J Appl Physiol
89: 891–898, 2000.
Effects of fatigue on sarcoplasmic reticulum and
myofibrillar properties of rat single muscle fibers
D. DANIELI-BETTO,1 E. GERMINARIO,1 A. ESPOSITO,1 D. BIRAL,2 AND R. BETTO2
Dipartimento di Anatomia e Fisiologia Umana, and 2Consiglio Nazionale delle Ricerche, Centro di
Studio per la Biologia e la Fisiopatologia Muscolare, I-35131 Padova, Italy
1
Received 15 November 1999; accepted in final form 11 April 2000
myofibrillar calcium sensitivity; chemically skinned fibers
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
PROLONGED STIMULATION OF SKELETAL
Address for reprint requests and other correspondence: D. DanieliBetto, Dipartimento di Anatomia e Fisiologia Umana, via Marzolo 3,
I-35131 Padova, Italy (E-mail: [email protected]).
http://www.jap.org
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 fasttwitch 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 slowtwitch 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
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
The costs of publication of this article were defrayed in part by the
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8750-7587/00 $5.00 Copyright © 2000 the American Physiological Society
891
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Danieli-Betto, D., E. Germinario, A. Esposito, D. Biral, and R. Betto. Effects of fatigue on sarcoplasmic reticulum and myofibrillar properties of rat single muscle fibers. J
Appl Physiol 89: 891–898, 2000.—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 䡠 min⫺1 䡠 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.
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SKELETAL MUSCLE FATIGUE
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
45
Ca2⫹ 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⫹ 䡠 light-scattering
unit⫺1 䡠 ␮g 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 con-
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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
SKELETAL MUSCLE FATIGUE
RESULTS
The occurrence and relevance of posttranslational
modifications during fatigue were investigated by using single muscle fibers chemically skinned immedi-
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.
ately 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.
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).
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⫹
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ditions 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 ␮l 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 ␮m thin) were dissolved in 100 ␮l 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 pCatension curve, which was normalized to 1, k is the pCa at 50%
of maximum tension (pCa50), and N is the Hill coefficient.
893
894
SKELETAL MUSCLE FATIGUE
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 fasttwitch 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 (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 soTable 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
Soleus
Rested (14)
Fatigued (17)
EDL
Rested (31)
Fatigued (31)
pCaTH
pCa50
N
6.80 ⫾ 0.04
6.66 ⫾ 0.04*
6.23 ⫾ 0.03
5.99 ⫾ 0.05*
2.49 ⫾ 0.16
2.81 ⫾ 0.12
6.26 ⫾ 0.03
6.37 ⫾ 0.03
5.91 ⫾ 0.02
5.88 ⫾ 0.04
4.04 ⫾ 0.25
4.07 ⫾ 0.35
Values are means ⫾ SE for the numbers of the analyzed single
fibers given in parentheses. EDL, extensor digitorum longus; pCaTH,
lowest pCa giving a detectable tension; pCa50, pCa value corresponding to 50% of maximum tension; N, Hill coefficient. Student’s t-test
was applied for comparison between groups. * Statistical differences
between the rested and the fatigued muscles for values of P ⬍ 0.01.
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).
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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.
leus 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).
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 fig-
895
SKELETAL MUSCLE FATIGUE
Table 2. Ca2⫹ transport activities of the sarcoplasmic reticulum of soleus and EDL rested and fatigued fibers
Soleus
Ca uptake capacity, ␮mol/mg
fiber protein
Rate of Ca2⫹ uptake,
nmol 䡠 min⫺1 䡠 mg fiber protein⫺1
Initial Ca2⫹ release rate,
nmol 䡠 min⫺1 䡠 mg fiber protein⫺1
EDL
Rested
Fatigued
Rested
Fatigued
0.249 ⫾ 0.016 (14)
0.276 ⫾ 0.069 (13)
0.531 ⫾ 0.085 (14)
0.512 ⫾ 0.091 (11)
14.2 ⫾ 1.0 (14)
19.6 ⫾ 2.5* (13)
43.0 ⫾ 5.9 (14)
41.5 ⫾ 6.01 (11)
77.4 ⫾ 13.6 (12)
75.0 ⫾ 9.8 (11)
2⫹
59.3 ⫾ 13.1 (14)
57.6 ⫾ 10.7 (13)
Values are means ⫾ SE; nos. of fibers are in parentheses. Ca uptake capacity, rate of Ca uptake and initial caffeine-induced Ca2⫹
release rate of rested and fatigued type 1 soleus fibers (i.e., fibers expressing type 1 MHC isoform only) and of rested and fatigued type 2B
(fibers expressing type 2B MHC isoforms plus fibers that, besides type 2B, contained traces of type 2X MHC isoforms) EDL fibers.
* P ⬍ 0.05 vs. resting fibers values.
2⫹
ure demonstrates that the caffeine contractures produced by both EDL and soleus muscles were significantly reduced by fatigue.
2⫹
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.
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
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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
896
SKELETAL MUSCLE FATIGUE
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 slowtwitch 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
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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).
SKELETAL MUSCLE FATIGUE
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.
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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).
897
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