Endospanin-2 enhances skeletal muscle energy metabolism and running endurance capacity.

Metabolic stresses such as dietary energy restriction or physical activity exert beneficial metabolic effects. In the liver, endospanin-1 and endospanin-2 cooperatively modulate calorie restriction-mediated (CR-mediated) liver adaptations by controlling growth hormone sensitivity. Since we found CR to induce endospanin protein expression in skeletal muscle, we investigated their role in this tissue. In vivo and in vitro endospanin-2 triggers ERK phosphorylation in skeletal muscle through an autophagy-dependent pathway. Furthermore, endospanin-2, but not endospanin-1, overexpression decreases muscle mitochondrial ROS production, induces fast-to-slow fiber-type switch, increases skeletal muscle glycogen content, and improves glucose homeostasis, ultimately promoting running endurance capacity. In line, endospanin-2-/- mice display higher lipid peroxidation levels, increased mitochondrial ROS production under mitochondrial stress, decreased ERK phosphorylation, and reduced endurance capacity. In conclusion, our results identify endospanin-2 as a potentially novel player in skeletal muscle metabolism, plasticity, and function.

Early postnatal motor experience shapes the motor properties of C57BL/6J adult mice.

This study aimed to evaluate the long-term consequences of early motor training on the muscle phenotype and motor output of middle-aged C57BL/6J mice. Neonatal mice were subjected to a variety of motor training procedures, for 3 weeks during the period of acquisition of locomotion. These procedures are widely used for motor training in adults; they include enriched environment, forced treadmill, chronic centrifugation, and hindlimb suspension. At 9 months, the mice reared in the enriched environment showed a slower type of fibre in slow muscles and a faster type in fast muscles, improved performance in motor tests, and a modified gait and body posture while walking. The proportion of fibres in the postural muscles of centrifuged mice did not change, but these mice showed improved resistance to fatigue. The suspended mice showed increased persistence of immature hybrid fibres in the tibialis, with a slower shift in the load-bearing soleus, without any behavioural changes. The forced treadmill was very stressful for the mice, but had limited effects on motor output, although a slower profile was observed in the tibialis. These results support the hypothesis that motor experience during a critical period of motor development shapes muscle phenotype and motor output. The different impacts of the various training procedures suggest that motor performance in adults can be optimized by appropriate training during a defined period of motor development.

Dendritic spine remodeling induced by hindlimb unloading in adult rat sensorimotor cortex.

A sensorimotor restriction, for instance in patients confined to bed, induces an impairment in motor function, which could be due to structural and functional reorganization of the sensorimotor cortex. Hindlimb unloading (HU) is a rodent model used to reproduce the chronic weightless bearing and reduction in hindlimb movement. In this study, we determined whether a 14-day period of HU in adult rats leads to dendritic spine plasticity. For this purpose, we visualized a large number of spines on pyramidal neurons located in superficial and deep layers of the cortex within the hindpaw representation area, by means of confocal microscopy. Spines were classified according to their shape, as stubby, thin, mushroom, or filopodium. Spine density was increased (+26%) after HU. The increase concerned mainly filopodium spines (+82%) and mushrooms (+33%), whereas no change was noticed for stubby and thin spines. Spine length was decreased, whatever their shape. Head diameter evolved differently depending on the layer: it was increased in superficial layers and decreased in deeper ones. These results indicate that morphological changes accompany functional reorganization of motor cortex in response to a decrease in sensorimotor function during adulthood.

Resveratrol prevents the wasting disorders of mechanical unloading by acting as a physical exercise mimetic in the rat.

Long-term spaceflight induces hypokinesia and hypodynamia, which, along microgravity per se, result in a number of significant physiological alterations, such as muscle atrophy, force reduction, insulin resistance, substrate use shift from fats to carbohydrates, and bone loss. Each of these adaptations could turn to serious health deterioration during the long-term spaceflight needed for planetary exploration. We hypothesized that resveratrol (RES), a natural polyphenol, could be used as a nutritional countermeasure to prevent muscle metabolic and bone adaptations to 15 d of rat hindlimb unloading. RES treatment maintained a net protein balance, soleus muscle mass, and soleus muscle maximal force contraction. RES also fully maintained soleus mitochondrial capacity to oxidize palmitoyl-carnitine and reversed the decrease of the glutathione vs. glutathione disulfide ratio, a biomarker of oxidative stress. At the molecular level, the protein content of Sirt-1 and COXIV in soleus muscle was also preserved. RES further protected whole-body insulin sensitivity and lipid trafficking and oxidation, and this was likely associated with the maintained expression of FAT/CD36, CPT-1, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in muscle. Finally, chronic RES supplementation maintained the bone mineral density and strength of the femur. For the first time, we report a simple countermeasure that prevents the deleterious adaptations of the major physiological functions affected by mechanical unloading. RES could thus be envisaged as a nutritional countermeasure for spaceflight but remains to be tested in humans.

Activity-dependent changes in the electrophysiological properties of regular spiking neurons in the sensorimotor cortex of the rat in vitro.

Sensorimotor performance is highly dependent on the level of physical activity. For instance, a period of disuse induces an impairment of motor performance, which is the result of combined muscular, spinal and supraspinal mechanisms. Concerning this latter origin, our hypothesis was that intrinsic properties and input/output coupling of cells within the sensorimotor cortex might participate to the alteration in cortical motor control. The aim of the present study was thus to examine the basic electrophysiological characteristics of cortical cells in control rats and in animals submitted to 14 days of hindlimb unloading, a model of sensorimotor deprivation. Intracellular recordings were obtained in vitro from coronal slices from cortical hindpaw representation area. We have also made an attempt to determine the morphological characteristics as well as the location of the investigated neurons by biocytin labelling. Passive properties of neurons were affected by hindlimb unloading: input resistance and time constant were decreased (-20%), the rheobase was increased (+34%), whereas the resting potential was unchanged. The frequency-current relationships were also modified, the curve being shifted towards right. The size of body area of recorded neurons was unchanged in unloaded rats. Taken together, these data reflect a decrease in excitability of cortical cells in response to a decreased cortical activation.

Activity-dependent regulation of myelin maintenance in the adult rat.

Hindlimb unloading (HU) is known to induce changes in the neuromuscular system. However, no data describing the effects of HU on morphological characteristics of peripheral nerve have been reported so far. Therefore, we used soleus and radial nerves obtained from control and rats submitted to 14 days of HU to study the consequences of a decrease (soleus) or an increase (radial) in neural activity on its morphology. The mean number of fibers was not changed after HU. The soleus nerve axon diameter was weakly affected after HU, whereas the myelin thickness was reduced. For the radial nerve, both axon and fiber diameter were increased, and the myelin thickness and internodal distance were higher in HU rats. These results suggest that regulation of myelin maintenance undergoes plastic mechanisms. Neural activity and/or neural pattern might be essential in the maintenance of myelin sheath in adults.

Concentration of amino acid neurotransmitters in the somatosensory cortex of the rat after surgical or functional deafferentation.

Hindlimb unloading is considered as a model of functional deafferentation, since in this situation the tactile information from the paw and the proprioceptive input from the limb are dramatically reduced. Unloading induces a shrinkage of the cortical representation of the affected body part associated to a reorganization of topographic maps and to an expansion of receptive fields. Previous studies have suggested that cortical plasticity was the result of a change in the balance of excitation and inhibition in the cortex. The aim of the present study was thus to determine whether deafferentation of the hindlimb representation in the somatosensory cortex, by 14 days of unloading or by surgical means (selective dorsal rhizotomy during 17 days), can change the concentration in various amino acid neurotransmitters in the deprived cortex. The present findings indicate that both types of deafferentation result in a decrease in inhibitory amino acids (GABA, taurine) without significant changes in the main excitatory amino acid (glutamate). In conclusion, the present results support the idea that cortical changes are more likely due to a release from inhibition than to an increased excitation.

The structure and response properties of Golgi tendon organs in control and hypodynamia-hypokinesia rats.

The Golgi tendon organs (GTOs) are encapsulated mechano-receptors that, in normal conditions, monitor via Ib afferent fibers the contractile force. A 14-day period of hypodynamia, absence of weight bearing and hypokinesia, and reduction of motor activity (HH) is known to induce changes in postural muscles such as the soleus. At present, there is no data available regarding the Ib afferent feedback in normal rats (CONT group) and in rats after a hypodynamia-hypokinesia (HH group) period. Consequently, the aim of our study was to determine the HH effects on the morphological (histochemistry on gross morphology) and electrophysiological properties of the GTOs in rat soleus muscle. In the histological study, nine CONT and nineteen HH GTOs of the soleus muscle were identified. The results demonstrated that HH GTOs were morphologically similar to the CONT GTOs. Regarding the electrophysiological study, a L2-L6 laminectomy was performed under deep anesthesia (sodium pentobarbital, 60 mg kg(-1)). Responses in single Ib fibers from the L5 dorsal root to the isometric twitch and tetanic fused contractions of "in-series" motor units (MUs) were recorded. Twenty-three and twenty-eight GTO/MU pairs were studied in the CONT and HH groups, respectively. In the HH group, the Ib afferent response exhibited a decrease in dynamic peak for the high stimulation frequencies and an increase in static sensitivity for all stimulation frequencies. Our results suggest that after an HH period, the GTOs continue to fulfil their mechano-sensory function to signal the contractile force but with a higher static sensitivity.

Hypergravity from conception to adult stage: effects on contractile properties and skeletal muscle phenotype.

This study examined the effects of an elevation of the gravity factor (hypergravity--2 g) on the molecular and functional characteristics of rat soleus and plantaris muscles. Long Evans rats were conceived, born and reared (CBR) continuously in hypergravity conditions until the age of 100 days. Whole muscle morphological parameters, Ca2+ activation characteristics from single skinned fibers, troponin (Tn) subunit and myosin heavy (MHC) and light (MLC) chains isoform compositions were examined in CBR and control muscles from age-paired terrestrial rats. Decreases in body and muscle mass in soleus and plantaris muscles were observed and associated, in the soleus, with a decrease in fiber diameter. The specific force of CBR soleus fibers was increased, and correlated with the elevation of Ca2+ affinity. This was accompanied by slow-to-slower TnC and TnI isoform transitions and a rearrangement in TnT fast isoform content. The MHC transformations of the soleus after hypergravity were associated with the up (down)-regulation of the MHCI (MHCIIa) mRNA isoforms. The MLC2 phosphorylation state remained unchanged in the soleus muscle. The results suggested that the gravity factor could interact with rat muscle development and that hypergravity experiments could provide good tools for the study of myofibrillar protein plasticity and their associated pathways of regulation.

Effects of unilateral and bilateral labyrinthectomy on rat postural muscle properties: the soleus.

The aim of our study was to determine whether the suppression of the vestibular inputs could have effects on the soleus muscle properties similar to the modifications observed after an episode of microgravity. The inner ear lesion was performed by surgical labyrinthectomy. Twenty-nine male Wistar rats were used for this study and were divided into three experimental groups: control (CONT, n=7), unilateral labyrinthectomized (UL, n=14) and bilateral labyrinthectomized (BL, n=8). Mechanical, histochemical and electrophoretic parameters were determined 17 days after the operation. Furthermore, electromyographic (EMG) activity of the soleus muscle was examined at 1 h, 1 day and 17 days. Our results showed that UL and BL groups did not present any sign of muscle atrophy when compared to CONT group. However, the contractile and phenotypical characteristics of UL and BL soleus muscles revealed that the muscle evolved from slow toward a slower type. This transition was correlated with a more tonic EMG activity pattern. To conclude, our data demonstrated that soleus muscle transformations observed after microgravity (muscle atrophy, slow-to-fast transition, phasic EMG activity) were not directly the consequence of a vestibular silence.

Expression of Myosin heavy chain isoforms in rat soleus muscle spindles after 19 days of hypergravity.

The aim of this study was to determine whether a period of 19 days in hypergravity was long enough to induce changes in the expression of myosin heavy chain (MyHC) isoforms in the muscle spindles. The soleus muscle of 10 male Wistar rats (control: CONT, n=5; hypergravity: HG, n=5) was frozen, cut into serial sections, and labeled with antibodies against MyHCs: I, IIA, IIA + IIX + IIB, slow-tonic, and alpha-cardiac. Forty CONT and 45 HG spindles were analyzed. The results from HG spindles compared to CONT showed that there was no change in the cross-sectional area of intrafusal fibers. However, along the entire length of B1 fibers, the expression of both MyHC I and alpha-cardiac was increased significantly, whereas the labeling against MyHC IIA and MyHC slow-tonic was decreased. In B2 fibers, the labeling against MyHC IIA (region A), slow-tonic (region A), and fast myosins (regions A-C) was statistically decreased. In chain fibers, the labeling against both MyHC IIA and fast MyHC was reduced significantly. We conclude that hypergravity has a real impact on the MyHC content in the muscle spindles and induces some inverse changes of those observed in hypogravity for MyHCs I, alpha-cardiac, and slow-tonic.

p53 inactivation leads to impaired motor synchronization in mice.

We have combined genetic and pharmacological approaches to investigate the behavioural consequences of inactivation of the murine p53 protein. Our behavioural analysis revealed that p53-null mice (p53KO) exhibit a very specific and significant motor deficit in rapid walking synchronization. This deficit, observed using the rotarod test, was the only behavioural defect of p53KO mice. We demonstrated that it was not due to an increase in neuronal number or abnormal connectivity in the olivo-cerebellar system, thought to control motor synchronization. In order to test the role of p53 in the central nervous system, we injected a pharmacological inhibitor of p53 activation, pifithrin-alpha, into the cerebellum of wild-type mice. This treatment mimicked the walking synchronization deficit of p53KO mice, suggesting that presence of p53 protein in the cerebellum is necessary to execute this synchronization of walking. Our investigation reveals a functional role of cerebellar p53 protein in adult walking synchronization.

Compared effects of hindlimb unloading versus terrestrial deafferentation on muscular properties of the rat soleus.

Hindlimb unloading is known to induce some neuromuscular changes especially in postural muscles such as the soleus. Our goal was to determine the role of proprioceptive inputs on these modifications by comparing soleus muscle properties of rats being either hindlimb unloaded or terrestrial deafferented. Under deep anesthesia, a first group of rats were submitted to a bilateral deafferentation (DEAF group, n = 6) performed by section of the dorsal roots L(3) to L(5) after laminectomy. A second group of rats was submitted to a hindlimb-unloading period (HU group, n = 6). After 14 days, the morphological and contractile properties as well as the content in myosin heavy-chain (MHC) isoforms were studied in the right soleus muscle in HU and DEAF groups. The results were compared to those obtained in control animals (CON group). After HU versus CON group, the soleus muscle was atrophied and presented a decrease in muscle forces in relation with a slow-to-fast transition characterized by a decrease in kinetic contractile parameters and by an overexpression in the fast MHC isoforms. The DEAF soleus muscle showed both a significant muscle atrophy and a loss of forces when compared with CON rats. The comparison between DEAF and HU rats indicated that some modifications occurring after HU are purely of motor origin (i.e., slow to fast transition), whereas muscle atrophy and decrease in muscle force are partly the result of an afferent silent. Our study underlined the importance of afferent input integrity in the maintenance of muscle characteristics.

Effects of hypodynamia-hypokinesia on the muscle spindle discharges of rat soleus muscle.

The aim of this study was to determine whether Ia and II fiber discharges of soleus muscle spindles were modified after a 14-day period of hypodynamia (absence of weight bearing) and hypokinesia (reduction of motor activity). Fifty-one and 38 afferent fibers were studied, respectively, in control and hypodynamia-hypokinesia (HH) groups. Under deep anesthesia (pentobarbital, 30 mg/kg), a L3-L6 laminectomy was performed. Unitary potentials from the L5 dorsal root were recorded in response to ramp-and-hold stretches applied at two stretch amplitudes (3 and 4 mm) and four stretch velocities (6, 10, 15, and 30 mm/s) and to sinusoidal stretches applied at four stretch amplitudes (0.12, 0.25, 0.5, and 1 mm) and six stretch frequencies (0.5, 1, 2, 3, 6, and 10 Hz). In both animal groups, the Ia fibers showed higher dynamic index values, smaller linear range, and higher vibration sensitivity than the II fibers. They also exhibited a pause in their discharges during the stretch release contrary to II fibers, which displayed no pause in their responses. After HH, our results showed that for both fiber types all parameters measured under ramp-and-hold stretches (except the static sensitivity) were significantly increased and under sinusoidal stretches, the vibration sensitivity increased, and the response amplitude only increased at 0.12-mm stretch amplitude. The linear range of Ia afferents was limited to 0.12 mm, whereas it was unchanged for the II fibers. After HH, the stretches could be better transmitted to the muscle spindles, probably resulting from changes in passive mechanical properties of the soleus.

Characterization of spindle afferents in rat soleus muscle using ramp-and-hold and sinusoidal stretches.

The discharge properties of 51 afferents were studied in the rat soleus muscle spindles. Under deep anesthesia using a pentobarbital sodium solution (30 mg/kg), a laminectomy was performed and the right L(4) and L(5) dorsal and ventral roots were transected near their entry into the spinal cord. In situ, the minimal (L(min)) muscle length [3 +/- 0.08 (SE) cm] of the soleus was measured at full ankle extension. Unitary potentials from the L(5) dorsal root were recorded in response to ramp-and-hold stretches applied at 3 mm (S3) and 4 mm (S4) amplitudes and four stretch velocities (6, 10, 15, and 30 mm/s), sinusoidal stretches performed at four amplitudes (0.12, 0.25, 0.5, and 1 mm) and six stretch frequencies (0.5, 1, 2, 3, 6, and 10 Hz), and vibrations applied at 50-, 100-, and 150-Hz frequencies. These two kinds of stretches were performed at three different muscle lengths (L(min+10%), L(min+15%), and L(min+20%)), whereas vibrations were applied at L(min+20%) muscle length. Conduction velocity of the fibers was calculated but did not allow to discriminate different fiber types. However, the mean conduction velocity of the first fiber group (43.3 +/- 0.8 m/s) was significantly higher than that of the second fiber group (33.9 +/- 0.9 m/s). Three parameters allowed to differentiate the responses of primary and secondary endings: the dynamic index (DI), the discharge during the stretch release from the ramp-and-hold stretches, and the linear range and the vibration sensitivity from sinusoidal stretches. The slope histogram of the linear regression based on the DI and the stretch velocity was clearly bimodal. Therefore the responses were separated into two groups. During the stretch release at a velocity of 3 mm/s, the first response group (n = 26) exhibited a pause, whereas the second (n = 25) did not. The linear range of the second ending group (0.12-1 mm) was broader than that of the first (0.12-0.25 mm). The first ending group showed a higher sensitivity to high-vibration frequencies of small amplitude than the second. In comparison with the literature, we can assert that the first and the second ending groups corresponded to the primary and secondary endings, respectively. In conclusion, our study showed that in rat soleus muscle spindles, it was possible to immediately classify the discharge of Ia and II fibers by using some parameters measured under ramp-and-hold and sinusoidal stretches.