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J Appl Physiol 99: 542-548, 2005. First published March 24, 2005; doi:10.1152/japplphysiol.00757.2004
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Dual effect of deafferentation on contractile characteristics and sarcoplasmic reticulum properties in rat soleus fibers

Y. Mounier, V. Montel, F. Picquet, L. Stevens, B. Bastide, and M. Falempin

Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France

Submitted 19 July 2004 ; accepted in final form 17 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The neural message is known to play a key role in muscle development and function. We analyzed the specific role of the afferent message on the functional regulation of two subcellular muscle components involved in the contractile mechanism: the contractile proteins and the sarcoplasmic reticulum (SR). Rats were submitted to bilateral deafferentation (DEAF group) by section of the dorsal roots L3 to L5 after laminectomy. Experiments were carried out in single skinned fibers of the soleus muscle. The maximal force developed by the contractile proteins was increased in the DEAF group compared with control, despite a decrease in muscle mass by 17%. The tension-pCa relationship was shifted toward lower calcium (Ca2+) concentrations. Different functional properties of the SR of DEAF soleus were examined by using caffeine-induced contractions. The caffeine sensitivity of the Ca2+ release was decreased after deafferentation and ryanodine receptor 1 isoform was expressed at a lower level. The rate of Ca2+ uptake was only slightly increased. The results underlined the dual effect of the afferent input on the functional regulation of both contractile proteins and SR.

neural message; muscular atrophy; contractile proteins; calcium activation properties; caffeine tensions

IT IS A CONCEPT NOW WELL ESTABLISHED and supported by numerous data that the type of neural message (tonic or phasic pattern) is a key factor for muscle development and function. The amount of neuromuscular activity, i.e., the amount of electrical activation, is the primary physiological signal that initiates a cascade of mechanical-biochemical responses. These eventually modulate gene expression and/or posttranscriptional mechanisms that may alter protein expression. All results agree that motoneurons play an essential role in determining the phenotype of mammalian skeletal muscle (for review, see Ref. 22). The reduction of motor activity by different disuse procedures, such as limb immobilization (3), hindlimb unloading (10, 19, 23, 38), spinal cord transection (4, 14, 27), or tenotomy (34), has a speeding effect on slow muscles. This results in faster twitch contraction kinetics or increase of maximal shortening velocity correlated with changes in the expression of different contractile proteins, especially myosin heavy chains (30, 32, 33). On the other hand, the abolition of motor activity by short-duration denervation (up to 2 wk) is classically reported to have a slowing effect on muscles. Therefore, although disuse and denervation both induce muscle atrophy, they produce opposite effects on some contractile properties and support the view that the neuromotor activity is not the only factor regulating those properties. Indeed, it is unquestionable that the motor activity is also dependent on the afferent message via the myotatic reflex. However, the distinctive influence of this afferent message, without alterations of the motoneurons, on the morphological, phenotypical, and functional muscle properties is poorly documented. A few papers have reported a role of the afferent message, under hindlimb unloading conditions, in participating in the regulation of muscle mass and forces (18, 24). The changes in force suggested that the intracellular calcium (Ca2+) level and the Ca2+ affinity of the contractile system might be modulated by the afferent message. Surprisingly no data were devoted to the mechanisms that might regulate muscle force under changes in afferent message control.

Therefore, the main purpose of this study was to analyze the regulation of force development by the afferent message. This was examined on skinned fibers to investigate the functional properties of two key subcellular structures involved in the contractile mechanism. The force production results from the actin-myosin attachment regulated by the binding of Ca2+ ions on the troponin C. The level of myoplasmic Ca2+ in skeletal muscle fibers is regulated by the equilibrium between passive leakage and Ca2+ release via the ryanodine receptor RyR1 and Ca2+ pumping by Ca2+-ATPases. Thus our objective was to determine in the soleus muscle the effect of a selective deafferentation, first on the force amplitude in relation with Ca2+ affinity of the contractile system and second on the capability of the SR to release and pump Ca2+ ions. We were next able to establish whether these structures participate in the adaptive properties of a deafferented muscle. These functional analyses will contribute to a better understanding of muscular dysfunctions such as sensorimotor pathologies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

The experiments were carried out on adult male Wistar rats (IFFA CREDO, l'Arbresle, France) weighing 280–300 g. They were divided into two groups housed in the same environment. A first group (n = 7) used as control (Cont) included nonoperated rats. A second group (n = 8) was submitted to deafferentation (DEAF). These animals were deeply anesthetized by intraperitoneal injection of pentobarbital sodium (35 mg/kg body wt). Under aseptic conditions, the midline dorsal musculature was retracted laterally and a laminectomy was performed between L2 and S1. After careful separation from the ventral roots with a small blunt hook, the bilateral dorsal roots L3, L4, and L5 were exposed. The dorsal roots were identified after recording of the afferent neurogram. For deafferentation, these dorsal roots were sectioned from their entry to spinal cord (including the ganglion) to their entry in the vertebra to avoid regeneration of nerve fibers. Dorsal musculature and skin incision were sutured (3–0 chronic gut). After recovery, the rats were kept in isolated normalized cages. An antiseptic (Betadine) was applied to the cutaneous incision area once a day to prevent skin infection. Antibiotics (Trisulmix, 100 µl/kg) and analgesic (Metacam, 100 µl/kg) were orally administered up to 6 days after surgery. Reflex testings on the hindlimbs were performed once a day, i.e., withdrawal reflex to verify the effectiveness of the bilateral deafferentation. Throughout the study, in this group, there was no response to toe pinching. Moreover, the postoperative gait pattern presented exaggerated plantar flexion of the feet during walking, which was an evidence of deafferentation, as previously described (13, 35). Daily consumption of rat chow and water was monitored. No debilitating problems related to the maintenance of general health were encountered in any of the rats used in this study.

Both groups of rats were anesthetized 14 days after surgery. The left soleus muscles were devoted to the experiments on single skinned fibers for contractile protein properties and sarcoplamic reticulum studies reported in this paper. A sham-operated group of four rats was also constituted to monitor the effects of the trauma induced by the surgical procedure, i.e., the different steps of the operation and both antibiotics and analgesic administrations described above, except laminectomy and deafferentation. No differences in morphological properties and tension amplitudes in skinned fibers were found between Cont and sham-operated groups.

The experiments as well as the maintenance conditions of the animals received authorizations from the Ministry of Agriculture and the Ministry of Education (veterinary service of health and animal protection, authorization 03805).

Skinning Preparation

Bundles of muscle fibers were biopsied from the excised soleus. These biopsies were immediately skinned according to a procedure previously described (17) and then stored at –20°C in a 50:50 glycerol-skinning solution (storage solution). The skinning procedure with EGTA, permeabilized the sarcolemmic and transverse tubular membranes. This allowed the application of activating solutions directly to the contractile proteins and the SR, whose structure was not affected by the treatment (7, 39). Protease inhibitor leupeptin was added to the storage solution (10 µg/ml) to prevent loss of contractile proteins and preserve the fiber tension (12).

Solutions

All reagents were provided by Sigma (St. Louis, MO). The concentrations of the different components were calculated using the program of Fabiato and Fabiato (9). The stability constants (21) used in the calculations were KCaEGTA 1.919 x 106 M–1, KCaATP 5.0 x 103 M–1, KMgEGTA 40 M–1, and KMgATP 1.0 x 104 M–1. All solutions were buffered with MOPS (10 mM) and contained ATP (2.5 mM). The pH of all the solutions was adjusted to 7.0 ± 0.02. The compositions of skinning solution also used as relaxing solution (R) and washing solution (W) were reported in Table 1. Phosphocreatine (10 mM) was added to W solution. The composition of the different pCa (or pSr) activating solutions consisted of potassium propionate (172 mM), MOPS (10 mM), ATP (2.5 mM), and MgAc2, which ranges from 2.39 mM (pCa 4.2) to 2.46 mM (pCa 7.0), and various levels of free Ca2+ (or Sr2+) from CaCO3 or SrCl2, respectively, buffered with 5 mM EGTA all included. CaEGTA and K2EGTA were mixed in adequate proportions to obtain the different pCa (7.0 to 4.2) or pSr values (5.0 and 3.4). For the composition of Sr solutions, KSrEGTA was 2 x 104 M–1 (15). Compositions of pCa 7.0, 5.0, 4.2, and pSr 5.0 solutions were reported in Table 1. Ionic strengths of the R and different activating solutions were in the range 200–205 mM. For SR experiments, the loading solution was made up with a solution of a low Ca2+ concentration (pCa 7.0) able to load the SR without inducing contraction. The caffeine solutions were obtained by dilution of caffeine with W solution.


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  		Table 1. Ionic composition of different solutions: W, R activating pCa (pCa = –log[Ca2+]), and pSr solutions

 
Experimental Procedure

For each experiment, a 2- to 2.5-mm single-fiber segment was isolated from the skinned biopsy. A silk thread was tied at each extremity, allowing the mounting of the fiber in an experimental chamber initially filled with R (relaxing) solution under constant stirring. The fiber was held at one end by small fixed forceps and at the other end by a clamp connected to a strain gauge [force transducer Fort 10 (World Precision Instruments); sensitivity 10 V/g]. The mounted fiber was viewed through a high-magnifying binocular (x80) with a micrometer, allowing fiber diameter measurements, assuming the cross-section was circular. Fibers with a high degree of ellipticity were discarded (about 5%). The resting sarcomere length was measured by means of a helium-neon laser (Spectra Physics) directed perpendicular to the long axis of the fiber. The fiber was then stretched to ~120% of resting length to allow maximal isometric tension development on ionic activation. The resulting sarcomere length (2.6 ± 0.04 µm) was subsequently regularly controlled and readjusted if necessary. The output of the force transducer was amplified and recorded on a graph recorder (Gould, model 6120) and simultaneously analyzed by computer software. All experiments were performed in a thermostatically controlled room (19 ± 1°C).

Tension-pCa Relationships

To eliminate a hypothetic influence of the SR on the tension developed by the myofilaments, each fiber was bathed for 20 min at the beginning of an experiment in a Brij solution made up of R solution with 2% Brij 58 (polyoxyethylene 20 cetyl ether). The nonionic Brij 58 detergent irreversibly eliminated the ability of the SR of skinned muscles to sequester and release Ca2+, without altering the actomyosin system (20). After this treatment, the fiber was washed in R and W solutions and a maximal tension (P0) was induced by applying a pCa 4.2 solution that contained enough calcium to saturate all TnC sites. An experimental sequence was defined as follows: after exposure to each pCa, a tension P was recorded and immediately followed by P0. This allowed the calculation of the relative isometric force (P/P0). Then, the R solution was applied, and the fiber was washed to eliminate the EGTA traces from the R solution before initiation of a new tension activation. The relations between P/P0 and the different pCa were given by the tension-pCa or T/pCa curves. The tension-pCa experimental data were fitted to the Hill equation: P/P0 = {([Ca2+]/K)nH/[1 + ([Ca2+]/K)nH]}, where P/P0 is the normalized tension, nH is the Hill coefficient, and K is the apparent dissociation constant (pK = –log K = pCa50). Three parameters can be deduced from the T/pCa curves: the pCa threshold (pCathr), defined as the lowest Ca2+ concentration that produced a detectable tension response; the pCa50 value, which corresponded to the pCa necessary to develop 50% of the maximal tension; and the Hill coefficient nH related to the steepness of the curve.

Functional Identification of Fiber Type

The criterion for functional fiber identification was based on the difference of Ca2+ and Sr2+ activation characteristics between fast and slow fibers. Indeed, it is generally assumed that fast muscle fibers are less sensitive to Sr2+ than slow fibers. To minimize the number of tensions developed by the fiber, only two Sr2+ solutions, pSr 3.4 and 5.0, were applied. The application of pSr 3.4 solution elicited the maximal Sr2+ tension. The pSr 5.0 solution produced tensions close to 95% P0 in slow soleus fibers and tensions ranging from 0 to 10% in fast fibers of different muscles (31, 37). Thus slow and fast fibers were clearly identified. This paper reports data obtained on slow fibers.

Ca2+ Movements Through the SR

The experiments of Ca2+ movements through the SR were conducted on fibers taken from the same biopsies as those used for the T/pCa studies. Measurements of Ca2+ uptake, Ca2+ release, and passive leakage of Ca2+ were conducted using the protocols described in detail in the appropriate sections of RESULTS. Before each protocol, maximal tension P0 and tension elicited by a pSr 5.0 solution for the fiber type identification were recorded. They were always immediately followed by application of a caffeine (12.5 mM) solution to ensure that SR was emptied before performing one of the three protocols. Then, Ca2+ movements through the SR were studied using the caffeine method previously described (8). Indeed, caffeine is well known to induce a contraction of the skinned fiber when applied at appropriate concentrations, and the caffeine-induced tensions were the consequence of Ca2+ release from the SR by the drug (36). Therefore, the amplitude of the tension was an index of the Ca2+ amount stored in the SR. A solution of 12.5 mM caffeine was used, because this concentration induced a complete release of Ca2+ from the SR in Cont and DEAF animals. In each experiment, two successive applications of 12.5 mM caffeine ensured that the release was complete from the first application (Fig. 1). Lower caffeine concentrations (10 mM) cannot be used because two or more applications are sometimes needed to empty the SR in DEAF fibers.



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  		Fig. 1. Tension elicited by a 12.5 mM caffeine solution (caf 12.5) in control (Cont) and deafferented (DEAF) fibers. Note that a second application was inefficacious. After relaxation in solution R and wash with solution W, maximal tension (P0) was recorded (solution pCa 4.2 + caf 12.5). Fast transient peaks on records were produced by exchange of solutions.

 
Maximum force P0 was induced after each caffeine tension (Tcaf) to compare its amplitude with the maximal contracting force of the fiber and thus to calculate a relative tension (Tcaf/P0). Moreover, in these experiments, each pCa 4.2 solution that elicited P0 received the concentration of caffeine used to induce the tension Tcaf. Thus amplitudes of caffeine-induced contractions when expressed relative to [P0 + caf] are corrected for the caffeine-induced changes in Ca2+ sensitivity.

Western Blot Analysis of RyR1 Isoform Expression

Skeletal soleus muscle samples were homogenized in ice-cold buffer (320 mM sucrose, 5 mM Na-HEPES, pH 7.4, and 0.1 mM phenylmethylsulfonyl fluoride) using a Dounce homogenizer. Homogenates were centrifuged at 7,000 g for 15 min at 4°C. The supernatant obtained was centrifuged at 20,000 g for 1 h at 4°C. The microsomes were resuspended in the buffer and stored at –80°C. The protein concentration of the microsomal fraction was quantified using the Lowry protein assay kit (Bio-Rad). Microsomal proteins were separated on a 5% SDS-PAGE. The gels were silver stained. The migration level of RyR1 isoform was determined according to previous reports (1), using RyR1 antibody provided by Dr. M. Vilaz (CEA, Grenoble, France). Relative proportions between Cont and DEAF samples were measured using integrating densitometry (Bio-Rad).

Statistical Analysis

Data are expressed as means ± SE. Student's t-test was used for statistical analysis, and significance was taken at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Muscular Atrophy and Fiber Type Identification

After deafferentation, the muscle wet weight decreased from 147 ± 3 mg in control soleus (n = 7) to 123 ± 3 mg (n = 8), without change in body weight. At the single-fiber level, the fiber type was identified, according to the differential sensitivity to Ca2+ and Sr2+ ions. In Cont fibers, tensions elicited by pCa 5.0 and pSr 5.0 solutions were equal to 95.8 ± 1.9 and 93.5 ± 1.8% P0 (n = 50), respectively. In DEAF fibers (n = 50), these values were 97.8 ± 0.8 and 94.8 ± 0.8% P0, respectively. These data indicated that all the fibers kept for this study were slow. After deafferentation, the fibers were atrophied, because their mean diameter decreased by 18% (Table 2).


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  		Table 2. Contractile parameters of soleus muscle fibers from Cont and DEAF groups

 
Maximal Tension and Tension-pCa Relationships

Figure 2A illustrates records of a tension P elicited by a pCa 5.8 solution followed by the maximal tension P0 elicited by a pCa 4.2 solution. The absolute P0 value (P0, µN) and relative P0 amplitude expressed per cross-sectional area (kN/m2) were clearly increased by 28 and 72%, respectively (Table 2). The T/pCa relationships for Cont and DEAF muscle fibers are illustrated in Fig. 2B. The different parameters derived from these relations are reported in Table 2. After DEAF, the sigmoid curve was shifted toward lower calcium concentrations. The pCa threshold and pCa50 values were significantly increased (P < 0.05). The slopes of the T/pCa curves (nH parameter) were not significantly different between Cont and DEAF fibers.



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  		Fig. 2. A: recordings of submaximal tensions elicited by pCa 5.8 solution followed by maximal tensions P0 induced by pCa 4.2 solution in Cont and DEAF muscle fibers. B: tension-pCa relationships of control ({bullet}, n = 18) and DEAF ({circ}, n = 18) single skinned soleus muscle fibers. Values are means ± SE.

 
Ca2+ Uptake

The control of Ca2+ uptake was carried out using the following method: the SR of the soleus fibers was preloaded with a pCa 7.0 solution (loading solution) for various periods of time (15, 30, 60, 180, 300, 600 s). Thus, for a given time, the SR accumulated a given amount of Ca2+. Then, after the fiber had been rinsed in W solution, the SR was emptied of its Ca2+ by applying the 12.5 mM caffeine solution (as justified in MATERIALS AND METHODS). The results are presented in Fig. 3 for Cont and DEAF muscles. To quantify and normalize the SR Ca2+ loading capability in both conditions, the caffeine response after each loading time was expressed as a percentage of P0 recorded in the presence of 12.5 mM caffeine. In both Cont and DEAF muscles, the amplitude of caffeine tension varied with the time of Ca2+ loading: the longer the time, the larger the tension. For Cont and DEAF animals, Tcaf was maximal and equal to 85.3 ± 3.2% P0 (n = 11 fibers) and 84.1 ± 4.1% P0 (n = 17 fibers), respectively. In both cases, maximal Ca2+ loading was reached after 3 min. However, after short loading durations, such as 15, 30, and 60 s, the ratio was higher for DEAF fibers, the difference with respective control values being significant. When the estimation of Ca2+ loading was made by measurements of caffeine tension areas normalized to the maximal one (instead of peak amplitudes), a similar qualitative result was obtained showing slightly higher values for DEAF fibers after short loading durations up to 60 s. Thus DEAF fibers took up Ca2+ more rapidly but the maximal loading was similar to control.



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  		Fig. 3. Ca2+ uptake by sarcoplasmic reticulum (SR) of fibers from Cont ({bullet}, n = 11) and DEAF ({circ}, n = 17) soleus. Amplitudes of caffeine tensions were expressed in percentage of P0 (in y-axis) after various times of SR Ca2+ load (in x-axis). Values are means ± SE. *Significantly different from Cont, P < 0.05.

 
Stimulation of Ca2+ Release by Caffeine

To test the stimulating effect of caffeine on the SR Ca2+ release mechanism, the following experiment was performed. Caffeine solutions at various concentrations (from 0.625 to 12.5 mM) were applied after the fiber had been exposed to the loading solution pCa 7.0 for 5 min, a time long enough to load maximally the SR of Cont and DEAF fibers as shown above. Each Tcaf was normalized to P0 obtained by application of the pCa 4.2 solution in which the corresponding caffeine concentration was added. The results were summarized in Fig. 4A. The caffeine activation threshold, defined as the lowest caffeine concentration that induced a detectable tension (6% P0), corresponded to 2.5 mM in DEAF fibers (n = 20), whereas the same concentration induced a tension of 36% P0 in Cont fibers (n = 16). For other caffeine concentrations that induced submaximal caffeine tensions, the sensitivity to the drug was lower after DEAF. A shift of the curve toward higher caffeine concentrations was also obtained when Ca2+ release was estimated by measurements of caffeine tension areas. Therefore, deafferentation induced a decrease in Ca2+ release.



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  		Fig. 4. A: stimulation of Ca2+ release by caffeine. Relationship between amplitude of relative caffeine tension response Tcaf (in percent of P0) and caffeine concentrations for Cont ({bullet}, n = 16) and DEAF ({circ}, n = 20) soleus fibers. Values are means ± SE. *Significant difference between Cont and DEAF, P < 0.05. B: representative illustration of RyR1 migration using 5–10% SDS-PAGE in microsomal vesicles prepared from soleus muscles of Cont and DEAF animals. Fifty micrograms of proteins were loaded for each lane. Only the region corresponding to RyR1 band is shown, the level of migration being identified according to previous reports (2).

 
To establish whether the decrease in Ca2+ release after deafferentation resulted from changes in the expression level of the ryanodine receptor, Western blots were performed. The expression of RyR1 isoform was measured in microsomes isolated from Cont and DEAF soleus muscles. Figure 4B is a representative determination obtained on one of the three different preparations made from Cont and DEAF muscles. A lower expression of RyR1 was found after deafferentation because the signal was 58 ± 1.5% of the control.

Passive Leakage of Ca2+ From the SR

In this experiment, the fiber was exposed to the Ca2+ loading solution (pCa 7.0–5 min). The fiber was then left in the R solution for various periods of time, allowing the leakage of calcium through the SR membrane. The results are described in Figure 5. The amount of Ca2+ remaining in the SR was tested, as previously explained, by the application of 12.5 mM caffeine. For Cont (n = 7) and DEAF (n = 7) fibers, the longer the time of exposure to the R solution, the smaller the amount of calcium remaining in the SR and, thus the lower the caffeine tension amplitude. However, it appeared that the amount of Ca2+ in the SR decreased slowly after DEAF because no tension response could be recorded after 5 min of exposure to the R solution for a Cont fiber, whereas a tension of 37% was still developed by a DEAF fiber. In the two cases, the Ca2+ leakage followed a single exponential time course because the two semilogarithmic analyses (inset in Fig. 5) described a linear relation. The time constant increased from 1 min 20 s in Cont to 5 min 5 s after DEAF.



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  		Fig. 5. Time course of passive release ("leakage") of SR-stored Ca2+ for Cont ({bullet}, n = 7) and DEAF ({circ}, n = 7) soleus fibers. Amplitudes of caffeine tensions (Tcaf) were reported in percent of Tcaf max normalized to P0 (taken as 100% and measured less than 5 s after maximal SR loading) and plotted against time of exposure to R solution. Inset: semilogarithmic analysis of time course of Ca2+ leakage for both studied conditions. Values are means ± SE. *Significantly different from Cont, P < 0.05.

 
To check that leakage occurred through ryanodine receptors, the same leakage protocol was used, except that ryanodine (200 µM) was added to R solution. In Cont fibers, after 1 min of R + ryanodine soaking, a caffeine tension could be recorded that reached 90% P0 compared with Cont caffeine tension (measured less than 5 s after maximal SR loading) equal to 82% P0. Thus leakage was blocked by ryanodine treatment, and a slightly larger caffeine tension could be obtained since Ca2+ uptake was maintained.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results presented in this paper demonstrate a dual effect of deafferentation on the functional properties of two subcellular components that are essential for the contractile function in muscle fibers. Ca2+ sensitivity and force developed by the contractile proteins increased whereas induced Ca2+ release was clearly decreased.

Deafferentation Effects on the Contractile Proteins

The present results show that the contractile properties of slow soleus muscle fibers are changed after 14 days of deafferentation. They were analyzed in single skinned fibers, which permitted a direct measurement of the myofibrillar performance and offered appropriate conditions for studying the interaction of Ca2+ with the contractile system. Our data indicate that the activation of the contractile proteins is enhanced after deafferentation. A 28% increase in maximal P0 amplitude was found. When P0 was expressed per cross-sectional area, this increase rose up to 72%, which cannot be explained by the atrophy (17% decrease in muscle mass and a 18% decrease in the fiber diameter) of the deafferented soleus. A similar decrease (11%) in muscle mass has previously been described for deafferented soleus (18). Therefore, atrophied deafferented fibers appear able to produce enhanced maximal tensions. The possible reasons for this apparent contradictory result need to be examined. The simplest explanation for the finding of enhanced maximal tensions in deafferented soleus fibers, which is consistent with other data presented in this study, is that deafferentation is associated with changes in the intrinsic cross-bridge properties (11) and kinetics (25), especially on a decrease in the dissociation rate (parameter identified as g in the Huxley model). Thus by a decrease in g, force generating cross bridges might retain Ca2+ on the thin filament. This could also explain the observed increase in both force and apparent Ca2+ sensitivity.

The steepness of the curve (nH coefficient) was not modified after deafferentation, suggesting a lack of effect of this process on the cooperativity of interaction between different components of the contractile apparatus and regulatory system.

Deafferentation Effects on SR Properties

Ca2+ uptake.   The SR capacity to accumulate Ca2+ as well as the kinetic of pumping are related to a number of factors, which include the quantity of SR membranes and the number of Ca2+ pumps per milligram of proteins. Our data show that in deafferented fibers, the SR accumulated Ca2+ slightly more rapidly than in control fibers, the maximal uptake being similar after 3 min. In hindlimb unloading conditions, which at least partly involve changes in the afferent message, an upregulation of SERCA1 mRNA and protein (28) in the soleus, associated with SR proliferation (1), appeared with an increase in SR Ca2+-dependent ATPase activity and a higher Ca2+ loading function (28, 29). Using here the same caffeine test as previously employed in hindlimb-unloading experiments, we found evidence that deafferentation has an effect on the Ca2+ pumping kinetics of the SR-ATPase.

Ca2+ release and leakage.   After deafferentation, SR presented a clear decrease in caffeine sensitivity for concentrations lower than 10 mM, i.e., in conditions that did not induce a complete Ca2+ emptying of the SR. This seems to indicate changes in the properties and/or expression levels of the Ca2+-release channels, which, together with other factors, would determine the amount of Ca2+ released during muscle activation. Moreover, the study of the Ca2+ leakage showed that the time required to empty the SR previously loaded with Ca2+ was increased by a factor of about four after deafferentation. The decrease and slowing in caffeine-induced Ca2+ release is also illustrated by the necessity to use high caffeine concentrations to produce in one step a total emptying of Ca2+ stored in the SR (see MATERIALS AND METHODS). One possible explanation for this finding is that the expression of caffeine-sensitive Ca2+ channels in the terminal cisternae of the SR, which can be assimilated to ryanodine receptors (RyR1) in striated skeletal muscle fibers, is clearly decreased in deafferented muscles. This possibility is supported by the data shown in Fig. 4B. Conversely, an increased Ca2+ release after denervation has been associated with an increase in RyR1 expression (6).

Moreover, because it has already been proposed that real or stimulated microgravity corresponds to a functional deafferentation (2, 5, 16, 26), it is interesting to compare the data from this study with those observed in hindlimb unloading. Thus the decreases in Ca2+ release and Ca2+ leakage reported here are in contrast with those previously observed after hindlimb unloading. In this latter situation, an increased Ca2+ release related to SR proliferation (1, 29) and a faster passive leakage (29), which became close to those found in fast muscles, have been demonstrated. Such a difference underlines the fact that some modifications occurring after hindlimb unloading have a motor origin (i.e., slow to fast transformation) other than the change in sensory message.

To conclude, the findings underline the importance of the afferent message in the control of some muscle characteristics (especially mass and force) and the implications of afferent input in the regulation of protein expression and functional properties.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from the "Centre National d'Etudes Spatiales" (3194) and the Association Française contre les Myopathies (Grant 8557).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Mounier, Université des Sciences et Technologies de Lille, Laboratoire de Plasticité Neuromusculaire-Bât SN4, 59655 Villeneuve d'Ascq Cedex, France (E-mail: Yvonne.Mounier{at}univ-lille1.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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