Journal of Applied Physiology Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 89: 891-898, 2000;
8750-7587/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Danieli-Betto, D.
Right arrow Articles by Betto, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Danieli-Betto, D.
Right arrow Articles by Betto, R.
Vol. 89, Issue 3, 891-898, September 2000

Effects of fatigue on sarcoplasmic reticulum and myofibrillar properties of rat single muscle fibers

D. Danieli-Betto1, E. Germinario1, A. Esposito1, D. Biral2, and R. Betto2

1 Dipartimento di Anatomia e Fisiologia Umana, and 2 Consiglio Nazionale delle Ricerche, Centro di Studio per la Biologia e la Fisiopatologia Muscolare, I-35131 Padova, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fatigue protocol. 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).

pCa-tension relationship. 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) nmol 45Ca2+ · 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 (r2 = 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-fiber SDS-PAGE. 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).


View larger version (121K):
[in this window]
[in a new window]
  		Fig. 1.   Identification of myosin heavy chain (MHC) isoform composition in single skeletal muscle fibers by SDS-PAGE analysis. Lane a: MHC isoforms from soleus and extensor digitorum longus (EDL) muscle cryostat sections containing all four isoforms; lanes b and c: MHC isoforms from soleus single muscle fibers (fibers like that shown in lane b, i.e., containing trace amounts of MHC2A isoform, were not included in the study); lanes d and e: MHC isoforms from EDL single muscle fibers.

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).

Statistical analysis. 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 = max xN/(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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

pCa-tension relationships. 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. 2A). 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 (Table 1). 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.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2.   pCa-tension relationships of rested and fatigued type 1 soleus fibers (A) and rested and fatigued type 2B (and 2B/2X) EDL fibers (B). Relative tension (P/Po) was plotted as a function of pCa. Values are means ± SE for the no. of fibers indicated in parentheses. * P < 0.05 and ** P < 0.01 vs. rested values.


                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Effect of fatigue on the pCa sensitivity of tension development of type 1 and type 2 rat fibers from soleus and EDL skeletal muscles

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. 2B); 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).


View larger version (106K):
[in this window]
[in a new window]
  		Fig. 3.   Two-dimensional analysis of myosin light chain isoform composition in rested and fatigued EDL and soleus muscles. Only the myosin light chain region is shown. Data shown are representative of 3 separate experiments. 1F, 2F, and 3F, myosin light chain fast isoforms; 2F-P, phosphorylated myosin light chain 2; 1Sa, 1Sb, and 2S, myosin light chain slow isoforms; PA, parvalbumin. As shown, EDL muscle cells, but not soleus, possess parvalbumin, as has been previously demonstrated (36).

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).

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Ca2+ transport activities of the sarcoplasmic reticulum of soleus and EDL rested and fatigued fibers

Fatigue did not modify the mean total SR Ca2+ uptake capacities of either EDL type 2B or soleus type 1 muscle fibers (Table 2). 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. 4A). No differences in Ca2+ uptake rate were evident in EDL type 2B fibers (Fig. 4B). The values of KCa, 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).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 4.   Caffeine contracture tension recording after sarcoplasmic reticulum Ca2+ loading for various time periods of soleus and EDL single muscle fibers (A and B, respectively). Values are means ± SE for number of examined fibers indicated in parentheses. * P < 0.05 vs. resting fibers values.

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.


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 5.   Caffeine contracture of rested (open symbols) and fatigued (solid symbols) soleus (circles) and EDL (triangles) whole rat muscles. Values are means ± SE for number of examined muscles indicated in parentheses; caffeine contractures in fatigued muscles were always significantly lower than in control muscles.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

We thank Prof. Roger A. Sabbadini for critical reading of the manuscript. The technical assistance of Luigi Beriotto is gratefully acknowledged.


    FOOTNOTES

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: danielid{at}ux1.unipd.it).

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. §1734 solely to indicate this fact.

Received 15 November 1999; accepted in final form 11 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andrade, FH, Reid MB, Allen DG, and Westerblad H. Effect of hydrogen peroxide and dithiothreitol on contractile function of single skeletal muscle fibers from the mouse. J Physiol (Lond) 509: 565-575, 1998[Abstract/Free Full Text].

2.   Balagopal, P, Roovackers OE, Asey DB, Ades PA, and Nair KS. Effects of aging on in vivo synthesis of skeletal muscle myosin heavy chain and sarcoplasmic reticulum protein in humans. Am J Physiol Endocrinol Metab 273: E790-E800, 1997[Abstract/Free Full Text].

3.   Biral, D, Damiani E, Volpe P, Salviati G, and Margreth A. Polymorphism of myosin light chains: an electrophoretic and immunological study of rabbit skeletal-muscle myosins. Biochem J 203: 529-540, 1982[Web of Science][Medline].

4.   Briggs, FN, Poland JL, and Solaro RJ. Relative capabilities of sarcoplasmic reticulum in fast and slow mammalian skeletal muscles. J Physiol (Lond) 266: 587-594, 1977[Abstract/Free Full Text].

5.   Byrd, SK, McCutcheon LJ, Hodgson DR, and Gollnick PD. Altered sarcoplasmic reticulum function after high-intensity exercise. J Appl Physiol 66: 1383-1389, 1989[Abstract/Free Full Text].

6.   Chin, ER, and Green HJ. Effects of tissue fractionation on exercise-induced alterations in SR function in rat gastrocnemius muscle. J Appl Physiol 80: 940-948, 1996[Abstract/Free Full Text].

7.   Danieli-Betto, D, Betto R, Megighian A, Midrio M, Salviati G, and Larsson L. Effects of age on sarcoplasmic reticulum properties and histochemical composition of fast- and slow-twitch rat muscles. Acta Physiol Scand 154: 59-64, 1995[Web of Science][Medline].

8.   Danieli-Betto, D, Betto R, and Midrio M. Calcium sensitivity and myofibrillar protein isoforms of rat skinned skeletal muscle fibres. Pflügers Arch 417: 303-308, 1990[Web of Science][Medline].

9.   Danieli-Betto, D, Zerbato E, and Betto R. Type 1, 2A and 2B myosin heavy chain electrophoretic analysis of rat muscle fibers. Biochem Biophys Res Commun 138: 981-987, 1986[Web of Science][Medline].

10.   Dossett-Mercer, J, Green HJ, Chin E, and Grange F. Preservation of sarcoplasmic reticulum Ca2+-sequestering function in muscle tissue of different type composition following sprint activity. Can J Physiol Pharmacol 72: 1231-1237, 1994[Web of Science][Medline].

11.   Favero, TG. Sarcoplasmic reticulum Ca2+ release and muscle fatigue. J Appl Physiol 87: 471-483, 1999[Abstract/Free Full Text].

12.   Fitts, RH. Cellular mechanisms of muscle fatigue. Physiol Rev 74: 49-94, 1994[Abstract/Free Full Text].

13.   Fitts, RH, Cortright JB, Kim DH, and Witzmann FA. Muscle fatigue with prolonged exercise: contractile and biochemical alterations. Am J Physiol Cell Physiol 242: C65-C73, 1982[Abstract/Free Full Text].

14.   Fryer, MW, Owen VJ, Lamb GD, and Stephenson DG. Effects of creatine phosphate and Pi on Ca2+ movements and tension development in rat skinned skeletal muscle fibers. J Physiol (Lond) 482: 123-140, 1995[Abstract/Free Full Text].

15.   Godt, RE, and Nosek TM. Changes on intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle. J Physiol (Lond) 412: 155-180, 1989[Abstract/Free Full Text].

16.   Hawkins, C, Xu A, and Narayanan N. Sarcoplasmic reticulum calcium pump in cardiac and slow-twitch skeletal muscle but not fast-twitch skeletal muscle undergoes phosphorylation by endogenous and exogenous Ca2+/calmodulin-dependent protein kinase. Characterization of optimal conditions for calcium pump phosphorylation. J Biol Chem 269: 31198-31206, 1994[Abstract/Free Full Text].

17.   Höök, P, Li X, Sleep J, Hughes S, and Larsson L. In vitro motility speed of slow myosin extracted from single soleus fibres from young and old rats. J Physiol (Lond) 520: 463-471, 1999[Abstract/Free Full Text].

18.   Knuth, ST, and Fitts RH. Effect of pH and temperature on the contractile properties of slow skinned skeletal muscle fibers (Abstract). Med Sci Sports Exerc 31: S222, 1999.

19.   Kugelberg, E, and Lindegren B. Transmission and contraction fatigue of rat motor units in relation to succinate dehydrogenase activity of motor unit fibers. J Physiol (Lond) 288: 285-300, 1979[Abstract/Free Full Text].

20.   Manning, DR, and Stull JT. Myosin light chain phosphorylation-dephosphorylation in mammalian skeletal muscle. Am J Physiol Cell Physiol 242: C234-C241, 1982[Abstract/Free Full Text].

21.   Metzger, JM, and Moss RL. Greater hydrogen ion-induced depression of tension and velocity in skinned single fibers of rat fast than slow muscles. J Physiol (Lond) 393: 727-742, 1987[Abstract/Free Full Text].

22.   Metzger, JM, and Moss RL. Myosin light chain 2 modulates calcium-sensitive cross-bridge transitions in vertebrate skeletal muscle. Biophys J 63: 460-468, 1992[Web of Science][Medline].

23.   Midrio, M, Danieli Betto D, Megighian A, and Betto R. Early effects of denervation on sarcoplasmic reticulum properties of slow-twitch rat muscle fibres. Pflügers Arch 434: 398-405, 1997[Web of Science][Medline].

24.   Movsesian, MA, Morris GL, Wang JH, and Krall J. Phospholamban-modulated Ca2+ transport in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum. Second Messenger Phosphoproteins 14: 151-161, 1992-93.

25.   Pate, E, Bhimani M, Franks-Skiba K, and Cooke R. Reduced effect of pH on skinned rabbit psoas muscle mechanics at high temperatures: implications for fatigue. J Physiol (Lond) 486: 689-694, 1995[Abstract/Free Full Text].

26.   Rossini, K, Rizzi C, Sandri M, Bruson A, and Carraro U. High resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins. Electrophoresis 16: 101-104, 1995[Web of Science][Medline].

27.   Salviati, G, Sorenson MM, and Eastwood AB. Calcium accumulation by the SR in two populations of chemically skinned human muscle fibers. J Gen Physiol 79: 603-632, 1982[Abstract/Free Full Text].

28.   Salviati, G, and Volpe P. Ca2+ release from sarcoplasmic reticulum of skinned fast- and slow-twitch muscle fibers. Am J Physiol Cell Physiol 254: C459-C465, 1988[Abstract/Free Full Text].

29.   Stephenson, DG, Lamb GD, and Stephenson GMM Events of the excitation-contraction-relaxation (E-C-R) cycle in fast- and slow-twitch mammalian muscle fibres relevant to muscle fatigue. Acta Physiol Scand 162: 229-245, 1998[Web of Science][Medline].

30.   Sweeney, HL, Bowman BF, and Stull JT. Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function. Am J Physiol Cell Physiol 264: C1085-C1095, 1993[Abstract/Free Full Text].

31.   Tessier, F, and Birlouez-Aragon I. Effect of pH, phosphate and copper on the interaction of glucose with albumin. Glycoconj J 15: 571-574, 1998[Web of Science][Medline].

32.   Thomson, LV, and Fitts RH. Muscle fatigue in the frog semitendinosus: role of the high-energy phosphates and Pi. Am J Physiol Cell Physiol 263: C803-C809, 1992[Abstract/Free Full Text].

33.   Trachez, MM, Sudo RT, and Suarez-Kurtz G. Alterations in the functional properties of skinned fibers from denervated rabbit skeletal muscle. Am J Physiol Cell Physiol 259: C503-C506, 1990[Abstract/Free Full Text].

34.   Westerblad, H, and Allen DG. The influence of intracellular pH on contraction, relaxation in fatigued single fibers from mouse skeletal muscle. J Physiol (Lond) 468: 729-740, 1993[Abstract/Free Full Text].

35.   Westerblad, H, Bruton JD, and Lannergren J. The effect of intracellular pH on contractile function of intact, single fibres of mouse muscle declines with increasing temperature. J Physiol (Lond) 500: 193-204, 1997[Abstract/Free Full Text].

36.   Westerblad, H, Lee JA, Lannergren J, and Allen DG. Cellular mechanisms of fatigue in skeletal muscle. Am J Physiol Cell Physiol 261: C195-C209, 1991[Abstract/Free Full Text].

37.   Williams, JH. Contractile apparatus and sarcoplasmic reticulum function: effects of fatigue, recovery, and elevated Ca2+. J Appl Physiol 83: 444-450, 1997[Abstract/Free Full Text].

38.   Williams, JH, Ward CW, and Klug GA. Fatigue-induced alterations in Ca2+ and caffeine sensitivities of skinned muscle fibers. J Appl Physiol 75: 586-593, 1993[Abstract/Free Full Text].

39.   Wilson, GJ, dos Remedios CG, Stephenson DG, and Williams DA. Effects of sulphydryl modification using 5,5'-dithiobis(2-nitrobenzoic acid) on skinned rat skeletal muscle fibres. J Physiol (Lond) 437: 409-430, 1991[Abstract/Free Full Text].

J APPL PHYSIOL 89(3):891-898
8750-7587/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Physiol.Home page
R. M. Murphy, T. L. Dutka, and G. D. Lamb
Hydroxyl radical and glutathione interactions alter calcium sensitivity and maximum force of the contractile apparatus in rat skeletal muscle fibres
J. Physiol., April 15, 2008; 586(8): 2203 - 2216.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
D. G. Allen, G. D. Lamb, and H. Westerblad
Skeletal Muscle Fatigue: Cellular Mechanisms
Physiol Rev, January 1, 2008; 88(1): 287 - 332.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
A. Esposito, E. Germinario, M. Zanin, P. T. Palade, R. Betto, and D. Danieli-Betto
Isoform switching in myofibrillar and excitation-contraction coupling proteins contributes to diminished contractile function in regenerating rat soleus muscle
J Appl Physiol, April 1, 2007; 102(4): 1640 - 1648.
[Abstract] [Full Text] [PDF]

Home page
 Br. J. Sports. Med.Home page
J P Weir, T W Beck, J T Cramer, T J Housh, T D Noakes, A St Clair Gibson, and E V Lambert
Is fatigue all in your head? A critical review of the central governor model * Commentary.
Br. J. Sports Med., July 1, 2006; 40(7): 573 - 586.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
D. Danieli-Betto, A. Esposito, E. Germinario, D. Sandona, T. Martinello, A. Jakubiec-Puka, D. Biral, and R. Betto
Deficiency of {alpha}-sarcoglycan differently affects fast- and slow-twitch skeletal muscles
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2005; 289(5): R1328 - R1337.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
D. Danieli-Betto, E. Germinario, A. Esposito, A. Megighian, M. Midrio, B. Ravara, E. Damiani, L. D. Libera, R. A. Sabbadini, and R. Betto
Sphingosine 1-phosphate protects mouse extensor digitorum longus skeletal muscle during fatigue
Am J Physiol Cell Physiol, June 1, 2005; 288(6): C1367 - C1373.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
E. Germinario, A. Esposito, A. Megighian, M. Midrio, D. Biral, R. Betto, and D. Danieli-Betto
Early changes of type 2B fibers after denervation of rat EDL skeletal muscle
J Appl Physiol, May 1, 2002; 92(5): 2045 - 2052.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Danieli-Betto, D.
Right arrow Articles by Betto, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Danieli-Betto, D.
Right arrow Articles by Betto, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online