not differ in I and SAL groups and were reduced with A or A+I. HR was lower at rest in D pretreated mice. In the treadmill test HR increase over speeds of 6, 12 and 18m/min was roughly 1/7-1/10 of HR increase observed after placing the mice on the treadmill. HR could not be increased with cholinolytic (I), but was reduced after sympatholytic (A) or A+I treatment. Anxiolytic (D) reduced heart rate at lower speeds of movement and its overall effect was to unmask the dependency of HR on running speed. During voluntary running in non-stressful conditions of the home cage HR in mice linearly increased with increasing running speeds. We conclude that in test situations cardiovascular reactions in mice are governed predominantly by stress-associated sympathetic activation, rendering efforts to evaluate HR and MAP reactions to workload unreliable.

[Dynamics of systemic and local physiological indices while changing intensity of physical load].

It is well known that metabolic, cardiovascular and respiratory indices during exercise of moderate intensity are linearly related to exercise power. After the load reaches the definite level this relationship changes for nonlinear. Different methods of evaluating the intensity of load at which this transition takes place are discussed. The methods for investigation of transient process dynamics in energy supply of muscle contractions with changing intensity of contractions is described. The dependence of dynamic characteristics of physiological indices from fitness level and in its turn from age and level of physical activity is discussed.

[The effects of aerobic exercise upon genes expression in skeletal muscle of trained and untrained men].

It is well recognized the PGC-1 alpha is a key regulator of mitochondrial biogenesis. Mechanical and metabolic perturbations in a skeletal muscle during and after aerobic exercise lead to increase PGC-1alpha expression. In addition to that an increase of PGC-1alpha expression after exercise depends on relative workload intensity and does not depend on fitness level. The aim of the study was to compare the expression of regulators of mitochondrial biogenesis PGC-1alpha, TFAM and TFB2M and of proteolysis-related genes FOXO1 and Atrogin-1 in skeletal muscle untrained and trained men after aerobic exercise with the same relative workload. The study showed that after exercise the PGC-1alpha expression did not differ between groups but TFAM and TFB2M expression was higher in untrained muscle than trained. On the contrary proteolysis-related genes FOXO1 and Atrogin-1 expression increased only in the muscle of trained men.

[Optimization of training: development of a new partial load mode of strength training].

Hypertrophic effect of strength training is known to originate from mechanical and metabolic stimuli. During exercise with restricted blood supply ofworking muscles, that is under conditions of intensified metabolic shifts, training effect may be achieved with much lower external loads (20% of one repetition maximum (1 RM)). The aim of the study was to compare the effects of 8 wks high-intensity (80-85% MVC) strength training and low-intensity (50% 1 RM) training without relaxation. The high-intensity strength training leads to somewhat higher increments in strength and size of trained muscles than training without relaxation. During high-intensity training an increase of area occupied by type II fibers at muscle cross section prevails while during training without relaxation - an increase of area occupied by type I fibers takes place. An exercise session without relaxation leads to a more pronounced increase in secretion of growth hormone, IGF-1 and cortisol. Expression of gene regulating myogenesis (Myostatin) is changed in different ways after high-intensity strength exercise session and after exercise session without relaxation. Low-intensity strength training (50% 1 RM) without relaxation is an effective way for inducing increases of strength and size of trained muscles. This low intensive type of training may be used in restorative medicine, sports and physical culture.

[Changes of rat respiratory and locomotory muscles during aerobic exercise training in continuous and interval regimens].

Male Wistar rats were treadmill-trained for 8 weeks using one of the two regimens: with the constant running speed or with alternating high-speed and low-speed intervals. Both training regimens led to an increase of rat aerobic capacities and to a higher citrate synthase activity in the medial head of gastrocnemius muscle. No differences between the effects of two training regimens were observed. However, in contrast to constant-speed training the interval one resulted in myocardium hypertrophy and also in less pronounced changes in diaphragm muscle, such as slow-direction shift of myosin phenotype and reduction of muscle fiber cross-sectional area. Neither of the training regimens had an effect on corticosterone and thyroid hormones levels in rat blood, whereas the interval training resulted in a higher level of testosterone. Anabolic influence of testosterone during interval aerobic training may be favorable for heart hemodynamic capacity and force characteristics of the diaphragm.

[Comparison of aerobic performance of leg and arm muscles in cross country skiers].

Recently there was a tendency to increase usage of double poling during cross country skiing and accordingly to increase loading of the arm muscles. The aim of the study was to compare the aerobic performance of cross country skiers in the incremental double poling and running tests till exhaustion. Eight junior subnational level cross country skiers ( V(O)2max 70 (66-72) ml/min/kg) have participated in the study. The rate of blood lactate increase during double poling test was higher than during running test. Double poling V(O)2peak was 88 (84-93)% from running V(O)2max. Relative anaerobic threshold (% V(O)2max at AT), closely linked to the current aerobic performance, was substantially lower in double poling test compared with running test: 79 (57-83)% and 94 (90-98)% respectively. We suggest that the main reserve for further increase of aerobic performance of cross country skiers lies in an increase of double poling aerobic capacity.

[Aerobic performance: role of oxygen delivery and utilization, glycolytic flux].

Oxygen delivery to muscle, its consumption and glycolytic flux, all of each affect and restrict aerobic performance, are discussed. Energy supply of intensive exercise till exhaustion lasting 3 to 4 min is provided mainly by oxidative metabolism, simultaneously glycolytic flux may be increased considerably. Other conditions being equal, capacity of oxygen delivery determines oxygen partial pressure in myoplasm of exercising/contracting muscle. With PO2 in myoplasm increasing from 0 to 1-2 mm Hg oxygen consumption (VO2) in mitochondria enhances dramatically, with further increase of PO2 its rise slows down. At the ascending part of VO2-PO2 relationship for mitochondria the increase of VO2 is noticeably restricted by oxygen delivery to contracting muscle. When PO2 approaches plateau of the VO2-PO2 relationship, an increase of VO2 is restricted by mitochondria capacity to accumulate oxygen and augmented oxygen delivery will not lead to a significant increase of muscle VO2. On the other hand considerable accumulation of glycolytic metabolites in contracting muscle causes a decrease of contractility which in its turn may restrict aerobic performance. Noteworthy no strict relationship between glycolytic flux and PO2 in myoplasm exists. That is why correct evaluation of factors limiting aerobic performance presupposes simultaneous evaluation of both glycolytic flux and oxygen consumption in muscle which in its turn depends on oxygen delivery to mitochondria and its utilization.

[Association of the muscle-specific creatine kinase (CKMM) gene polymorphism with physical performance of athletes].

The aim of the study was to investigate allele and genotype distributions of the muscle-specific creatine kinase gene (CKMM) A/G polymorphism in athletes (n = 384) and controls (n = 1116), and to find interrelation between genotypes and aerobic capacity in rowers (n = 85). Genotyping was performed by restriction fragment length polymorphism analysis. Aerobic capacity (maximal oxygen consumption (VO2(max)) and maximal power production capacity (W(max was determined using an incremental test to exhaustion by rower ergometer. The frequencies of CKMM A allele and AA genotype were significantly higher in endurance-oriented athletes (n = 176) than in controls (A allele: 78.7% vs. 65.4%; p < 0.0001; AA genotype: 59.7% vs. 44.2%; p = 0.0003). On the other hand, GG genotype was more prevalent in weightlifters (n = 74) in comparison with controls (31.1% vs. 13.4%; p = 0.0001). Furthermore, CKMM AA genotype was associated with high values of VO2(max) (AA - 58.98 (3.44) ml/kg/min, GA - 56.99 (4.36) ml/kg/min, GG - 52.87 (4.32) ml/kg/min, p = 0.0097). Thus, CKMM gene A/G polymorphism is associated with physical performance of athletes.

[Comparative analysis of gene expression in rat locomotor muscles and diaphragm].

Gene expression profile in diaphragm in comparison to three principally different hindlimb muscles (soleus, red and white gastrocnemius) was studied using quantitative PCR. Expression levels of PGC-1alpha mRNA and myogenin mRNA in diaphragm were in accordance with its myosin phenotype and citrate synthase activity. However, diaphragm was characterised by atypically high content of MyoD mRNA as well as high content of IGF-1 mRNA and low content of myostatin mRNA. The latter two findings suggest high intensity of protein synthesis in diaphragm muscle fibers, although they have smaller cross sectional area than fibers in locomotor muscles.

[Dynamics of heart rate changes in rats following stepwise change of treadmill running speed].

Amplitude and temporal responses of heart rate to stepwise increase or decrease of treadmill running intensity were investigated in rats. Heart rate amplitude response was shown to be connected mainly with the change of sympathetic nervous activity whereas heart rate temporal response was shown to be determined predominantly by parasympathetic cardiotrophic influences.

[Wavelet analysis of m. vastus lateralis surface EMG activity in incremental tests till exhaustion using bicycle and knee extension exercises].

Continuous wavelet analyses was applied to investigate the spectral characteristics of m. vastus lateralis EMG activity in two incremental tests till exhaustion: rhythmic knee-joint extensions and cycling. Wavelet analysis of surface EMG revealed differences in the dynamics of time-frequency characteristics of the signal during single cycle of two types of movements with different loads, as well as differences in the slow variations of spectral characteristics associated with the development of muscle fatigue during the tests. It was shown that during cycling with low loads (beginning of the test) maximum of EMG activity was confined within the second half of muscle contraction (the angle in the knee joint approximately 140 degrees), increase of load at the end of the test led to a shift of the peak to the beginning of the active phase of movement, while the median frequency of the "instant" wavelet spectra during muscle contraction remained almost unchanged. During knee-joint extensions the maximum of EMG activity was observed at the end of the active phase of movement for all loads, median frequency increased significantly with increasing the angle at the knee joint. Long-term dynamics of EMG intensity growth during these tests differed as well, whereas dynamics of wavelet-spectra median frequencies were practically the same--during both tests their growths were observed.

[Effect of strength-building training on manifestation of pressor reflex from working muscles receptors].

Ten young normal volunteers and 8 armrestlers worked with forearm muscles till refusal at 30% of maximal arbitrary force. Work was either static or rhythmic with alternation of 20-s period of contraction and relaxation and followed by post-work arterial occlusion of the forearm muscles (PWAO). Heart rate (HR), blood pressure (BP) and muscular vessels-related sympathetic activity (MRSA) were measured continuously. MRSA was registered in n. peroneus using the microneurographic technique. Static work and subsequent PWAO produced different BP and MRSA neither in sportsmen nor amateurs. On the contrary, rhythmic work followed by PWAO suppressed the muscle pressor reflex in sportsmen significantly. The authors consider possible origination of the effect by change in energy supply to working muscles, enhanced extraction of metabolites, and sensory decrement of sportsmen's muscular receptors.