Changes in cell respiration of postural muscle fibers under long-term gravitational unloading after addition of succinate into diet

The intensity of cell respiration of the rat m. soleus, gastrocnemius c.m., and tibialis anterior fibers during 35-day gravitational unloading, with the addition of succinate in the diet at a dosage rate of 50 mg per 1 kg animal weight has been investigated. The gravitational unloading was modeled by antiorthostatic hindlimb suspension. The intensity of cell respiration was estimated by polarography. It was shown that the rate of oxygen consumption by soleus and gastrocnemius fibers on endogenous and exogenous substrates and with the addition of ADP decreases after the unloading. This may be associated with the transition to the glycolytic energy pathway due to a decrease in the EMG activity. At the same time, the respiration rate after the addition of exogenous substrates in soleus fibers did not increase, indicating a disturbance in the function of the NCCR-section of the respiratory chain and more pronounced changes in the structure of muscle fibers. In tibialis anterior fibers, no changes in oxygen consumption velocity were observed. The introduction of succinate to the diet of rats makes it possible to prevent the negative effects of hypokinesia, although it reduces the basal level of intensity of cell respiration.     

Parameters of fibers 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 fibers 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 the 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 intracellular localization of mitochondria.     

Role of systemic control of postural muscle hydration under conditions of gravitational unloading

The purpose of our research was to investigate a role of systemic mechanisms of regulation of hydration in postural muscles of mammals under conditions of gravitational unloading. It was shown that administration of desmopressin in hindlimb suspended rats led to systemic hyperhydration and amelioration of soleus muscle water loss. However in desmopressin administered and unloaded animals the soleus fiber size and soleus dry weight reduction turned out to be non significant.     

Changes in the functional state of spinal-cord cell structures under gravitational unloading

The functional properties of the spinal-cord structures of experimental rats under a 7-day gravitational unloading were assessed using the method of transcranial magnetic stimulation. Hypogravity was modeled by hanging the animals by their tails in an antiorthostatic position. The gastrocnemius muscle potentials evoked by magnetic stimulation of the efferent structures of the spinal cord were registered. We found that gravitational unloading causes significant changes in motor-potential parameters and the central motor transmission time. We propose that the cause of the revealed transformations is afferent inflow limitation, first of all the motor type, as well as adaptation of the central nervous system to new conditions of motor activity.     

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.     

Substrate profile in rat soleus muscle fibers after hindlimb unloading and fatigue.

Limb muscles from rats flown in space and after hindlimb unloading (HU) show an increased fatigability, and spaceflight has been shown to result in a reduced ability to oxidize fatty acids. The purpose of this investigation was to determine the effects of HU on the substrate content in fast- and slow-twitch fibers and to assess the substrate utilization patterns in single slow type I fibers isolated from control and HU animals. A second objective was to assess whether HU altered the ability of the heart or limb muscle to oxidize pyruvate or palmitate. After 2 wk of HU, single fibers were isolated from the freeze-dried soleus and gastrocnemius muscles. HU increased the glycogen content in all fiber types, and it increased lactate, ATP, and phosphocreatine in the slow type I fiber. After HU, the type I fiber substrate profile was shifted toward that observed in fast fibers. For example, fiber glycogen increased from 179 +/- 16 to 285 +/- 25 mmol/kg dry wt, which approached the 308 +/- 23 mmol/kg dry wt content observed in the post-HU type IIa fiber. With contractile activity, the type I fiber from the HU animal showed a greater utilization of glycogen and accumulation of lactate compared with the control type I fiber. HU had no effect on the ability of crude homogenate or mitochondria fractions from the soleus or gastrocnemius to oxidize pyruvate or palmitate. The increased fatigability after HU may have resulted from an elevated glycolysis producing an increased cell lactate and a decreased pH.     

Mechanical and electrical adaptation of skeletal muscle to gravitational unloading

The physiological and biochemical properties of limb skeletal muscle have been shown to adapt to variety of experimental conditions. Among these is the microgravity encountered with spaceflight. It is adaptations accompanying skeletal muscle disuse atrophy. Foremost among these changes is a reduction in the force-generating capacity, which is presumably a direct result of decrease in fiber number and diameter. These changes suggest a spaceflight-induced reduction in muscle work capacity. The interesting finding that the reduction of the mechanical tension is not proportional to the reduction of muscle weight, fiber diameter, and concentration of contractile protein suggested that changes of electrical activity might contribute to the reduction of the contraction force in disused muscle. The purpose of our study was to assess the effects of a 7-d "dry" immersion on the contractile properties of the triceps surae muscle.     

Human soleus fibers contractile characteristics and sarcomeric cytoskeletal proteins after gravitational unloading. Contribution of support stimulus

The effects of support withdrawal and support stimulation on the contractile characteristics of human soleus fibers and cellular factors which influence them were studied. The experimental model of the "dry" head-out water immersion was used in the study. In this model, the hydrostatic pressure on different sites of the body surface are equal so that the experimental conditions are close to the complete supportlessness. A 7-day exposure to dry immersion resulted in a decrease in the maximal isometric tension of the skinned fibers, a decline in the myofibrillar Ca2+-sensitivity, and the relative loss of the titin and nebulin content. A significant decrease in the percentage of fibers containing slow myosin heavy chains was also observed after dry immersion. The application of the mechanical stimulator influencing the plantar support zones with a pressure of 0.2 +/- 0.15 kg/cm2 6 times a day for 20 minutes of each hour brought about a complete prevention of the above listed effects of dry immersion. The data obtained allow one to conclude that the decline in maximal tension and Ca2+-sensitivity as well as myosin shift and loss of sarcomeric cytoskeletal proteins are associated with the support withdrawal during the exposure to dry immersion.     

Changes in the activity of succinate dehydrogenase in muscle fibers of functionally different muscles in the rat after a reduction in exertion--hypodynamia

In 105 male rats of Wistar strain distal parts of one of the thoracic extremities are amputated with keeping intact the places where the brachial muscle is fixed. This does not restrict the volume of the brachium movements but essentially decreases their dynamic component (power loading). For 45 days dynamics of succinate-dehydrogenase (SDG) activity is being revealed in muscle fibers (MF) of functionally different muscles: m. brachialis, m. serratus ventralis and m. triceps brachii (the medial head). Average tendency of the process, changes in the distribution margins, asymmetry and kurtosis are taken into account. Under hypodynamia reconstruction of the MF has a wavy character with a gradually longer period of fluctuations. In all the muscles appear MF with a greater than in the control SDG activity. In the medial head of the m. triceps brachii the fibers with the lowest SDG activity disappear. The amount of MF with the lowest activity decreases, while those with the higher--increases, the process being more pronounced in the m. triceps brachii. The amount of MF with middle activity of the enzyme remains nearly unchanged. MF with different initial enzymatic activity do not change simultaneously. The degree of the changes in the fibers and the power leading are connected with each other, the fibers with the low initial SDG activity including into the reaction at a sharper decrease of the latter. The changes in quantitative ratio of MF with different SDG activity are not the same in every muscle studied.     

Mitochondrial adaptations in skeletal muscle cells in mammals exposed to gravitational unloading

It is known that exposure to actual or simulated weightlessness is often accompanied by decreased muscle dynamic performance, and increased level of blood lactate accumulation. Decreased mitochondrial content found in fibers of the working muscles is considered to be one of the possible causes for those changes. Studies on oxidative potential of the muscle cell (i.e. capacity of the cell to oxidative energy production) under conditions of altered gravity have been carried out since late 70-ties. It was shown that the relatively short term spaceflight and hindlimb suspension induced significant decrease oxidative enzyme activities and mitochondrial volume density in rat fast muscle. However postural soleus muscle failed to exhibit similar changes, although the absolute mitochondrial content was found to be sufficiently lower after exposure to simulated microgravity. This phenomenon allowed to conclude that the pronounced soleus fiber atrophy masked the proportional absolute decrease in oxidative potential which failed to be revealed as subsequent changes in mitochondrial volume density and oxidative enzyme activity. It is also important, that biosatellite studies exposed considerable changes in mitochondria distribution pattern inside m. soleus fibers: volume density of mitochondria (and, correspondingly, activity of oxidative enzymes) increases (or does not change) in the center of fiber, and decreases at its periphery, in subsarcolemmal area. However the time course of mitochondrial alterations development (particularly during long-duration exposures to real or simulated microgravity) and some peculiarities of the mitochondria distribution were not described yet. Also, materials dealing with simultaneous time-course comparative analysis of mitochondrial characteristics and indices of physiological cost of submaximal exercise are very rare. The present paper is purposed to compare the data, obtained in several experimental studies, allowed to analyze the possible contribution of muscle mitochondria changes to changes in metabolic cost of submaximal exercise and the time-course dynamics of mitochondrial characteristics under conditions of actual or simulated gravitational unloading.     

Afferent and peripheral control of muscle fiber properties during gravitational unloading

The present paper covers two series of the experiment studies performed in attempt to analyze the support-triggered cellular mechanisms, controlling the maintenance of tonic muscle fiber characteristics. Exposure to 7 day dry immersion induced significant decline of the human soleus single fiber peak isometric tension and the Ca(2+)-sensitivity of myofibrils. 30-40% losses of the relative content of titin and nebulin were found after immersion. The application of the plantar support stimulation device prevented all these alterations. In the second experimental series the treatment of hindlimb suspended rats with the Ca(2+)-binding agent (EGTA) allowed to prevent or attenuate all the above mentioned unloading-induced soleus fiber alterations. Thus it is concluded that resting Ca2+ accumulation in the unloaded fibers may be among the mechanisms involved in the changes of fiber properties during unloading.     

Measuring mitochondrial respiration in intact single muscle fibers

Measurement of mitochondrial function in skeletal muscle is a vital tool for understanding regulation of cellular bioenergetics. Currently, a number of different experimental approaches are employed to quantify mitochondrial function, with each involving either mechanically or chemically induced disruption of cellular membranes. Here, we describe a novel approach that allows for the quantification of substrate-induced mitochondria-driven oxygen consumption in intact single skeletal muscle fibers isolated from adult mice. Specifically, we isolated intact muscle fibers from the flexor digitorum brevis muscle and placed the fibers in culture conditions overnight. We then quantified oxygen consumption rates using a highly sensitive microplate format. Peak oxygen consumption rates were significantly increased by 3.4-fold and 2.9-fold by simultaneous stimulation with the uncoupling agent, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and/or pyruvate or palmitate exposure, respectively. However, when calculating the total oxygen consumed over the entire treatment, palmitate exposure resulted in significantly more oxygen consumption compared with pyruvate. Further, as proof of principle for the procedure, we isolated fibers from the mdx mouse model, which has known mitochondrial deficits. We found significant reductions in initial and peak oxygen consumption of 51% and 61% compared with fibers isolated from the wild-type (WT) animals, respectively. In addition, we determined that fibers isolated from mdx mice exhibited less total oxygen consumption in response to the FCCP + pyruvate stimulation compared with the WT mice. This novel approach allows the user to make mitochondria-specific measures in a nondisrupted muscle fiber that has been isolated from a whole muscle.     

Quantitative succinate dehydrogenase analysis in normal and ragged-red muscle fibers

Summary Single fiber analyses were performed in normal and diseased muscle by means of a high-resolution microphotometric method. We investigated the activity distribution of a mitochondrial marker enzyme, succinate dehydrogenase, within single muscle fibers. We differentiated between the central and the subsarcolemmal region. Both normal muscle fibers, and ragged-red fibers from patients with a mitochondrial myopathy showed significantly higher succinate dehydrogenase activities in the subsarcolemmal region. Since the fibers' supply of oxygen is accomplished by diffusion from capillaries located close to the sarcoplasmic membrane our results are of functional importance.     

Quantitative succinate dehydrogenase analysis in normal and ragged-red muscle fibers

Single fiber analyses were performed in normal and diseased muscle by means of a high-resolution microphotometric method. We investigated the activity distribution of a mitochondrial marker enzyme, succinate dehydrogenase, within single muscle fibers. We differentiated between the central and the subsarcolemmal region. Both normal muscle fibers, and ragged-red fibers from patients with a mitochondrial myopathy showed significantly higher succinate dehydrogenase activities in the subsarcolemmal region. Since the fibers' supply of oxygen is accomplished by diffusion from capillaries located close to the sarcoplasmic membrane our results are of functional importance.     

Expression and functional behavior of troponin C in soleus muscle fibers of rat after hindlimb unloading.

Troponin C (TnC) plays a key role in the regulation of muscle contraction, thereby modulating the Ca(2+)-activation characteristics of skinned muscle fibers. This study was performed to assess the effects of a 15-day hindlimb unloading (HU) period on TnC expression and its functional behavior in the slow postural muscles of the rat. We investigated the TnC isoform expression in whole soleus muscles and in single fibers. The latter were also checked for their Ca(2+) activation characteristics and sensitivity to bepridil, a Ca(2+) sensitizer molecule. This drug has been described as exerting a differential effect on slow and fast fibers, depending on the TnC isoform. With regard to TnC expression, three populations were found in control muscle fibers: slow, hybrid slow, and hybrid fast fibers, with the TnC fast being always coexpressed with TnC slow. In the whole muscle, TnC fast expression increased after HU because of the increase in the proportion of hybrid fast fibers. The HU hybrid fast fibers had properties similar to those of control hybrid fast fibers. The fibers that remained slow after HU exhibited similar bepridil and Sr(2+) properties as control slow fibers. Therefore, in these fibers, the changes could not be related to the TnC molecule.     

Effect of deafferentation on the size and myosin phenotype of muscle fibers during stretching of rat m. soleus and gravitational unloading

The study was purposed to evaluate the contribution of the reflectory and local components during the chronic stretch of the postural muscle to the attenuation of the unloading-induced fiber size reduction and changes in the myosin heavy chain (MHC) profile. The surgical unilateral deafferentation (dorsal rhizotomy) was used. It was shown that unilateral deafferentation didn't influence on the amelioration of unloading-induced fiber size reduction in chronically stretched soleus muscle. Thus, the results obtained in the present study don't confirm the hypothesis, supposing the predominant contribution of the muscle afferent activation to the attenuation of unloading-induced fiber atrophy. Deafferentation of unloaded as well as control rats leads to the increase of the percentage of fibers expressing slow MYC isoforms.     

Energetic driving forces are maintained in resting rat skeletal muscle after dietary creatine supplementation

The total creatine(TCr) pool of skeletal muscle is composed of creatine (Cr) andphosphocreatine (PCr). In resting skeletal muscle, the ratio ofPCr to TCr (PCr/TCr; PCr energy charge) is ~0.6-0.8, dependingon the fiber type. PCr/TCr is linked to the cellular free energy of ATPhydrolysis by the Cr kinase equilibrium. Dietary Cr supplementationincreases TCr in skeletal muscle. However, many previous studies havereported data indicating that PCr/TCr falls after supplementation,which would suggest that Cr supplementation alters the restingenergetic state of myocytes. This study investigated the effect of Crsupplementation on the energy phosphates of resting skeletal muscle.Male rats were fed either rodent chow (control) or chow supplementedwith 2% (wt/wt) Cr. After 2 wk on the diet, the gastrocnemius andsoleus muscles were freeze clamped and removed from anesthetizedanimals. Cr supplementation increased TCr, PCr, and Cr levels in thegastrocnemius by 20, 22, and 17%, respectively (P < 0.05). A numerical 6% higher mean soleus TCr in Cr-supplemented ratswas not statistically significant. All other energy phosphate concentrations, free energy of ATP hydrolysis, and PCr/TCr were notdifferent between the two groups in either muscle. We conclude that Crsupplementation simply increased TCr in fast-twitch rat skeletal musclebut did not otherwise alter resting cellular energetic state.