B. S. Shenkman

PPARα gene variation and physical performance in Russian athletes

Peroxisome proliferator-activated receptor α (PPARα) regulates genes responsible for skeletal and heart muscle fatty acid oxidation. Previous studies have shown that the PPARα intron 7 G/C polymorphism was associated with left ventricular growth in response to exercise. We speculated that GG homozygotes should be more prevalent within a group of endurance-oriented athletes, have normal fatty acid metabolism, and increased percentages of slow-twitch fibers. We have tested this hypothesis in the study of a mixed cohort of 786 Russian athletes in 13 different sporting disciplines prospectively stratified by performance (endurance-oriented athletes, power-oriented athletes and athletes with mixed endurance/power activity). PPARα intron 7 genotype and allele frequencies were compared to 1,242 controls. We found an increasing linear trend of C allele with increasing anaerobic component of physical performance (P=0.029). GG genotype frequencies in endurance-oriented and power-oriented athletes were 80.3 and 50.6%, respectively, and were significantly (P<0.0001) different compared to controls (70.0%). To examine the association between PPARα gene variant and fiber type composition, muscle biopsies from m. vastus lateralis were obtained and analyzed in 40 young men. GG homozygotes (n=25) had significantly (P=0.003) higher percentages of slow-twitch fibers (55.5±2.0 vs 38.5±2.3%) than CC homozygotes (n=4). In conclusion, PPARα intron 7 G/C polymorphism was associated with physical performance in Russian athletes, and this may be explained, in part, by the association between PPARα genotype and muscle fiber type composition.

Transversal Stiffness and Young's Modulus of Single Fibers from Rat Soleus Muscle Probed by Atomic Force Microscopy

The structural integrity of striated muscle is determined by extra-sarcomere cytoskeleton that includes structures that connect the Z-disks and M-bands of a sarcomere to sarcomeres of neighbor myofibrils or to sarcolemma. Mechanical properties of these structures are not well characterized. The surface structure and transversal stiffness of single fibers from soleus muscle of the rat were studied with atomic force microscopy in liquid. We identified surface regions that correspond to projections of the Z-disks, M-bands, and structures between them. Transversal stiffness of the fibers was measured in each of these three regions. The stiffness was higher in the Z-disk regions, minimal between the Z-disks and the M-bands, and intermediate in the M-band regions. The stiffness increased twofold when relaxed fibers were maximally activated with calcium and threefold when they were transferred to rigor (ATP-free) solution. Transversal stiffness of fibers heavily treated with Triton X-100 was about twice higher than that of the permeabilized ones, however, its regional difference and the dependence on physiological state of the fiber remained the same. The data may be useful for understanding mechanics of muscle fibers when it is subjected to both axial and transversal strain and stress.

Mice in Bion-M 1 Space Mission: Training and Selection

After a 16-year hiatus, Russia has resumed its program of biomedical research in space, with the successful 30-day flight of the Bion-M 1 biosatellite (April 19–May 19, 2013). The principal species for biomedical research in this project was the mouse. This paper presents an overview of the scientific goals, the experimental design and the mouse training/selection program. The aim of mice experiments in the Bion-M 1 project was to elucidate cellular and molecular mechanisms, underlying the adaptation of key physiological systems to long-term exposure in microgravity. The studies with mice combined in vivo measurements, both in flight and post-flight (including continuous blood pressure measurement), with extensive in vitro studies carried out shortly after return of the mice and in the end of recovery study. Male C57/BL6 mice group housed in space habitats were flown aboard the Bion-M 1 biosatellite, or remained on ground in the control experiment that replicated environmental and housing conditions in the spacecraft. Vivarium control groups were used to account for housing effects and possible seasonal differences. Mice training included the co-adaptation in housing groups and mice adaptation to paste food diet. The measures taken to co-adapt aggressive male mice in housing groups and the peculiarities of “space” paste food are described. The training program for mice designated for in vivo studies was broader and included behavioral/functional test battery and continuous behavioral measurements in the home-cage. The results of the preliminary tests were used for the selection of homogenous groups. After the flight, mice were in good condition for biomedical studies and displayed signs of pronounced disadaptation to Earths gravity. The outcomes of the training program for the mice welfare are discussed. We conclude that our training program was effective and that male mice can be successfully employed in space biomedical research.

Effect of short-term gravitational unloading on rat and mongolian gerbil muscles

Gravitational unloading leads to destructive changes in the structure and function of muscle fibers. However, the role of the EMG activity level is still unclear. We measured changes caused by one- and three-day hypogravity in the following muscles: Soleus (Sol), Tibialis anterior (TA) and Gastrocnemius c.m. (MG). We used Wistar rats and Mongolian gerbils. The following parameters were assessed: the specific force of contraction of isolated fibers by tensometry, the transverse stiffness of the contractile apparatus by atomic force microscopy, and the calcium content by Fluo-4. We detected the accumulation of calcium ions in all muscles even after one-day unloading. In Sol this effect was more significant than in other muscles. After one-day of hypogravity we detected an increase in the specific force in all muscle types and species. Meanwhile, the transverse stiffness of the contractile apparatus, M-band and Z-disc increased only in fast muscles but not in Sol. After three-days of unloading, the specific force in Sol decreased, and the transverse stiffness of the contractile apparatus behaved in the same way as the force. The specific tension of fast muscle fibers decreased significantly in comparison with one-day unloading. In addition, the transverse stiffness of some areas of MG had a tendency to decrease in comparison to “one-day” unloading, although there was no such a tendency in the fibers of TA. In Mongolian gerbils the tendencies were the same as in the rats, but showed less dramatic changes. The reduction in the magnitude of changes in the Sol–MG–TA series correlates with EMG activity.

Attenuation of unloading-induced rat soleus atrophy with the heat-shock protein inducer 17-(allylamino)-17-demethoxygeldanamycin

We hypothesized that pharmacological induction of HSP70 would attenuate soleus atrophy development under 3 d of rat hindlimb unloading. Male Wistar rats were divided into control (C; n=7), 3‐d hindlimb unloading (HUL; n=7), HUL with HSP90 inducer administration, 17‐allylamino‐17‐emethoxygel‐danamycin (17‐AAG; 60 mg/kg, HUL+17‐AAG, n=8). The relative weight of soleus muscle to body weight [soleus wt (mg)/body wt (g)] in the HUL group was less than that of the C and HUL + 17‐AAG groups (P<0.05). We revealed HSP90, HSP70 mRNA decrease in the HUL group (but not the HUL+ 17‐AAG group) vs. C (P<0.05). The unloading resulted in significant increases of μ‐calpain and conjugated ubiquitin (Ub) levels (proteins as well as mRNAs) vs. the C group, whereas 17‐AAG administration prevented these alterations (studied by SDS‐PAGE and RT‐PCR). pFOXO3 protein was decreased in the HUL group vs. C, but not in HUL + 17‐AAG. Content of E3‐lygase (MuRF‐1, MAFbx) mRNA was increased in both suspended groups. In summary, 17‐AAG administration attenuates soleus muscle atrophy, μ‐calpain, and Ub increases under hindlimb unloading as well as decrease of pFOXO3.—Lomonosova, Y. N., Shenkman, B S., Nemirovskaya, T. L. Attenuation of unloading‐induced rat soleus atrophy with the heat‐shock protein inducer 17‐(allylamino)‐17‐demethoxygeldanamycin. FASEB J. 26, 4295–4301 (2012). www.fasebj.org

Effect of support stimulation on unloaded soleus in rat.

Abstract. We studied the efficacy of plantar support in the prevention of atrophy in a disused soleus muscle during hindlimb suspension (HS). The 14-day investigation involved three groups of hindlimb-suspended male Wistar rats and a group of control rats (C). In all HS groups, the left hindlimbs (L) of the animals were left free. As for the right hindlimbs (R), they were either provided support by an adjustable platform (Sup), or immobilized at the ankle joint in a neutral position (Im), or both supported by the platform and immobilized (Sup+Im). Mass, cross-sectional area (CSA) and slow twitch (ST) fiber percentage (ST%) in the R soleus muscle were similar in the Sup and control groups. In the Sup+Im group, these parameters were significantly lower than in the Sup R and C groups. However, the CSA of ST fibers in the Sup+Im R soleus was significantly higher than in those hindlimbs that were left hanging free. Succinate dehydrogenase activity in ST fibers, and α-glycerophosphate dehydrogenase activity in fast-twitch fibers had decreased in the Sup R as compared with the controls. The maximal rate of ADP-stimulated mitochondrial respiration was increased in the free-hanging Sup L hindlimb in comparison with the control. In conclusion, during HS: (1) hindlimb support prevents slow-to-fast fiber transformation and losses in muscle mass and fiber CSA, but brings about a decrease in metabolic enzyme activity, and (2) hindlimb plantar support attenuates but does not fully prevent ST fiber atrophy in the immobilized soleus.

The role of L-type calcium channels in the accumulation of Ca2+ in soleus muscle fibers in the rat and changes in the ratio of myosin and serca isoforms in conditions of gravitational unloading

Gravitational unloading is known to produce changes in the expression of a number of contractile and regulatory proteins in the soleus muscle. This applies particularly to isoforms of myosin heavy chains (MHC) and SERCA sarcoplasmic reticulum calcium pumps. Unloading increases the resting levels of extracellular calcium in soleus muscle fibers. The present study addresses verification of the hypothesis that changes in the expression of MHC and SERCA isoforms in gravitational unloading are linked with the accumulation of calcium ions in the myoplasm of muscle fibers. It is suggested that specific blockade of L-type calcium channels using nifedipine decreases the myoplasmic calcium ion concentration, thus preventing the development of changes in the expression of MHC and SERCA isoforms. A total of 36 male Wistar rats were divided into three groups: a control group, an unloading group using the Morley-Holton soleus muscle functional unloading model, and an unloading + nifedipine group, where animals received daily nifedipine (7 mg/kg/day) with their drinking water on the background of suspension. The results showed that blockade of L-type calcium channels on the background of gravitational unloading significantly decreased the extent of calcium ion accumulation in the myoplasm of soleus muscle fibers, which partly prevented the transformation of muscle fibers (in relation to the fast and slow isoforms of MHC and SERCA) to the rapid type. There was no nuclear translocation of the greater part of transcription factor NFATc1, as seen on unloading.

Decrease in the electrogenic Contribution of Na,K-ATPase and the resting membrane potential as a possible mechanism of Ca2+ accumulation in rat soleus muscle in a short-term gravity unloading

The resting membrane potential and electrogenic contribution of α1- and α2-isoforms of Na+/K+-ATPase in the rat soleus muscle at early stages of gravity unloading were analyzed. The role of L-type calcium channels in accumulation of calcium ions in the myoplasm under these conditions was estimated. After 3-day antiorthostatic suspension, the resting membrane potential of the muscle fibers decreased from −71.0 ± 0.5 to −66.8 ± 0.7 mV, the muscle excitability reduced, and a trend of muscle fatigue acceleration appeared. The electrogenic contribution of ouabain-sensitive α2-isoform of Na+/K+-ATPase, determined as the depolarization caused by 1μM ouabain, decreased after suspension from 6.2 ± 0.6 to 0.5 ± 0.8 mV. The contribution of ouabain-resistant α1-isoform of Na+/K+-ATPase, determined as an additional depolarization after addition of 500 μM ouabain, decreased from 4.6 ± 0.6 to 2.6 ± 0.6 mV. The intensity of Fluo-4AM fluorescence in individual muscle fibers increased after suspension more than fourfold, which suggests an elevated calcium concentration in the myoplasm. A local delivery of nifedipine, a blocker of the L-type calcium channels, to the muscle removed this effect. The existence of a selective mechanism suppressing the electrogenic contribution of Na+/K+-ATPase α2-isoform, which is the main cause of the muscle fiber membrane depolarization after 3-day suspension, is postulated. The depolarization can activate part of potential-sensitive L-type Ca2+ channels, causing the accumulation of calcium ions in the muscle fiber myoplasm.

Parameters of fiber cell respiration and desmin content in rat soleus muscle at early stages of gravitational unloading

The aim of the work was to study the parameters of fiber cell respiration and desmin content in Wistar rat soleus muscle after 1, 3, 7 and 14 days of gravitational unloading. Gravitational unloading was simulated by antiorthostatic hindlimb suspension. The parameters of cell respiration were determined using polarography, and desmin content was assessed by means of Western blotting. The results showed that the intensity of cell respiration is reduced after three days of gravitational unloading, reaches a minimum level after seven days and slightly increases by the fourteenth day of hindlimb unloading, as well as the content of desmin, which, however, to the fourteenth day returns to the control level. Taking into account that mitochondrial function depends on the state of cytoskeleton, the data allow us to assume that early reduction of the intensity of cell respiration under unloading could be caused by degradation of the protein desmin that determines the intracellular localization of mitochondria.

The contents of desmin and α-actinin-1 in the human soleus muscle after seven-day “dry” immersion

Measurement of the transversal stiffness in various regions of the muscle fiber allows the state of local structures to be assessed at the level of both the con tractile apparatus and the sarcolemma [6]. Earlier studies of this type on the effect of seven day “dry” immersion on the state of the human soleus muscle fibers [7] have demonstrated that the changes in the contractile apparatus can be prevented by a plantar mechanical stimulator compensating for gravitational unloading, developed by Kozlovskaya et al. [10]. Elec tromyostimulation is another yet least efficient method.