Force decline during fatigue in skeletal muscle is attributed mainly to progressive alterations of the intracellular milieu. Metabolite changes and the decline in free myoplasmic calcium influence the activation and contractile processes. This study was aimed at evaluating whether fatigue also causes persistent modifications of key myofibrillar and sarcoplasmic reticulum (SR) proteins that contribute to tension reduction. The presence of such modifications was investigated in chemically skinned fibers, a procedure that replaces the fatigued cytoplasm from the muscle fiber with a normal medium. Myofibrillar Ca2+ sensitivity was reduced in slow-twitch muscle (for example, the pCa value corresponding to 50% of maximum tension was 6.23 ± 0.03 vs. 5.99 + 0.05, P < 0.01, in rested and fatigued fibers) and not modified in fast-twitch muscle. Phosphorylation of the regulatory myosin light chain isoform increased in fast-twitch muscle. The rate of SR Ca2+ uptake was increased in slow-twitch muscle fibers (14.2 ± 1.0 vs. 19.6 ± 2.5 nmol · min−1 · mg fiber protein−1, P < 0.05) and not altered in fast-twitch fibers. No persistent modifications of SR Ca2+ release properties were found. These results indicate that persistent modifications of myofibrillar and SR properties contribute to fatigue-induced muscle force decline only in slow fibers. These alterations may be either enhanced or counteracted, in vivo, by the metabolic changes that normally occur during fatigue development.
- myofibrillar calcium sensitivity
- chemically skinned fibers
prolonged stimulation of skeletal muscle causes the well-known phenomenon of fatigue. The mechanisms underlying skeletal muscle fatigue are, however, not yet completely understood. Many factors appear to contribute to force decline, the most relevant being 1) reduction of Ca2+ release from sarcoplasmic reticulum (SR),2) reduction of myofibrillar Ca2+sensitivity, and 3) reduction of maximum Ca2+-activated tension (11, 12,29, 36). In addition, mammalian skeletal muscles are composed of variable mixtures of fibers with oxidative and/or glycolytic capabilities, and, consequently, they exhibit different fatigue resistance (19). During sustained exercise, the myoplasmic concentration of several metabolites varies greatly, thus influencing the activity of some of the proteins directly involved in the control of the contractile machinery. Both acidic pH and high Pi concentrations, for example, are known to affect force production as well as Ca2+ uptake and release by the SR (14, 15, 21), even though the effective role of acidosis at physiological temperature is less evident (25, 35) than at lower temperature (18).
It has been recently suggested that, in addition to the well-known changes in muscle metabolites, tension fall during fatigue can also be correlated with posttranslational modification of myofibrillar and/or SR proteins. Significantly, it has been demonstrated with chemically skinned fibers that, after replacement of the fatigued myoplasm with an environment that simulates the cytoplasm of a rested cell, fatigue alterations are still evident (37, 38). In those studies, frog fast-twitch semitendinosus muscle exposed to repetitive stimulation exhibited increased myofibrillar sensitivity to Ca2+ and a reduction of Ca2+ uptake and caffeine sensitivity of the SR.
The present study was undertaken to investigate whether fatigue causes similar persistent modifications also in mammalian skeletal muscles. Because of well-known differences in fatigability between muscle fiber types, fatigue was induced in both fast- and slow-twitch rat muscles by a prolonged tetanic stimulation. Whole muscles were chemically skinned before or immediately after fatigue. The SR Ca2+ uptake and Ca2+ release properties, SR caffeine sensitivities, and myofibrillar Ca2+ sensitivities were investigated on single skinned fibers for both muscle fiber types. Our results show that fatigue induces persistent modifications of the myofibrillar and SR properties in mammalian muscle, particularly in slow-twitch fibers.
The study was approved by the Ethical Committee of the Medical Faculty of the University of Padova. Soleus (140 ± 6 mg) and extensor digitorum longus (EDL, 130 ± 3 mg) muscles isolated from Wistar male rats (2–3 mo old) were used. The animals were killed under ether anesthesia. The muscles were dissected and immediately placed in a Ringer solution containing (in mM) 120 NaCl, 4.7 KCl, 2.5 CaCl2, 3.15 MgCl2, 1.3 NaHPO3, 25 NaHCO3, 11.1 glucose, and 3.75 × 10−3 d-tubocurarine. The solution was continuously bubbled with O2 (95%) and CO2 (5%); the pH was 7.2–7.4. One muscle from each animal was used as control, and the contralateral was stimulated to fatigue. The muscle was mounted vertically and connected to an isometric force transducer (Harvard 50–7947, South Natick, MA) and stimulated by applying supramaximal stimuli delivered by an electronic stimulator (Grass S44, Quincy, MA) (23). Fatigue was induced at room temperature by a tetanizing stimulation (25 Hz for soleus and 40 Hz for EDL), frequencies known to produce 50% of peak force, of 300-ms duration, repeated every 3 s. Electric stimulation was prolonged for 30 min in soleus and 10 min in EDL until the tetanic force declined to a plateau level that was ∼30 and 15% of the initial value, respectively.
Chemical skinning of muscle fibers.
Resting and fatigued muscles were tied to a wooden stick and quickly immersed into an ice-cold skinning solution containing (in mM) 170 potassium propionate, 2.5 magnesium propionate, 2.5 ATP, 5 EGTA, and 10 imidazole, pH 7.0. Chemical skinning was carried out at 0–4°C as previously described (8, 28). At the first, second, fourth, and twenty-third hour, the skinning solution was replaced with fresh solution. After 24 h, skinned muscles were transferred to a skinning solution supplemented with 50% (vol/vol) glycerol and stored at −20°C. Skinned fibers were used within 2–3 wk of preparation. The skinning procedure, by permeabilizing the sarcolemma, allows the complete removal of the myoplasm, but it preserves the SR and myofilaments (27).
Single fiber segments were isolated under a dissecting microscope and transferred to a chamber containing 0.8 ml of a relaxing (Ca2+-free) solution containing (in mM) 170 potassium propionate, 2.5 magnesium propionate, 5 ATP, 5 EGTA, and 10 imidazole, pH 7.0. The fiber segments were inserted between two clamps (the mean fiber segments' length between the clamps was ∼1.5 mm), one of which was connected to a tension transducer (AK Sensonor, Horten, Norway), and stretched up to 30% of their slack length (8). pCa-tension curves (in which pCa indicates −log of Ca2+concentration) were obtained by exposing the fibers sequentially to solutions of different free calcium concentrations at room temperature (22–24°C), as previously described (8). The isometric tension generated in each solution was continuously recorded, and the baseline tension was established as the steady-state voltage output recorded with the fiber in relaxing solution. Specific tension for each single fiber was calculated by normalizing the maximum tension measured at pCa 5 to the fiber cross-sectional area, as calculated by three different diameter determinations along the fiber length, considering the fiber immersed in solution as a cylinder. For rested and fatigued fibers, maximum tension developed in the presence of pCa 5 was determined before and after each experimental protocol. Only fibers showing no significant differences between initial and final values were utilized.
Ca2+ uptake and Ca2+ release measurements.
Ca2+ uptake by the SR was measured at room temperature (22–24°C) either by the light-scattering method (27), as previously described (7,23), or by a caffeine contracture method (37). With the light-scattering method, fibers were mounted in a chamber containing relaxing solution and stretched to 180% of slack length to avoid interference in light-scattering measurements caused by actin-myosin interactions (27). Fibers were then incubated in a Ca2+ loading solution (pCa 6.4) containing (in mM) 170 potassium propionate, 5 Na2K2ATP, 2.5 magnesium propionate, 5 K2EGTA, 2.15 Ca2+, and 10 imidazole buffer, pH 7.0. Ca2+-loading activity of the SR was measured by the fiber light-scattering increase after the addition of 5 mM oxalate, which is proportional to the increase in Ca2+ content, with the plateau level of light scattering representing the maximum capacity for Ca2+ uptake of SR (27). The calibration procedures for converting the light-scattering signal to fiber Ca2+ concentration by using 45Ca2+were described in detail elsewhere (27). The relative increase in light scattering was proportional to the Ca2+concentration inside the fiber. The proportionality constants for type 1 and type 2 fibers were 0.260 ± 0.035 (n = 6 fibers) and 0.200 ± 0.031 (n = 6 fibers) nmol45Ca2+ · light-scattering unit−1 · μg fiber protein−1, respectively.
When the light-scattering signal reached a plateau level, Ca2+ release from the SR was initiated by rapidly exchanging the Ca2+-loading solution with a relaxing solution containing (in mM) 170 potassium propionate, 5 K2EGTA, 10 caffeine, and 10 imidazole buffer, pH 7.0. To prevent SR Ca2+ uptake by the Ca2+ pumps, the releasing solution did not contain Mg2+ and ATP. Caffeine-induced Ca2+ release from the SR exhibited an exponential decay from which the initial efflux rates were calculated. To evaluate the possible contribution of SR leakage to the caffeine-induced Ca2+ release, spontaneous release of the SR-stored Ca2+ was analyzed before and after fatigue by use of the method described by Trachez et al. (33). Fibers were maximally loaded with Ca2+ (3 min in pCa 7.0) and then soaked in the relaxing solution for variable periods of time (1, 3, and 5 min). Ca2+ released as a consequence of spontaneous leakage from the SR was buffered by the EGTA present in relaxing solution. After two washings with the washing solution, 20 mM caffeine was added to promote release of Ca2+ remaining in the SR. The peak amplitude of caffeine-induced tension was used to estimate leakage of Ca2+ from SR during the exposure to relaxing solution. However, no SR leakage was found in rested and fatigued muscle fibers (not shown).
The caffeine contracture method is an indirect technique that allows estimation of the amount of Ca2+ stored in the SR after various periods of Ca2+ loading (37). Fibers were first exposed to the relaxing solution containing 20 mM caffeine to deplete the SR of Ca2+. After being rinsed in the relaxing solution, the fiber was incubated in pCa 7.0 loading solution for 15, 30, 60, 120, and 180 s. After each loading period, the fiber was immersed in a relaxing solution deprived of EGTA (washing solution) and exposed to 20 mM caffeine, and the tension developed was measured. For each fiber, the contracture force was plotted against loading duration, and data were fitted, via nonlinear regression (r 2 = 0.93–0.99), to the equation F = 100(1−e −kt), where F is the measured tension normalized to the maximum tension developed,k is the rate constant for Ca2+ uptake, and t is the loading duration (37).
Caffeine sensitivity of the SR.
Caffeine sensitivity of SR Ca2+ release was also analyzed indirectly by following the minimal caffeine-induced tension development (7, 28). Fibers were allowed to accumulate Ca2+ into the SR by incubation in a pCa 7.0 loading solution (same composition of relaxing solution with 0.8 mM Ca) for 30 s at room temperature. After Ca2+ loading, fibers were immersed in the washing solution (see above) and then challenged in a stepwise manner with increasing concentrations of caffeine until tension was recorded. Caffeine threshold was defined as the lowest concentration of caffeine that was able to induce an appreciable tension (28).
Caffeine contracture experiments were performed by exposing the whole muscle to a 30 mM caffeine solution and measuring the subsequent contracture, both in resting conditions and 30 s after the fatiguing protocol. The contracture tension was expressed as percentage of twitch tension.
Single chemically skinned fibers were identified by their myosin heavy chain (MHC) composition (9). At the end of each experiment, the fiber segment was dissolved with 20 μl of SDS-PAGE solubilization buffer (62.5 mM Tris, pH 6.8, 2.3% SDS, 5% 2-mercaptoethanol, 10% glycerol) and analyzed by 7% PAGE (26) to identify the MHC isoform composition. The evaluation of the experimental data was limited to type 1 (slow-twitch, fatigue-resistant) fibers from soleus muscle containing only the MHC1 isoform (Fig. 1,lane c) and from type 2 (fast-twitch, fatigue-sensitive) EDL muscle fibers containing only the MHC2B isoforms (Fig. 1,lane d), or those fibers of EDL that besides the MHC2B contained traces of MHC2X only (Fig. 1,lane e).
Myosin light chain isoform analysis.
Myosin light chain composition was analyzed by two-dimensional gel electrophoresis as previously described (3). About 20 cryostat muscle sections (20 μm thin) were dissolved in 100 μl of 9.5 M urea, 2% (vol/vol) Nonidet NP-40, 5% (vol/vol) 2-mercaptoethanol, 1.0% (vol/vol) Ampholine (LKB) of pH range 5–7, and 1.0% (vol/vol) Ampholine (LKB) of pH range 3.5–10 and subjected to isoelectric focusing. SDS-PAGE in the second dimension was performed in 15% (wt/vol) polyacrylamide slab gels. The relative amounts of phosphorylated and nonphosphorylated myosin light chain bands were determined by densitometry of SDS-PAGE slab gels by using a Bio-Rad imaging densitometer (GS-670).
Means and SE were calculated from individual values by standard procedures. Results were analyzed by one-way ANOVA performing multiple comparisons against the control group (SigmaStat, Jandel Scientific). The 0.05 level of probability was established for statistical significance. pCa-tension data from each muscle fiber were fitted by a least squares method using the Table Curve fitting program (Jandel Scientific) according to the equation y = maxx N/(xN +kN ) where max is the maximal value of pCa-tension curve, which was normalized to 1, k is the pCa at 50% of maximum tension (pCa50), and N is the Hill coefficient.
The occurrence and relevance of posttranslational modifications during fatigue were investigated by using single muscle fibers chemically skinned immediately after the fatiguing protocol and by comparing their contractile properties with those of resting fibers. The chemical skinning procedure allows the complete removal of fatigue milieu and, by replacement with a physiological medium, should reintroduce the original capacity of the fiber to produce 100% of tension (36).
The pCa-tension relationship of chemically skinned type 1 fibers from soleus muscle was significantly shifted to the right after fatigue compared with rested fibers (Fig.2 A). In fact, the pCa threshold, i.e., the lowest concentration of calcium inducing a detectable tension, and the pCa50 were significantly lower in fatigued than in rested fibers (Table1). The Hill coefficient, an estimate of the cooperativity among the elements participating to the activation of the contractile apparatus (8), was unmodified by fatigue (Table 1), suggesting that only calcium sensitivity of myofilaments was altered.
In comparison with the behavior of soleus fibers, the pCa-tension relationship of fast-twitch fibers isolated from EDL muscle was only modestly affected by fatigue. In fact, minor, not significant, increases in the pCa threshold for tension development were observed in the EDL fatigued fibers (Fig. 2 B); no modifications of the pCa50 and of the Hill coefficient were evident (Table 1).
Two-dimensional analysis of myosin light chains.
The myofibrillar calcium sensitivity of skeletal and cardiac muscles may be influenced by the state of phosphorylation of the regulatory myosin light chains, which are phosphorylated by a specific Ca2+/calmodulin-dependent kinase and dephosphorylated by a type 1 myofibrillar phosphatase (30). We investigated whether the changes in the pCa-tension relationship of fatigued muscle fibers were attributable to changes in the phosphorylation states of the regulatory light chains. Accordingly, two-dimensional analysis of myosin light chains was performed to identify changes in protein phosphorylation resulting from fatigue. As shown in Fig.3, the regulatory light chains (labeled 2F) of EDL fibers were present as two distinct protein bands with the same molecular weight but different isoelectric point, both in rested and fatigued muscles. After fatigue, the EDL muscle exhibited a significant (P < 0.05) increase in the amount of the phosphorylated regulatory light chains (2F-P), which changed from 44.3 ± 3.3% (n = 4) on rested muscles to 61.3 ± 3.8% (n = 4) on fatigued muscles (Fig. 3,left). Conversely, the regulatory myosin light chains (2S) of soleus fibers were not phosphorylated in the rested muscle and were not phosphorylated after fatigue (Fig. 3, right).
SR Ca2+ uptake and release.
It has been previously demonstrated, using purified SR vesicles (4) and skinned fiber preparations (27), that nonfatigued fast-twitch muscle fibers possess mean SR Ca2+ uptake capacities that are at least double those of slow-twitch muscle fibers. We have confirmed those initial observations and extended them to include other measures of SR function and, most importantly, changes in SR function resulting after fatigue (Table 2). The initial Ca2+release rate induced by 10 mM caffeine was 30% higher in EDL type 2B fibers than in soleus type 1 fibers, whereas in isolated SR vesicles it is reported that the Ca2+ release rate of fast SR is at least four times that of slow SR (29). However, this apparent difference is attributed to the well-known higher sensitivity to caffeine of slow-twitch muscle fibers compared with fast-twitch fibers (28, 29; see also the caffeine threshold data shown below).
Fatigue did not modify the mean total SR Ca2+ uptake capacities of either EDL type 2B or soleus type 1 muscle fibers (Table2). On the other hand, the rate of Ca2+ uptake by the SR measured by the light-scattering method was significantly increased in fatigued soleus fibers, whereas it was unmodified in fast EDL fibers (Table 2). Similar results were obtained by using the caffeine contracture procedure (37), in which the SR of fatigued soleus type 1 fibers accumulated calcium at a higher rate than did rested fibers (Fig. 4 A). No differences in Ca2+ uptake rate were evident in EDL type 2B fibers (Fig. 4 B). The values of K Ca, i.e., the rate constant for Ca2+ uptake, were 2.08 ± 0.29 in rested type 1 fibers and 3.06 ± 0.28 in fatigued fibers (P < 0.05), whereas the values in type 2B fibers were 1.66 ± 0.11 and 1.78 ± 0.38, respectively. It is worth noting that, because of the higher sensitivity to caffeine of slow than of fast muscles (see above), the method used in Fig. 4(37) does not allow appreciation of the known higher Ca2+ uptake rate of fast muscle than of slow muscles (28, 29).
With the light-scattering method, it is also possible to evaluate the SR Ca2+ release properties of single muscle fibers by stimulating Ca2+ release with 10 mM caffeine after Ca2+ filling of the SR (23, 27). The initial SR Ca2 release rates of chemically skinned type 1 soleus and of type 2 EDL muscle fibers were not modified after fatigue (Table 2).
In this preparation, we also analyzed the caffeine sensitivity of SR Ca2+ release. The mean caffeine threshold concentrations capable of inducing significant tension were 1.30 ± 0.22 and 7.89 ± 0.51 mM, respectively, for type 1 and type 2 nonfatigued fibers. The corresponding values for fatigued fibers were 2.03 ± 0.06 and 7.93 ± 0.54 mM, demonstrating no significant alterations in caffeine threshold in mammalian muscle fibers as a function of fatigue status.
Whereas fatigue did not cause persistent modifications in caffeine sensitivity of SR Ca2+ release, the caffeine sensitivity of the whole muscle contractility was dramatically affected by fatigue (Fig. 5). This figure demonstrates that the caffeine contractures produced by both EDL and soleus muscles were significantly reduced by fatigue.
The decline in force induced by fatigue in skeletal muscle is ascribed mainly to the accumulation of metabolites and to decreases in the free calcium concentration of the myoplasm (11,12, 29, 36). Recovery of a fatigued muscle takes variable time, depending on the ability of the muscle to restore the normal ionic and metabolite levels. One would predict that chemically skinned fibers, which have carefully controlled metabolic and ionic environments, should demonstrate no evidence of fatigue if alterations in soluble metabolites were the only factors responsible for fatigue. However, previous results on frog muscle (37, 38) and the present results on mammalian muscle fibers demonstrate that fatigue-related changes of myofibrillar protein properties and of SR activities are still evident in the chemically skinned muscle fiber preparation devoid of metabolite perturbations. Moreover, our results show that, in mammalian muscles, the effects of fatigue that persisted after chemical skinning were different in fast- and slow-twitch fibers.
The persistent changes that we demonstrated in chemically skinned fibers could be attributed to posttranslational modifications of proteins, which include enzymatic and nonenzymatic modifications. Enzymatic posttranslational modification of proteins comprises, for example, phosphorylation and dephosphorylation, cleavage, methylation, glycosylation, and ADP-ribosylation. Nonenzymatic modifications, instead, involve chemical-physical perturbations of proteins such as, for example, oxidation, glycation, and deamination.
Myofibrillar properties of fatigued fibers.
For skinned slow-twitch rat muscle fibers, fatigue causes a significant reduction in myofibrillar protein sensitivity to calcium, indicating that, to produce the same tension as in rested fibers, a higher free calcium concentration is needed. In contrast to slow-twitch fibers, fast-twitch fibers did not show a fatigue-dependent right shift of pCa-tension curves.
Even though calcium sensitivity may be influenced by fatigue, we observed that the maximal Ca2+-activated tension of fatigued skinned fibers was identical to that of rested fibers. This result indicates that changes in myofibrillar calcium sensitivity caused by fatigue in soleus skinned fibers reside in modifications of regulatory proteins, which reduce the number of cross bridges at a given pCa, but not when myoplasmic calcium concentration is above that for saturation of troponin C. However, in intact fibers, a reduced maximal calcium activated force, as well as a reduced myofibrillar calcium sensitivity, has been observed (36). Thus, besides the changes in the regulatory proteins, other factors may influence the number of or the tension developed by cross bridges, such as, for example, reduced intracellular Ca2+ and the accumulation of myoplasmic Pi, known to influence maximal Ca2+-activated tension (14, 15, 21).
On the basis of results with skinned fibers, repetitive stimulation of fast-twitch muscle fibers is known to cause a leftward shift in the pCa-tension relationship as a consequence of myosin light chain-2 phosphorylation, and it is also known that this is mediated by a specific Ca2+/calmodulin-dependent endogenous protein kinase (20, 30). Phosphorylation of the regulatory light chain affects Ca2+ sensitivity of fast-twitch fibers prevalently at high and moderate pCa values (30). Phosphorylation of the regulatory light chain may represent a mechanism activated by mammalian skeletal muscle to counteract the effects of fatigue. However, this adaptation is true only for fast-twitch myosin. In fact, the regulatory light chain of slow-twitch muscles is not phosphorylated in the resting state, and stimulation does not modify this condition (see Fig. 3). On the other hand, it is possible that fatigue causes a right shift also in fast-twitch fibers, but this occurrence may be counteracted by light chain phosphorylation.
A possible mechanism operating in slow-twitch muscle to account for changes in myofibrillar calcium sensitivity during fatigue is oxidation of SH groups, which has been shown to modify Ca2+sensitivity (1, 39). However, this mechanism has been shown to also reduce maximal Ca2+-activated tension. Because we did not observe significant modification of maximal tension, either this mechanism is not working or its effect is not relevant. An additional possible mechanism operating during fatigue is glycation (17) and/or deamination (2) of myofibrillar proteins. In particular, the glycation mechanism appears to be plausible during fatigue, because it has been observed that both pH and phosphate affect glycation of proteins (31). Finally, Williams (37) hypothesized also that extensive stimulation might produce some transient disarrangement of myofilaments that could involve regulatory proteins. In fact, removal, even partial, of regulatory proteins strongly affects myofilaments calcium sensitivity (22).
SR Ca2+ flux properties of fatigued fibers.
Tension decline during fatigue is associated with substantial changes in the intracellular milieu, which, in turn, are mainly responsible for the progressive ineffective delivery of calcium to the myofilaments, likely attributable to an altered excitation-contraction coupling mechanism and changes in SR calcium content and Ca2+ release (11, 12,29, 36).
The present results showed that the SR of slow-twitch fibers chemically skinned immediately after fatigue accumulates Ca2+ at a higher rate than that of fibers skinned before fatigue, whereas no appreciable modifications were evident in fast-twitch fibers. Moreover, the SR Ca2+ release properties of both fast- and slow-twitch chemically skinned fibers were not modified by fatigue. In a fast-twitch frog skeletal muscle, a significant reduction both in the rate constant of SR Ca2+ uptake and in the caffeine sensitivity of SR Ca2+ release was reported (37, 38). The discrepancy between these data and ours may be ascribed to species-specific mechanisms.
Studies on isolated SR demonstrate that strenuous exercise causes either reduction of SR Ca2+ uptake (5,6, 13) or no modifications (10). It is possible that these conflicting results may be due to different SR isolation techniques and/or to differences in the type of exercise and fiber population of the muscles studied. In addition, calcium phosphate precipitation within the SR occurring in late fatigue (14) may alter the properties of SR membranes isolated from fatigued muscles that, in turn, could be responsible for the lower Ca2+ uptake capacity observed.
SR Ca2+ uptake activity is attributed to a specific Ca2+ pump, which is located in the SR. The activity of cardiac and slow-twitch skeletal muscles Ca2+ pumps can be modulated by phosphorylation. Direct phosphorylation by a Ca2+/calmodulin-dependent protein kinase activates the Ca2+ pump (16), and phosphorylation by cAMP-dependent and Ca2+/calmodulin-dependent protein kinases of phospholamban, a regulatory protein associated with the pump protein, further stimulates the Ca2+ pump (24). Thus the higher SR Ca2+ uptake rate observed in fatigued slow-twitch fibers is consistent with the possible activation of the Ca2+ pump by phosphorylation either directly or indirectly through phospholamban.
In intact single muscle fiber fatigue, however, a marked reduction of the rate of Ca2+ removal from cytoplasm has been observed (36). It is worth noting that the incubation of skinned fibers in conditions that mimic those produced by fatigue have been demonstrated to influence the Ca2+ uptake rate. For example, high Pi concentration stimulates (14, 32), whereas acidosis reduces, the SR Ca2+ uptake (34). Thus the slowing in the Ca2+ uptake rate observed in intact fibers is likely the result of the effects of fatigue metabolites combined with those of posttranslational modifications of the Ca2+ pump and/or phospholamban. Additionally, it appears that fatigue causes a number of modifications, some metabolic and others not, that lead to the slowing of the net Ca2+ uptake rate.
Even though we have demonstrated fatigue-dependent alterations in calcium uptake properties, our results show that SR Ca2+release properties of skinned fibers were not significantly modified by fatigue. Thus any calcium release defects seen in intact muscles should be ascribed to changes either in metabolite levels (36), in action potential characteristics, or in the activation process of the T-tubular charge sensor (12, 29,36).
In conclusion, these results demonstrate that posttranslational enzymatic and/or nonenzymatic modifications of proteins responsible for myofibrillar and SR properties contribute to force decline caused by fatigue in mammalian slow-twitch muscle fibers. Conversely, posttranslational changes of proteins appear not to play a role in fatigue of fast-twitch muscle.
We thank Prof. Roger A. Sabbadini for critical reading of the manuscript. The technical assistance of Luigi Beriotto is gratefully acknowledged.
This work was supported by grants from Telethon Italy (Grant no. 620 to D. Danieli-Betto and no. 629 to R. Betto), “Cofinanziamento Ministero dell'Universitá della Ricerca Scientifica e Tecnologica 1999” (D. Danieli-Betto), and Consiglio Nazionale delle Ricerche institutional funds (R. Betto).
Address for reprint requests and other correspondence: D. Danieli-Betto, Dipartimento di Anatomia e Fisiologia Umana, via Marzolo 3, I-35131 Padova, Italy (E-mail:).
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- Copyright © 2000 the American Physiological Society