Rat Fast Muscles Research Articles

Motor Unit Force Potentiation and Calcium Handling Protein Concentration in Rat Fast Muscle After Resistance Training.

Post-tetanic potentiation (PTP) of force depends on intramuscular Ca2+ levels and sensitivity and may be affected by fatigue. The aim of this study was to determine the ability of isolated fast fatigue-resistant (FR) and fast-fatigable (FF) motor units (MUs) to potentiate force evoked with single and 40-Hz electrical stimulation after 5 weeks of voluntary weight-lifting training. Tetanic contractions evoked by gradually increasing (10–150 Hz) stimulation frequency served as conditioning stimulation. Additionally, the concentration of myosin light chain kinase and proteins engaged in calcium handling was measured in rat fast medial gastrocnemius muscle. After the training, the potentiation of twitch force and peak rate of force development was increased in FF but not FR MUs. Force potentiation of 40-Hz tetanic contractions was increased in both fast MU types. After the training, the twitch duration of FR MUs was decreased, and FF MUs were less prone to high-frequency fatigue during conditioning stimulation. Muscle concentration of triadin was increased, whereas concentrations of ryanodine receptor 1, junctin, FKBP12, sarcoplasmic reticulum calcium ATPase 1, parvalbumin, myosin light chain kinase, and actomyosin adenosine triphosphatase content were not modified. After short-term resistance training, the twitch contraction time and twitch:tetanus force ratio of FR MUs are decreased, and PTP ability is not changed. However, PTP capacity is increased in response to submaximal activation. In FF MUs increase in PTP ability coexists with lesser fatigability. Further work is required to find out if the increase in triadin concentration has any impact on the observed contractile response.

The effect of modelling of hypogravity on postsynaptic acetylcholine receptors and activity of acetylcholinesterase in neuromuscular synapses of fast and slow muscles of rat

Rats were subjected to antiorthostatic hindlimb unloading for 35 days. Using immunofluorescent techniques, we found increased intensity of fluorescence and decreased area of staining of acetylcholine receptors and the increased intensity and area of staining of acetylcholinesterase in neuromuscular synapses of “fast” and “slow” muscles. Changes in the ratio of the number of acetylcholine receptors on the postsynaptic membrane and acetylcholinesterase and the alterations in their spatial location relative to each other in neuromuscular synapses of “fast” and “slow” muscles were also observed. These modifications are in accordance with the electrophysiological data on the decreased amplitudes of miniature end plate currents in both muscles. They were accompanied by a decrease in the volume of muscle fibers. Antiorthostatic unloading, a model of hypogravity, resulted in increased functional activity of acetylcholinesterase associated with decreased area of the postsynaptic membrane occupied by acetylcholine receptors, which led to a decrease in the amplitude of postsynaptic excitatory potentials and thus, to the decreased reliability threshold of excitation transmission from a nerve to a muscle.

The role of chloride ions in the maintenance of resting membrane potential in rat fast and slow muscles during hypogravity modeling.

Antiorthostatic hindlimb suspension reduces resting membrane potential of rat fast and slow muscles within 7 days. Changes in Na+/K+ pump activity and shifts of the equilibrium potential for chloride ions are the main mechanism of the changes in the resting potential of muscle fibers. The latter is presumably associated with increased intracellular ion current due to activation of the second active Na+, K+, 2Cl- symport. Reduction of the membrane potential is related to muscle denervation. However, membrane depolarization of muscle fibers during antiorthostatic suspension cannot be explained solely by changes in the mechanisms of neurotrophic control from motor neurons.

Collective effects of low cell pH and inorganic phosphate on force, velocity, stiffness, and power in rat slow and fast muscle fibers (1102.7)

Muscle fatigue is a loss of power resulting from a decline in both force and velocity. Intense exercise induces high rates of ATP hydrolysis and glycolysis, increasing inorganic phosphate (Pi) and H+. These factors are thought to induce fatigue by interfering with key steps in the cross bridge cycle, including ATP hydrolysis, ADP release, and rate and extent of cross‐bridge transition from the low‐ to high‐force state. Skinned fibers from rat soleus (slow) and gastrocnemius (fast) were isolated, and the combined effects of elevated H+ (pH 6.2) and Pi (30 mM) on force, velocity, stiffness, and power at low (15°C) and high (30°C) temperatures studied. At 30°C, pH 6.2 plus 30 mM Pi depressed force by 39% (158 to 97 kN/m2) in slow fibers and 42% (189 to 111 kN/m2) in fast fibers. Velocity was depressed by 17% (Vmax 4.23 to 3.56 Fl/s) in slow fibers and 44% (6.91 to 3.88 Fl/s) in fast fibers. The a/Po ratio was depressed in slow fibers but increased in fast fibers in pH 6.2, 30 mM Pi conditions, resulting in a greater power loss in slow (67%) versus fast (58%) fibers. Force‐stiffness plots normalized to rigor tension and stiffness show a higher number of low‐force cross bridges in fast versus slow fibers in pH 6.2, 30 mM Pi conditions. Our results show that the effects of elevated H+ and Pi are fiber‐type specific, and the larger decrease in force and velocity in type II fibers may in part be explained by a higher population of low‐force cross bridges.Grant Funding Source: Supported by a Marquette University Way Klinger Fellowship to RH Fitts

The Response of Denervated Muscle to Long-Term Electrical Stimulation.

Adapted from: Lømo T, Westgaard RH, Hennig R, Gundersen K. The response of denervated muscle to long-term electrical stimulation, In: Carraro U, Angelini C, eds. Proceedings of the First Abano Terme Meeting on Rehabilitation, 1985 August 28-30, Abano Terme, Padova, Italy, An International Symposium, Satellite Meeting of the XIII World Congress of Neurology, Hamburg 1985. Cleup Padova 1985. pp 81–90.

Expression of microRNA-1/133a in denervated skeletal muscle preliminary study

Objective To investigate the expression of MicroRNA-1/133a in denervated skeletal muscle at different periods.Methods From April 2012 to December 2012,a total of 54 Sprague-Dawle rats weighted (200 ± 10) g were used.They were randomly divided into 9 groups,each group of 6.The right sciatic nerve was cut off in rats (experimental group),with the left one being sham operation(control group)at various time points(0 h,8 h,1 d,3 d,1 weeks,2 weeks,3 weeks,4 weeks,8 weeks).Then after rats were sacrificed by spinal dislocation,the muscle sample(gastrocnemius,extensor digitorum longus and soleus)were harvested and weighed,then stored at-70 degrees.MicroRNA-1/133a were detected in each muscle specimens by RT-PCR.The ultrastructural changes of each muscle specimens at different periods were observed by electron microscopy.Results The muscle wet weight ratio of experimental group decreased gradually over time.Observed by electron microscopy,the different types of muscle fiber were disordered arrangement at different time points.RT-PCR was performed to quantitatively analyze miRNA-1/133a expressions of the fast-twitch muscles (i.e.,gastrocnemius and extensor digitorum longus)and slow-twitch muscle (soleus).In the control group,miRNA-133 and miRNA-1 expression in the soleus muscles were maintained at a certain level,whereas in the experimental group,these first decreased and then increased,with the lowest at 4 weeks and the highest at 8 weeks.For the gastrocnemius and extensor digitorum longus muscles in the experimental group,the expressions changed with time,whereas in the control group,these were maintained at a certain level.In the experimental group,miRNA-133 at 1 week was the lowest and was always low for all time points,whereas in the experimental group,miRNA-1 expression at 3 days was at a minimum level.The expression of MicroRNA-1/133a in the experimental group had significant differences than the control group (P ＜ 0.05).Conclusion MicroRNA-1/ 133a have differrent expression in rat fast and slow denervation muscle atrophy,presumably.It is a key point in the regulation of fast-twitch and slow-twitch muscle aspects. Key words: MicroRNA-1/133a; Denervation; Muscle atrophy; Rat

Influence of chronic food deprivation on structure–function relationship of juvenile rat fast muscles

In the present study, we analyze the influence of chronic undernutrition on protein expression, muscle fiber type composition, and fatigue resistance of the fast extensor digitorum longus (EDL) muscle of male juvenile rats (45 ± 3 days of life; n = 25 and 31 rats for control and undernourished groups, respectively). Using 2D gel electrophoresis and mass spectrometry, we identified in undernourished muscles 12 proteins up-regulated (8 proteins of the electron transport chain and the glycolytic pathway, 2 cross-bridge proteins, chaperone and signaling proteins that are related to the stress response). In contrast, one down-regulated protein related to the fast muscle contractile system and two other proteins with no changes in expression were used as charge controls. By means of COX and alkaline ATPase histochemical techniques and low-frequency fatigue protocols we determined that undernourished muscles showed a larger proportion (15% increase) of Type IIa/IId fibers (oxidative-glycolytic) at the expense of Type IIb (glycolytic) fibers (15.5% decrease) and increased fatigue resistance (55.3%). In addition, all fiber types showed a significant reduction in their cross-sectional area (slow: 64.4%; intermediate: 63.9% and fast: 61.2%). These results indicate that undernourished EDL muscles exhibit an increased expression of energy metabolic and myofibrillar proteins which are associated with the predominance of oxidative and Type IIa/IId fibers and to a higher resistance to fatigue. We propose that such alterations may act as protective and/or adaptive mechanisms that counterbalance the effect of chronic undernourishment.

Suppression of collagen Q expression in the extrajunctional regions of rat fast muscles is encoded in their stem cells (satellite cells)

In rat fast muscles, collagen Q (ColQ) expression is restricted to the neuromuscular junctions. In contrast, it is high also extrajunctionally in the slow soleus muscles. Fast muscles activated by chronic low-frequency electrical stimulation, similar to neural activation of the soleus muscles, did not increase their extrajunctional expression of ColQ. We assumed that the myogenic stem cells (satellite cells) in fast and slow muscles were intrinsically different in regard to the capacity that they convey to their respective muscle fibers to increase the extrajunctional ColQ expression upon innervation.ColQ mRNA levels were determined by quantitative real-time PCR. Extensive neural suppression of the extrajunctional ColQ expression in regenerating fast muscles during maturation is a very slow process requiring 30–60days. If the immature regenerating fast EDL muscles were indirectly or directly electrically stimulated immediately after innervation by chronic low-frequency impulse pattern for 8days, no significant increase of the extrajunctional ColQ mRNA levels was observed in stimulated regenerates in comparison to non-stimulated ones. In contrast, the extrajunctional ColQ mRNA levels in the regenerates of the soleus muscles, trans-innervated by the EDL nerve at the time of muscle injury, increased 4- to 5-fold after 8days of the same chronic low-frequency electrical stimulation in comparison to those in the stimulated EDL regenerates. Since both fast and slow muscles completely regenerated only from their own myogenic stem cells and were innervated by the same nerve and later activated by the same tonic pattern of impulses, these results demonstrated that the mechanism causing incapacity of regenerating fast muscles to increase their extrajunctional ColQ expression upon tonic activation is encoded in their satellite cells, which in this respect differ from those in the slow muscles.

Mechanism of force enhancement during and after lengthening of active muscle: a temperature dependence study

The aim of the present study was to examine the temperature dependence of active force in lengthening and shortening muscle. Experiments were done, in vitro, on bundles of intact fibres (fibre length L(0) ~2 mm; sarcomere length ~2.5 μm) isolated from a rat fast muscle (flexor hallucis brevis) and a ramp length change of 5-7% L(0) was applied on the plateau of an isometric tetanic contraction. Ramp lengthening increased and ramp shortening decreased the muscle tension to new approximately steady levels in a velocity-dependent way. The isometric tension and the lower steady tension reached at a given shortening velocity, increased with warming from 10 to 35 °C and the relation between tension and reciprocal absolute temperature was sigmoidal. However, the tension-temperature curve of shortening muscle was sharper and shifted to higher temperature with increased velocity. In contrast, the enhanced steady tension during lengthening at a given velocity was largely temperature-insensitive within the same temperature range; we hypothesize that the tension-temperature curve may be shifted to lower temperatures in lengthening muscle. Consequently, when normalised to the isometric tension at each temperature, the tension during lengthening at a given velocity decreased exponentially with increase of temperature. The residual force enhancement that remains after ramp lengthening showed a similar behaviour and was markedly reduced in warming from 10 to 35 °C. The findings are consistent with the thesis that active force generation in muscle is endothermic and strain-sensitive; during shortening with a faster crossbridge cycle it becomes more pronounced, but during lengthening it becomes depressed as the cycle slows in a velocity-dependent way. The residual force enhancement may be caused by the same process in addition to non-crossbridge mechanism(s).

PLASTICITY OF SKELETAL MUSCLE STUDIED BY STEREOLOGY

The present contribution provides an overview of stereological methods applied in the skeletal muscle research at the Institute of Anatomy of the Medical Faculty in Ljubljana. Interested in skeletal muscle plasticity we studied three different topics: (i) expression of myosin heavy chain isoforms in slow and fast muscles under experimental conditions, (ii) frequency of satellite cells in young and old human and rat muscles and (iii) capillary supply of rat fast and slow muscles. We analysed the expression of myosin heavy chain isoforms within slow rat soleus and fast extensor digitorum longus muscles after (i) homotopic and heterotopic transplantation of both muscles, (ii) low frequency electrical stimulation of the fast muscle and (iii) transposition of the fast nerve to the slow muscle. The models applied were able to turn the fast muscle into a completely slow muscle, but not vice versa. One of the indicators for the regenerative potential of skeletal muscles is its satellite cell pool. The estimated parameters, number of satellite cells per unit fibre length, corrected to the reference sarcomere length (Nsc/Lfib) and number of satellite cells per number of nuclei (myonuclei and satellite cell nuclei) (Nsc/Nnucl) indicated that the frequency of M-cadherin stained satellite cells declines in healthy old human and rat muscles compared to young muscles. To access differences in capillary densities among slow and fast muscles and slow and fast muscle fibres, we have introduced Slicer and Fakir methods, and tested them on predominantly slow and fast rat muscles. Discussing three different topics that require different approach, the present paper reflects the three decades of the development of stereological methods: 2D analysis by simple point counting in the 70's, the disector in the 80's and virtual spatial probes in the 90's. In all methods the interactive computer assisted approach was utilised.

Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that β2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9days of treatment, while hypertrophy was observed only in EDL after 9days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.

Effect of neurogenic inactivity on posttetanic responses of rat fast muscle

At a short-term tetanic stimulation of fast muscle in response to subsequent single stimulation there is recorded a temporary increase of the strength of single contractions that returns to the initial background after 6--10 min. This phenomenon is called posttetanic potentiation (PTP) and is recorded only in fast muscles. The goal of the present work was a study of effect of motor innervation on the course of PTP in rat m. extensor digitorum longus (m. Edl). It has been established that the first signs of effect of motor denervation on the PTP course after section of sciatic nerve in the area of popliteal fossa are recorded as early as at the 4th day after denervation and are expressed in a decrease of strength of single contractions after cessation of tetanic stimulation. These changes reach its maximum at the 14--15th day after denervation when effect of PTP in denervation muscle does not appear at all. Pharmacological analysis of the studied phenomenon has shown that dantrolene (10 microM) suppresses amplitude of the single contraction, but does not prevent the appearance of PTP in intact muscle. In the denervation of m. Edl, instead of the appearance of PTP, after a brief slight increase, a gradual decrease of the strength of contraction is recorded. Thus, it can be concluded that no significant PTP changes are present under action of dantrolene. It has been established that after the 10-min muscle incubation in Ringer's solution with caffeine (4 mM), strength of the single contraction in intact and denervated muscles increases by approximately equal value. Tetanization of intact muscle increases strength of the single contraction approximately by 7 % more than this occurs after incubation with caffeine, i. e., this substance reduces the capability of muscle for the appearance of PTP. On denervation muscle, caffeine increases strength of singly contraction, but does not potentiate development of PTP. The obtained data allow concluding about the existence of different mechanisms underlying the pretetatnic contraction and posttetanic potentiation of the single contraction. The main difference between two types of contractions can be recruiting of additional DICR-channels in the process of contraction under conditions of PTP.

Effect of hypothyroidism on actin subdomain-1 movement induced by myosin subfragment-1 binding in fast and slow rat skeletal muscles

Orientation and mobility of fluorescent probe N-((iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine)(1.5-IAEDANS)) specifically bound to Cys-374 of actin in ghost muscle fibers isolated from fast and slow rat muscles were studied by polarized fluorimetry in the absence and presence of myosin subfragment-1 (S1) in intact rats and in the animals with gradual (during 2-5 weeks) reduction of thyroid hormones synthesis (hypothyreosis development). S1 binding to F-actin of ghost muscle fibers was shown to induce changes in orientation of the dipoles of the fluorescent probe 1.5-IAEDANS and in the relative amount of the randomly oriented fluorophores that indicated changes in actin subdomain-1 orientation and mobility resulting from the formation of its strong binding with S1. This effect is markedly inhibited by hypothyreosis development. The maximal effect of hypothyreosis is observed after 34 days of disease development. It is suggested that the change of thyroid status in the muscle inhibits the ability of F-actin to form strong binding with myosin which is essential for force generation.

Resting membrane potential and Na+,K+-ATPase of rat fast and slow muscles during modeling of hypogravity

Antiorthostatic hindlimb suspension (unloading) decreased the resting membrane potential (RMP) of skeletal muscle fibers in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle of the rat by about 10% within 7 days and more. Inactivation of the membrane Na+,K+-pump by ouabain brought about similar depolarization as unloading. The increased sodium permeability of the membrane was excluded as the major cause of this depolarization by experiments in which TRIS was substituted for Na+ in the medium. On the other hand, the decrease in the electrogenic participation of the Na+,K+-pump is apparently one of the causes of RMP decrease during hypogravity, in EDL muscle in particular.

Muscle fatigue examined at different temperatures in experiments on intact mammalian (rat) muscle fibers

In experiments on small bundles of intact fibers from a rat fast muscle, in vitro, we examined the decline in force in repeated tetanic contractions; the aim was to characterize the effect of shortening and of temperature on the initial phase of muscle fatigue. Short tetanic contractions were elicited at a control repetition rate of 1/60 s, and fatigue was induced by raising the rate to 1/5 s for 2–3 min, both in isometric mode (no shortening) and in shortening mode, in which each tetanic contraction included a ramp shortening at a standard velocity. In experiments at 20°C (n = 12), the force decline during a fatigue run was 25% in the isometric mode but was significantly higher (35%) in the shortening mode. In experiments at different temperatures (10–30°C, n = 11), the tetanic frequency and duration were adjusted as appropriate, and for shortening mode, the velocity was adjusted for maximum power output. In isometric mode, fatigue of force was significantly less at 30°C (∼20%) than at 10°C (∼30%); the power output (force × velocity) was >10× higher at 30°C than at 10°C, and power decline during a fatigue run was less at 30°C (∼20–30%) than at 10°C (∼50%). The finding that the extent of fatigue is increased with shortening contractions and is lower at higher temperatures is consistent with the view that force depression by inorganic phosphate, which accumulates within fibers during activity, may be a primary cause of initial muscle fatigue.

An analysis of the temperature dependence of force, during steady shortening at different velocities, in (mammalian) fast muscle fibres

We examined, over a wide range of temperatures (10–35°C), the isometric tension and tension during ramp shortening at different velocities (0.2–4 L 0/s) in tetanized intact fibre bundles from a rat fast (flexor hallucis brevis) muscle; fibre length (L 0) was 2.2 mm and sarcomere length ~2.5 μm. During a ramp shortening, the tension change showed an initial inflection of small amplitude (P 1), followed by a larger exponential decline towards an approximate steady level; the tension continued to decline slowly afterwards and the approximate steady tension at a given velocity was estimated as the tension (P 2) at the point of intersection between two linear slopes, as previously described (Roots et al. 2007). At a given temperature, the tension P 2 declined to a lower level and at a faster rate (from an exponential curve fit) as the shortening velocity was increased; the temperature sensitivity of the rate of tension decline during ramp shortening at different velocities was low (Q 10 0.9–1.5). The isometric tension and the P 2 tension at a given shortening velocity increased with warming so that the relation between tension and (reciprocal) temperature was sigmoidal in both. In isometric muscle, the temperature T 0.5 for half-maximal tension was ~10°C, activation enthalpy change (∆H) was ~100 kJ mol−1 and entropy change (∆S) ~350 J mol−1 K−1. In shortening, these were increased with increase of velocity so that at a shortening velocity (~4 L 0/s) producing maximal power at 35°C, T 0.5 was ~28°C, ∆H was ~200 kJ mol−1 and ∆S ~ 700 J mol−1 K−1; the same trends were seen in the tension data from isotonic release experiments on intact muscle and in ramp shortening experiments on maximally Ca-activated skinned fibres. In general, our findings show that the sigmoidal relation between force and temperature can be extended from isometric to shortening muscle; the implications of the findings are discussed in relation to the crossbridge cycle. The data indicate that the endothermic, entropy driven process that underlies crossbridge force generation in isometric muscle (Zhao and Kawai 1994; Davis, 1998) is even more pronounced in shortening muscle, i.e. when doing external work.

Comparison of the tension responses to ramp shortening and lengthening in intact mammalian muscle fibres: crossbridge and non-crossbridge contributions

We examined the tension responses to ramp shortening and lengthening over a range of velocities (0.1-5 L(0)/s) and at 20 degrees C and 30 degrees C in tetanized intact fibre bundles from a rat fast (flexor hallucis brevis) muscle; fibre length (L(0)) was 2.2 mm and sarcomere length approximately 2.5 microm. The tension change during ramp releases as well as ramp stretches showed an early transition (often appearing as an inflection) at 1-4 ms; the tension change at this transition and the length change at which it occurred increased with velocity. A second transition, indicated by a more gradual reduction in slope, occurred when the length had changed by 14-28 nm per half-sarcomere; the tension at this transition increased with lengthening velocity towards a plateau and it decreased with shortening velocity towards zero tension. The velocity dependence of the time to the transitions and the length change at the transitions showed some asymmetries between shortening and lengthening. Based on analyses of the velocity dependence of the tension and modelling, we propose that the first transition reflects the tension change associated with the crossbridge power stroke in shortening, or with the reversal of the power stroke in lengthening. Modelling shows that the reduction in slope at the second transition occurs when most of the crossbridges (myosin heads) that were attached at the start of the ramp become detached. After the second transition, the tension reaches a steady level in the model whereas the tension continues to increase during lengthening and continues to decrease during shortening in the experiments; this continuous tension change is seen at a wide range of initial sarcomere lengths and when active force is reduced by the myosin inhibitor, BTS. The continuous tension decline during shortening is not abolished by caffeine, but the rate of decline is reduced when the active force is depressed by BTS. We propose that stiffening of non-crossbridge visco-elastic elements upon activation contributes to the continuous tension rise during lengthening and the release of such tension and Ca-insensitive deactivation contribute to the tension decline during shortening in muscle fibres.

Effects of 14 Days of Microgravity on Rat Fast Hindlimb Muscles

Effects of 14 Days of Microgravity on Rat Fast Hindlimb Muscles

Junctional and extrajunctional acetylcholinesterase in skeletal muscle fibers

The asymmetric A 12 acetylcholinesterase (AChE) molecular form, consisting of three tetrameric catalytic oligomers and three non-catalytic subunits of collagen Q (ColQ), is the functional AChE form in the neuromuscular junction. Its extremely high concentration and sharp localization in the junction is mostly due to the binding of this AChE form to perlecan in the synaptic basal lamina. In the rat neuromuscular junctions, about two-thirds of AChE molecules appear to be bound by ionic interactions involving calcium and the rest is probably bound covalently. In immature rat muscles, the A 12 AChE forms are expressed also extrajunctionally. During the early post-natal period, this expression is completely suppressed in rat fast muscles, whereas it extends into adulthood in the slow soleus muscles because of the differences in the extrajunctional expression of ColQ between fast and slow muscles. The level of the A 12 molecular forms of AChE in the extrajunctional muscle regions is regulated by the motor nerve, probably via the pattern of muscle fibre activations triggered by the nerve. The pattern of muscle activations also regulates the extrajunctional expression of the catalytic subunits of AChE: phasic, infrequent, high frequency activations enhance expression, whereas prolonged tonic low-frequency activations tend to decrease it. Calcineurin signalling pathway seems to be involved.

Ubiquitin targeting of rat muscle proteins during short periods of unloading

The ubiquitin-proteasome system is known to be involved in many situations leading to skeletal muscle atrophy. However, the cellular mechanisms triggering the atrophic process initiation are still poorly understood. For short periods of rat hindlimb unloading, we assessed the specific ubiquitin targeting of sarcoplasmic or myofibrillar proteins in slow and fast rat muscle types. Adult Sprague Dawley rats were randomly assigned to three groups: control, hindlimb-unloaded for 4 days (HU4) and hindlimb-unloaded for 8 days (HU8). In fractionated extracts from soleus (SOL) and Extensor Digitorum Longus (EDL) muscles, the relative contents of free and conjugated ubiquitin were quantified by immunoblotting. Hindlimb unloading of short durations resulted in a preferential atrophy of slow-twitch fibres and bound ubiquitin levels were increased by 37 and 68% in the soleus myofibrillar fraction after respectively 4 and 8 days. The ubiquitin conjugation was shown to principally affect the high molecular weight proteins. Free and conjugated ubiquitin levels remained unchanged in sarcoplasmic fraction from SOL muscle after 8 days HU. For the fast muscle (EDL), ubiquitin contents were approximately twofold lower in control conditions, and did not significantly change during the hindlimb unloading periods considered. The postural SOL muscle was shown to contain higher constitutive sarcoplasmic ubiquitin levels than the phasic EDL. The high response to unloading of the slow twitch fibres rich SOL muscle was accompanied by a specific conjugation of its myofibrillar proteins that may participate in the initiation of skeletal muscle remodelling consequent to disuse.