Download Improved fatigue resistance in Gs

J Appl Physiol 111: 834–843, 2011.
First published June 16, 2011; doi:10.1152/japplphysiol.00031.2011.
Improved fatigue resistance in Gs␣-deficient and aging mouse skeletal
muscles due to adaptive increases in slow fibers
Han-Zhong Feng,1 Min Chen,2 Lee S. Weinstein,2 and J.-P. Jin1
1
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan; 2Metabolic Diseases Branch,
National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland
Submitted 10 January 2011; accepted in final form 11 June 2011
Gs␣ deficiency; aging; skeletal muscle adaptation; myosin and troponin isoforms
SKELETAL MUSCLES ARE COMPOSED of slow and fast types of
muscle fibers with different contractile and metabolic properties. The slow-twitch type I muscle fibers are rich in mitochondria and possess high oxidative capacity and are resistant to
fatigue, whereas the fast-twitch type II muscle fibers have high
glycolytic metabolism and fatigue more easily. Skeletal muscle is
also an important tissue in glucose and energy metabolism due to
its large requirement for nutrients and function as a major site of
acute disposal of glucose load. Disruption of glucose uptake in
skeletal muscle due to deletion of glucose transporter-4 leads to
glucose intolerance and insulin resistance (33). Gs␣ is a ubiquitously expressed G protein ␣-subunit that couples receptors to
adenylyl cyclase and is required for receptor-mediated intracellular cAMP generation. Genetically modified mice with Gs␣ deficiency in skeletal muscle (MGsKO) showed reduced glucose
Address for reprint requests and other correspondence: J.-P. Jin, Dept. of
Physiology, Wayne State Univ. School of Medicine, Detroit, Michigan 48201
(e-mail: [email protected]).
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tolerance, low muscle mass, and decreased contractile force, along
with a fast-to-slow-fiber-type switch (9).
Fiber-type switches indicate an adaptation of skeletal muscle to
functional and metabolic demands. In Type 2 diabetic patients, the
number of slow oxidative fibers is reduced, whereas the number
of fast glycolytic fibers increased (35, 36). This replacement of
oxidative fibers by glycolytic fibers is also found in obese patients
(41). The muscle fiber-type switching toward low oxidative capacity may contribute to the pathogenesis of metabolic disorders,
since it was found to precede the development of obesity and
diabetes in animals (38). Exercise results in a shift in changes in
the type II fibers from type IIb to IIx/a (10). Endurance training,
on the other hand, has been shown to be beneficial in diabetic
patients by improving muscle quality and increased cross-sectional area of both type I and type II fibers (6).
Progressive losses of locomotor’s function and muscle mass
occur in aging. Sarcopenia, the age-related loss of muscle
mass, leads to declined muscle strength with reduction of fiber
size and number (27). Histological studies of human muscle
biopsies reported an increased ratio of type I to type II fibers in
aging (26). Considering the established function of type I slowmuscle fibers in the resistance to fatigue (31, 44, 46), maintenance
of slow type of fibers would have a benefit to the quality of life of
the elderly, with reduced muscle mass in age-associated sarcopenia, as well as diabetic and obese patients.
In the present study, we investigated the hypothesis that the
switching to more slow fibers is an adaptive response with
functional benefits. Studies of MGsKO mouse soleus muscle
showed that, corresponding to the switch of myosin isoforms,
the thin-filament regulatory proteins troponin T (TnT) and
troponin I (TnI) also had significant changes to their slow
isoforms. This fiber-type switching involving both thick- and
thin-myofilament protein progressed in the soleus muscles of
aging MGsKO mice to express solely slow isoforms of myosin
and troponin. Functional characterization found slower contractile and relaxation kinetics and lower force production, but
increased fatigue resistance, followed by better recovery in
MGsKO soleus muscle, correlating to the higher slow-fiber
contents. Since the fiber-type switching did not start in the
muscles of neonatal and 3-wk-old MGsKO mice, the fast-toslow-fiber-type switch appears to be an adaptation in metabolic
abnormality and in aging, providing a beneficial mechanism to
sustain skeletal muscle function by improving fatigue resistance.
MATERIALS AND METHODS
MGsKO mice. As described previously (9, 11), mice with floxed
Gs␣ exon 1 allele (E1fl) (7) were bred with transgenic mice bearing a
muscle creatine kinase (MCK)-cre allele (Taconic, Hudson, NY) to
induce striated muscle-specific disruption of the Gs␣ gene (MCK-cre,
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Feng HZ, Chen M, Weinstein LS, Jin JP. Improved fatigue resistance in Gs␣-deficient and aging mouse skeletal muscles due to adaptive
increases in slow fibers. J Appl Physiol 111: 834 – 843, 2011. First
published June 16, 2011; doi:10.1152/japplphysiol.00031.2011.—Genetically modified mice with deficiency of the G protein ␣-subunit (Gs␣)
in skeletal muscle showed metabolic abnormality with reduced glucose tolerance, low muscle mass, and low contractile force, along with
a fast-to-slow-fiber-type switch (Chen M, Feng HZ, Gupta D, Kelleher J, Dickerson KE, Wang J, Hunt D, Jou W, Gavrilova O, Jin JP,
Weinstein LS. Am J Physiol Cell Physiol 296: C930 –C940, 2009).
Here we investigated a hypothesis that the switching to more slow
fibers is an adaptive response with specific benefit. The results showed
that, corresponding to the switch of myosin isoforms, the thin-filament
regulatory proteins troponin T and troponin I both switched to their
slow isoforms in the atrophic soleus muscle of 3-mo-old Gs␣-deficient
mice. This fiber-type switch involving coordinated changes of both
thick- and thin-myofilament proteins progressed in the Gs␣-deficient
soleus muscles of 18- to 24-mo-old mice, as reflected by the expression of solely slow isoforms of myosin and troponin. Compared with
age-matched controls, Gs␣-deficient soleus muscles with higher proportion of slow fibers exhibited slower contractile and relaxation
kinetics and lower developed force, but significantly increased resistance to fatigue, followed by a better recovery. Gs␣-deficient soleus
muscles of neonatal and 3-wk-old mice did not show the increase in
slow fibers. Therefore, the fast-to-slow-fiber-type switch in Gs␣ deficiency at older ages was likely an adaptive response. The benefit of
higher fatigue resistance in adaption to metabolic deficiency and aging
provides a mechanism to sustain skeletal muscle function in diabetic
patients and elderly individuals.
MUSCLE FIBER-TYPE SWITCH IMPROVES FATIGUE RESISTANCE
J Appl Physiol • VOL
forms by glycerol-SDS-PAGE (12). Briefly, the SDS-PAGE samples
were resolved on 8% polyacrylamide gel with acrylamide-to-bisacrylamide ratio of 50:1, prepared in 200 mM Tris base, 100 mM
glycine (pH 8.8), containing 0.4% SDS and 30% glycerol. The
stacking gel contained 4% polyacrylamide with acrylamide-to-bisacrylamide ratio of 50:1, 70 mM Tris·HCl (pH 6.7), 4 mM EDTA,
0.4% SDS, and 30% glycerol. The upper cathode running buffer was
composed of 100 mM Tris base, 150 mM glycine, 0.1% SDS, and
0.01 mM 2-mercaptoethanol. The lower anode running buffer was
50% dilution of the upper running buffer without 2-mercaptoethanol.
The 0.75-mm-thick Bio-Rad minigels were run at 100 V in an icebox
for 24 h. The resolved protein bands were visualized with Coomassie
blue staining.
Immunohistochemistry. Immunohistochemical examination of mouse
soleus muscles was performed as described (12). The isolated muscle
tissues were mounted between two pins to keep the original slack
length in a small drop of optimum cutting temperature compound
(Sakura, Tissue-Tek) and rapidly frozen in isopentane at ⫺159°C. The
frozen muscle tissue was then embedded in optimum cutting temperature compound before cryo-sectioning. Thin cross sections of the
soleus muscles were fixed in 75% acetone/25% ethanol for 5 min.
After being blocked in phosphate-buffered saline (PBS) containing
0.05% Tween 20 (PBS-T) and 1% BSA for 30 min, the sections were
incubated with 1% H2O2 in PBS for 10 min to inactivate endogenous
peroxidases. The sections were then washed with PBS-T three times
and incubated with anti-MHC I MAb FA2 or SP2/0 myeloma culture
supernatant at 4°C overnight. After being washed with PBS-T to
remove unbound MAb, the sections were incubated with horseradish
peroxidase-labeled anti-mouse second antibody at room temperature
for 1 h, washed again, and developed in 3,3=-diaminobenzidine-H2O2
substrate solution in a dark box for 30 – 60 s. The substrate reaction
was stopped by washes in 20 mM Tris·HCl, pH 7.6, for six changes.
After counterstaining with hematoxylin for 5 min and washing with
water, the sections were immersed in a drop of 50% glycerol in PBS
and mounted with Cytoseal (Thermo Scientific). The slides were
examined using a Zeiss Observer microscope and photographed.
Data analysis. Densitometry analysis of SDS gels and Western
blots was performed on images scanned at 600 dots/in. resolution
using National Institutes of Health Image 1.61 software. Quantitative
data were documented as means ⫾ SE. Statistical significance of
differences between the mean values was analyzed using two-tail
unpaired Student’s t-test, unless specified in the figure legends.
RESULTS
Fast-to-slow switch of TnI and TnT isoforms in soleus
muscle of MGsKO mice corresponding to the switch from type
II to type I fibers. Mouse soleus muscle is a slow-type muscle
containing a large proportion of type I slow fibers mixed with
significant amount of type IIa fibers. Corresponding to the
previously reported switch to a higher level of type I isoform
of MHC in MGsKO mouse soleus muscle with increased
slow-fiber contents (9), the expression of TnI and TnT also
switched to more slow isoforms in 3-mo-old MGsKO mouse
soleus muscle compared with that in age-matched controls
(Fig. 1). The concurring isoform switch of both thick- and
thin-filament proteins (i.e., myosin and troponin) indicated a
coordinated fast-to-slow-fiber-type switch in MGsKO mouse
soleus muscle.
Minimum phenotype changes in EDL muscles of 3-mo-old
MGsKO mice. The fast-twitch muscle EDL of 3-mo-old
MGsKO mice had normal mass, and the myofilament protein
contents indicated an unaffected pure fast-fiber type (Fig. 2).
No significant change of overall protein profile was found in
SDS-PAGE (Fig. 2A). No type I MHC was detected by
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E1fl/fl; MGsKO). Genotyping of wild-type (E1⫹) and E1fl alleles was
performed by polymerase chain reaction using primers flanking the
downstream loxP site of the E1fl allele (8). The presence or absence of
the MCK-cre transgene was determined by polymerase chain reaction
using cre-specific primers (8). The E1fl allele has no effect on Gs␣
expression or phenotype (8), and, therefore, MCK-cre⫺ or E1⫹/⫹
littermates were used as controls. Animals were maintained on a
12:12-h light-dark cycle (6:00 AM/6:00 PM) and standard pellet diet.
The experimental protocols were approved by the Institutional Animal
Care and Use Committee and were conducted in accordance with the
Guiding Principles in the Care and Use of Animals, as approved by
the Council of the American Physiological Society.
Contractility measurement in isolated mouse soleus muscle. Contractile function of soleus muscle was analyzed using a protocol
modified from our laboratory’s previous studies (12) in female control
and MGsKO mice at 3 mo (n ⫽ 3 each) and 18 –24 mo (n ⫽ 6 and 7,
respectively) of age. The mice were anesthetized with pentobarbital
sodium (0.1 mg/g body wt intraperitoneally). Intact soleus muscles
were isolated from tendon to tendon, with care to avoid stretch or
tissue damage. The muscle was mounted vertically to a dual-mode
lever arm force transducer (model 300B, Aurora Scientific) by tying
the tendons with no. 3– 0 sutures in an organ bath containing 100 ml
modified Kreb’s solution (118 mM NaCl, 25 mM NaHCO3, 4.7 mM
KCl, 1.2 mM KH2PO4, 2.25 mM MgSO4, 2.25 mM CaCl2, and 11
mM D-glucose, continuously gassed with 95% O2-5% CO2, pH 7.4).
Contractions were elicited with bipolar pulse electrical stimulation via
platinum plate electrodes (1 ⫻ 5 cm), positioned 0.75 cm apart
flanking the muscle strip using a stimulator (model 701B, Aurora
Scientific).
Twitch contractions were elicited with supramaximal pulses (0.1
ms, 28 V/cm), unless specified otherwise. Tetanic contractions were
elicited with a train of the same pulses at 120 Hz for 0.7 s. Isometric
force data were collected via a digital controller A/D interface (model
604C, Aurora Scientific) and recorded using Chart software (ASI,
Aurora Scientific). The assays were carried out at 25°C. Developed
twitch and tetanic forces were determined at the optimal muscle
length that gave the highest twitch force and calculated as the
difference between the maximum contractile force and the baseline
tension that was constant throughout all experiments. After 20-min
equilibration with one tetanic contraction per minute, a 300-s fatigue
protocol was applied with intermittent tetani of 700 ms every second.
SDS-polyacrylamide gel electrophoresis and Western blot analysis.
Total protein extracts were made by homogenizing MGsKO and
control mouse soleus, extensor digitorum longus (EDL) and diaphragm muscle tissues in SDS-polyacrylamide gel electrophoresis
(PAGE) sample buffer containing 2% SDS using a high-speed mechanical homogenizer. After heating at 80°C for 5 min, the samples
were clarified by centrifugation and resolved on 14% Laemmli gels
with an acrylamide-to-bis-acrylamide ratio of 180:1 and visualized
using Coomassie blue staining or transferred to nitrocellulose membranes using a Bio-Rad semidry electrotransfer apparatus for Western
blot analysis with anti-TnI (TnI-1) and anti-TnT (CT3 and T12)
monoclonal antibodies (MAbs) (19, 30). As described previously (12),
the MAbs were diluted in Tris-buffered saline (TBS) containing 0.1%
bovine serum albumin (BSA) and incubated with the nitrocellulose
membranes at 4°C overnight. After high-stringency washes with TBS
containing 0.5% Triton X-100 and 0.05% SDS, the membranes were
incubated with alkaline phosphatase-labeled goat anti-mouse IgG
second antibody (Sigma) in TBS-BSA and washed as above. The blots
were then developed in 5-bromo-4-chloro-3-indolylphosphate nitro
blue tetrazolium substrate solution to reveal the protein bands recognized by the anti-TnI or anti-TnT MAb. All muscle tissues used in the
contractility studies were recovered for examination by Western
blotting to verify the expression of fiber-type-specific TnT and TnI
isoforms.
Glycerol-SDS-PAGE analysis of myosin heavy chain isoforms.
Muscle tissues were examined for myosin heavy chain (MHC) iso-
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MUSCLE FIBER-TYPE SWITCH IMPROVES FATIGUE RESISTANCE
glycerol-SDS gel. While the major MHC isoform remained to
be IIb, there was a detectable increase in MHC IIx in 3-mo-old
MGsKO EDL muscle compared with the controls. TnI and
TnT also maintained predominantly in fast isoforms, although
slow TnI and slow TnT became barely detectable in the
3-mo-old MGsKO EDL muscle (Fig. 2A).
There was a trend of decreased EDL muscle weight-to-body
weight ratio in 3-mo-old MGsKO mice, but no statistical
significance was established (Fig. 2B). Tibial length was measured to evaluate the body size and overall growth of the mice.
The results showed no difference between MGsKO and control
mice (18.9 ⫾ 0.1 vs. 18.7 ⫾ 0.2 mm). When the muscle weight
was normalized by tibial length to obtain a muscle crosssectional area index, no atrophic trend was found in the
MGsKO EDL muscle (Fig. 2B). The slight fast-to-slow-fibertype switch did not have direct functional effect on the
Fig. 2. Minimum phenotypic change in extensor digitorum
longus (EDL) muscle of 3-mo-old MGsKO mice. A: SDSPAGE, glycerol SDS gel, and Western blots showed that EDL,
a normally fast-fiber muscle, of 3-mo-old MGsKO mice had
slightly increased MHC IIx and barely detectable slow TnI and
slow TnT, in contrast to solely fast myofilament protein isoforms in age-matched controls. SOL, soleus muscle. B: the ratio
of EDL muscle weight to body weight or to tibial length
showed no statistically significant change in MGsKO vs. control groups (P ⱖ 0.097). C: twitch contraction showed shorter
time to develop peak tension (TPT) in 3-mo-old MGsKO
mouse EDL muscle compared with the control, but no difference in the time from maximum twitch force to half-relaxation,
T1/2. Values are means ⫾ SE; n ⫽ 4 mice in control and n ⫽
5 mice in MGsKO groups. *P ⬍ 0.05 vs. control in Student’s
t-test.
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Fig. 1. Fast-to-slow isoform switches of both thick- and
thin-filament proteins in soleus muscle of 3-mo-old genetically modified mice with Gs␣ deficiency in skeletal muscle
(MGsKO). A: glycerol-SDS gel was employed to identify
myosin heavy chain (MHC) isoforms, and regular SDSPAGE/Western blots using anti-troponin I (TnI) monoclonal
antibody (MAb) TnI-1, anti-slow and cardiac troponin T
(TnT) MAb CT3, and anti-fast TnT MAb T12 were used to
identify troponin isoforms. The results showed increases in
MHC I, slow TnI, and slow TnT, accompanied by decreases
in MHC IIx, fast TnI, and fast TnT in MGsKO soleus muscle
compared with age-matched controls. MLC, myosin light
chain. B: the fast-to-slow isoform changes in thick- and
thin-filament proteins were quantified using densitometry
analysis. Values are means ⫾ SE; n ⫽ 5 mice each in control
and MGsKO groups. *P ⬍ 0.05, **P ⬍ 0.01 vs. control in
Student’s t-test.
MUSCLE FIBER-TYPE SWITCH IMPROVES FATIGUE RESISTANCE
MGsKO EDL muscle, as the contractile and relaxation time
was not elongated (Fig. 2C).
Higher fatigue resistance of 3-mo-old MGsKO mouse soleus
muscle. The 300-s intermittent fatigue protocol with 70% duty
cycle (700-ms tetanic contraction in each 1,000-ms cycle)
showed that MGsKO soleus muscle had less decrease in
contractile force from the prefatigue maximum tension than
that in the control group (Fig. 3A). During the recovery period,
tetanic contractile force returned faster and to a higher level in
MGsKO soleus muscle than the controls (Fig. 3B). These
results indicated higher fatigue resistance in MGsKO soleus
muscle, corresponding to increased slow fibers, which suggests
a beneficial adaption.
Normal expression of myofilament protein isoforms in soleus
muscle of infantile MGsKO mice. To evaluate whether the
fiber-type switch was a direct consequence of the defect in Gs␣
signaling in muscle cells or a secondary response to the
primary changes, such as muscle atrophy and weakness,
MGsKO and control mouse soleus muscles were examined on
postnatal days 1, 7, and 21 for the expression of fast- and
slow-fiber-specific myofilament protein isoforms. The results
in Fig. 4 demonstrated similar patterns of MHC, TnI, and TnT
isoform expression during the early postnatal development of
MGsKO and control soleus muscles. Normal developmental
changes were observed in both groups, including downregulation of embryonic MHC and MHC IIx, appearance of MHC
IIa, and upregulation of MHC I. Slow TnI and slow TnT were
upregulated, whereas fast TnI and fast TnT downregulated in
soleus muscle during postnatal development. Fast TnT also
switched to have more of the alternatively spliced low-molecular-weight variants (43) (Fig. 4). Together with our laboratory’s previous finding of no difference between the fiber types
in soleus and gastrocnemius muscles of 3.5-wk-old MGsKO
and control mice (9), the results suggest that Gs␣ deficiency did
not change muscle fiber-type de novo. Our laboratory previously showed that soleus muscle of 3-mo-old MGsKO mice
had decreased mass, as measured by the ratio of muscle weight
to body weight and muscle cross-sectional area index derived
from the muscle weight vs. tibial length (9). Therefore, the
fast-to-slow-fiber-type switch in MGsKO soleus muscle could
be an adaptive response to low muscle mass, corresponding to
less total contractile force.
Soleus muscle of aging MGsKO mice switched to contain
solely slow fibers. Since fast-to-slow-fiber-type switch is also
a natural process in aging muscles, we further studied
fiber-type-specific myofilament protein isoforms and con-
Fig. 4. Normal expression of myofilament protein
isoforms in soleus muscle of infantile MGsKO
mice. SDS-PAGE, glycerol-SDS gel, and Western
blots showed that days 1, 7, and 21 postnatal
MGsKO and control mouse soleus muscles had
similar expression and developmental patterns of
MHC, TnI, and TnT isoforms. n ⫽ 3 mice per time
point in each group.
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Fig. 3. Better fatigue resistance and recovery of MGsKO mouse soleus muscle.
A: normalized to prefatigue developed tension, the fatigue treatment produced
less decrease of force in MGsKO soleus muscle than that in the control group.
The absolute forces normalized to muscle mass were 25.42 ⫾ 1.69 mN/mg for
the MGsKO and 29.70 ⫾ 0.69 mN/mg for the control groups (9). B: MGsKO
soleus muscle recovered faster and to a higher level than the controls in the first
15 min postfatigue treatment. Values are means ⫾ SE; n ⫽ 5 mice in control
and n ⫽ 6 mice in MGsKO groups. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs.
control in Student’s t-test.
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MUSCLE FIBER-TYPE SWITCH IMPROVES FATIGUE RESISTANCE
of age compared with age-matched controls (9). The tibial length
did not change between 3 and 18 –24 mo of age (Fig. 8A).
Normalized to tibial length, aging produced a significant loss of
mass in control soleus muscles (Fig. 8A). The normalized mass of
MGsKO soleus muscle at 18 –24 mo of age did not further
decrease from the level of 3-mo-old MGsKO soleus muscle and
was similar to that of age-matched controls (Fig. 8A).
In the mean time, the switch to more type I slow fibers in
aging soleus muscle (Fig. 5) elongated the duration of twitch
contraction and relaxation in both control and MGsKO groups
compared with the 3-mo-old groups (Fig. 8B). This is consistent with the previous observation that MHC II produced
higher contractile velocity than that of MHC I (18). Our results
showed that soleus muscles of aging MGsKO and control mice
both produced lower twitch (Fig. 8C) and tetanic (Fig. 8D)
forces than that of the 3-mo-old groups, which may have
resulted from other factors other than the change of myosin
isoenzymes. Since the tibial lengths that determine the in situ
muscle lengths are similar in the control and MGsKO groups,
force normalization to muscle mass should yield the same
results as that of normalization to muscle cross-sectional area
calculated from muscle weight vs. muscle length.
Supporting the hypothesis that the enhanced fast-to-slowfiber-type switch in soleus muscle of aging MGsKO mice may
have a benefit by functionally compensating for the decreased
force production, aging MGsKO soleus muscles exhibited
higher resistance to fatigue with better recovery than that of
age-matched controls (Fig. 9). Corresponding to the naturally
occurring fast-to-slow-fiber-type switch in aging soleus muscles, the controls also showed higher fatigue resistance and
recovery in the aging group than that in the 3-mo-old group
(Fig. 9).
Fig. 5. Aging MGsKO mice contained pure slow
fibers in soleus muscle. A: SDS-PAGE, glycerolSDS gel, and Western blots showed that soleus
muscles of 18- to 24-mo-old MGsKO mice expressed solely MHC I, slow TnI, and slow TnT.
Aging also produced switches to more slow isoforms of myofilament proteins in control soleus
muscle compared with that in the 3-mo-old control
group (Fig. 1A). B: immunohistochemistry using
MAb FA2 against MHC I showed 100% type I fibers
in the soleus muscle of aging MGsKO mice.
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tractile function of muscles from control and MGsKO mice
at 1.5–2 yr of age.
Soleus muscles of aging control mice exhibited a switch to
express more slow-type isoforms of myofilament proteins,
MHC, TnI, and TnT than that in the 3-mo-old control muscles
(Fig. 5A and Fig. 1A). This change was much more advanced
in aging MGsKO soleus muscles that became expressing solely
slow-type MHC I, slow TnI, and slow TnT (Fig. 5A). Consistently, immunohistochemistry using anti-MHC I MAb showed
100% type I slow fibers in the aging MGsKO soleus muscle
(Fig. 5B), confirming the switch to purely slow fibers.
The soleus muscle weight normalized to body weight did not
show statistical difference between aging MGsKO and agematched controls, likely due to the trend of lower body weight
in the MGsKO group (Fig. 6A). However, the muscle crosssectional area index suggested atrophy of MGsKO soleus
muscle in aging mice compared with age-matched controls
(Fig. 6B).
Aging MGsKO mouse soleus muscle had decreased contractile force and kinetics, but had higher resistance to fatigue.
Twitch and tetanic forces normalized to muscle weight were
lower in soleus muscle of aging MGsKO mice than that of
age-matched controls (Fig. 7A). The time parameters of twitch
contractions were longer in aging MGsKO soleus muscle than
that in age-matched controls (Fig. 7B). Compared with the
3-mo-old groups, the twitch kinetics of aging MGsKO and
control soleus muscles are correlated to the degrees of fast-toslow switch in muscle fiber types (Fig. 7C).
The effects of aging on the loss of muscle mass and on the
fast-to-slow-muscle-type switch were further compared between
control and MGsKO mouse soleus muscles. As shown previously,
MGsKO mice had severely decreased soleus muscle mass at 3 mo
MUSCLE FIBER-TYPE SWITCH IMPROVES FATIGUE RESISTANCE
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on improving fatigue resistance had the following findings for
the role of slow fibers in skeletal muscle function.
Fast-to-slow-fiber-type switch in soleus muscle improves
fatigue resistance. Fatigue resistance is a crucial feature of
weight-bearing skeletal muscles, such as the soleus. This function is especially important in the elderly and other individuals
with skeletal muscle weaknesses (37). Our laboratory’s previous studies showed slow-to-fast-fiber-type switches in rat soleus muscle treated with hindlimb unloading (14) and in
genetically modified mouse diaphragm muscle in which the
expression of slow skeletal muscle TnT was knocked down
(12). Both models resulted in decreased fatigue resistance.
Further supporting the critical role of slow fibers in weight
bearing and respiratory functions, the loss of slow fibers was
found in a human nemaline myopathy with lethal respiratory
failure (20, 21).
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Fig. 6. Decrease in soleus muscle mass of aging MGsKO mice. A: aging (18to 24-mo-old) MGsKO mice had a trend of lower body weight than agematched controls, and the weight of MGsKO and control soleus muscles
normalized to body weight showed no statistical difference. B: no difference
was found in tibial length (an indicator of the in vivo muscle length) between
MGsKO and control mice at 18 –24 mo of age. Muscle cross-sectional area
index calculated as the ratio of soleus muscle weight vs. tibial length detected
a lower soleus muscle mass in aging MGsKO mice vs. the control with
statistical significance (*P ⬍ 0.05 vs. control in Student’s t-test). Values are
means ⫾ SE; n ⫽ 6 mice for control and n ⫽ 7 mice for MGsKO groups.
Fast-to-slow-fiber-type switch in diaphragm but not EDL
muscle of aging MGsKO mice. Slow fibers play critical functions in slow- and mixed-fiber-type skeletal muscles. Therefore, we also examined diaphragm as a representative of
mixed-fiber muscle. In contrast to the prominent fiber-type
switch in aging soleus muscles (Fig. 5), the aging diaphragm
muscle of control mice expressed only minimal amounts of
slow isoforms of myofilament proteins (Fig. 10A). However,
slow isoforms of MHC, TnI, and TnT were clearly detectable
in aging MGsKO diaphragm muscle (Fig. 10A). For the continuous contractile activity of the diaphragm muscle in sustaining respiration, the increase in slow-fiber contents in MGsKO
diaphragm may as well be a beneficial adaptation to increase
the resistance to fatigue against the reduction of muscle mass
and power. In contrast, no slow-type myofilament protein
isoforms were detectable in aging control or MGsKO EDL
muscles (Fig. 10B).
DISCUSSION
The MGsKO mouse model provided an experimental system
to investigate pathophysiological adaptation of skeletal muscle
and functional significance. Our studies of the fast-to-slowfiber-type switch in MGsKO muscle and the beneficial effect
J Appl Physiol • VOL
Fig. 7. Lower force and contractile kinetics of aging MGsKO mouse soleus
muscle. A: twitch and tetanic forces normalized to muscle weight were lower
in aging MGsKO soleus muscle compared with age-matched controls. B and
C: compared with age-matched controls, kinetics of twitch contraction were
also lower in aging MGsKO mouse soleus muscle, as shown by the elongated
time parameters (TPT and T1/2). Values are means ⫾ SE; n ⫽ 6 mice for
control and n ⫽ 7 mice for MGsKO groups. *P ⬍ 0.05 vs. controls in
Student’s t-test.
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840
MUSCLE FIBER-TYPE SWITCH IMPROVES FATIGUE RESISTANCE
Fig. 9. Increased fatigue resistance of aging mouse soleus muscle enhanced in
MGsKO. Control aging mouse soleus muscles showed increased fatigue
resistance (A) and improved recovery (B) compared with that of 3-mo-old
controls. Compared with age-matched controls, aging MGsKO soleus muscles
exhibited further higher fatigue resistance (A) and better recovery (B). Values
are means ⫾ SE; n ⫽ 6 mice for control and n ⫽ 7 mice for MGsKO groups.
*P ⬍ 0.05, **P ⬍ 0.01 vs. aging controls in two-tail Student’s t-test. #P ⬍
0.05 vs. aging controls in one-tail Student’s t-test.
J Appl Physiol • VOL
Our present study demonstrated a unique example in which
the deficiency of Gs␣, the predominant G␣ isoform coupled to
␤-adrenoceptors in skeletal muscles, caused low muscle mass
and a fast-to-slow-fiber-type switch in soleus and diaphragm
muscles of MGsKO mice. Consistent with the role of ␤2adrenergic signaling in determining skeletal muscle fiber types,
chronic administration of ␤2-agonist was shown to increase
muscle mass and induced slow-to-fast-muscle fiber switches in
rat and mouse (1, 16, 45). As a consequence of the slow-tofast-fiber-type switch, ␤2-agonist-induced muscle hypertrophy
was accompanied with decreased fatigue resistance (4).
The previous studies were all based on conditions involving
slow-to-fast-fiber-type switching. To provide a strong positive
evidence for the role of slow fibers in the resistance of skeletal
muscle to fatigue, our present study employed a model of
fast-to-slow-fiber-type switch and demonstrated that increases
in slow fibers resulted in higher resistance to fatigue and better
recovery (Figs. 3 and 9).
The fast-to-slow-fiber-type switch is an adaptive response to
reduced muscle mass. Skeletal muscle exhibits a high plasticity
in response to chronic changes in physiological environment
and activity. Reduction of skeletal muscle mass occurs in
aging, starvation, physical inactivity, and various metabolic
and neuromuscular disorders (5, 13, 15, 24, 42). Following the
observation that skeletal muscles in diabetes undergo loss
of mass (40), our laboratory previously demonstrated that
MGsKO mice at 3.5 mo of age had impaired glucose
tolerance with low muscle mass and low mitochondria
content in soleus and gastrocnemius muscles (9).
The low-mass soleus muscle, but not the normal-mass EDL
muscle, of 3-mo-old MGsKO mice had significant fast-toslow-fiber-type switch (Figs. 1 and 2). Compared with that of
control mice, MGsKO mice did not have fiber-type change in
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Fig. 8. Soleus muscle of aging MGsKO mice
had compensated mass but slower contraction and lower developed force. A: 3-mo-old
and aging control and MGsKO mice showed
similar tibial length that was used to normalize the weight of soleus muscle to show an
aging-related decrease in soleus muscle
mass. While 3-mo-old MGsKO soleus muscle showed significantly lower mass compared with young controls, no further decrease was found in MGsKO soleus muscle
during aging. B: twitch contractile time parameters (TPT, T1/2) were elongated in aging
control and MGsKO soleus muscles. Twitch
(C) and tetanic (D) forces normalized to
muscle weight were lower in aging control
and MGsKO soleus muscles. Although the
aging MGsKO soleus muscles did not show
aging-related decrease in mass, their normalized force was lower than that of agematched control. CSA, cross-sectional area.
Values are means ⫾ SE; n ⫽ 5 mice each for
young control and young MGsKO groups,
n ⫽ 6 mice for aging control groups, and n ⫽
7 mice for aging MGsKO group. *P ⬍ 0.05,
**P ⬍ 0.01, ***P ⬍ 0.001 vs. young controls. #P ⬍ 0.05 vs. aging controls in Student’s t-test. &P ⬍ 0.05, &&&P ⬍ 0.001 vs.
young MGsKO mice in one-way ANOVA
Fisher test.
MUSCLE FIBER-TYPE SWITCH IMPROVES FATIGUE RESISTANCE
841
Fig. 10. Fast-to-slow-fiber-type switch in diaphragm
containing mixed types of fibers, but not in EDL
muscle containing only fast fibers of aging MGsKO
mice. A: SDS-PAGE, glycerol-SDS gel, and Western
blots showed that aging MGsKO, but not control,
diaphragm muscle had increased MHC I, slow TnI,
and slow TnT, with diminished MHC IIb, and decreases in fast TnI and fast TnT. B: no slow-type
myofilament protein isoform was detected in EDL
muscles of aging control or MGsKO mice.
J Appl Physiol • VOL
enhanced in MGsKO mice, resulting in solely slow fibers in
aging soleus muscle (Fig. 5). Whereas one study reported a
replacement of slow oxidative type I fibers with fast glycolytic
type II fibers during aging in the setting of caloric restriction
(3), other studies showed increases in the proportion of type I
fibers in aging human muscle (2, 22), corresponding to slower
(34) and more economical contractility (39) than that of type II
fibers. Consistent with the increased fatigue resistance in aging
muscle of healthy human subjects (23), our data showed that
aging control mouse soleus muscle had greater fatigue resistance than that of young control soleus. This effect was more
profound in aging MGsKO soleus muscle, corresponding to the
switch to purely type I fibers (Fig. 9).
As discussed above, reduced muscle mass occurs in aging
(37). Our data showed that soleus muscle of aging control
mouse had reduced mass compared with the 3-mo-old group.
Although low mass was found in soleus muscles of young
MGsKO mice compared with age-matched control, it did not
advance in aging, and the muscle mass became similar to that
of aging controls (Fig. 8). Therefore, the enhanced fast-toslow-fiber-type switch in MGsKO soleus muscle may not be a
simple adaptation to low muscle mass.
Our laboratory has shown that the MGsKO mice have
decreased cardiac function with occult heart failure (11). Severe heart failure was found to decrease skeletal muscle function without significant change in muscle fiber-type composition (28). Those changes were only found in glycolytic fast
fibers, but not in oxidative slow fibers, and the Gs␣-deficient
mice studied here did not have clinical signs of severe heart
failure. Therefore, the characteristic switch to more slow fibers
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infantile soleus muscles (Fig. 4) or in 3.5-wk-old soleus muscles when the lower muscle mass began to be detectable (9).
These observations suggest that the fast-to-slow-fiber-type
switch was not a primary cell signaling effect of Gs␣ deficiency, but rather a secondary adaptive change in response to
low muscle mass and the subsequently less force production.
The fast-to-slow-fiber-type switch that accompanies decreased
muscle mass during normal aging also supports the hypothesis
that fast-to-slow-muscle-type switch is an adaptive response to
low muscle mass and muscle weakness. This observation is
consistent with the report of Kugelberg (25) that, during the
growth of rat soleus muscle, a fast-to-slow-fiber-type switch
was in response to the functional overload due to an increasing
body-to-muscle weight ratio.
The fast-to-slow-fiber-type switch in soleus muscle of
MGsKO mice improved resistance to fatigue, providing a
plausible functional compensation for reduced force production. Whereas the muscles becoming slower would possibly
impair the quick response to loss of balance, the higher resistance to fatigue remains to be beneficial in weight-bearing
muscles. Although our laboratory’s previous study (9) demonstrated increased glucose intolerance in MGsKO mice, this
diabetes-related systemic defect is unlikely a consequence of
increased slow fibers, since mice have very few slow-fiber
muscles. Altogether, this compensatory mechanism is especially attractive in diabetic subjects, who tend to have a switch
toward more fast-muscle fibers (9, 10, 14, 46).
The fast-to-slow-fiber-type switch in aging muscles was
significantly enhanced in MGsKO mice. An intriguing finding
in the present study was that the fast-to-slow-fiber-type switch
in normal mouse soleus muscle in aging was significantly
842
MUSCLE FIBER-TYPE SWITCH IMPROVES FATIGUE RESISTANCE
J Appl Physiol • VOL
work for further studies on the molecular mechanisms underlying muscle-fiber differentiation and function.
ACKNOWLEDGMENTS
We thank Hui Wang for technical assistance and Dr. Jim Lin, University of
Iowa for the T12 MAb.
GRANTS
This study was supported by National Institutes of Health (NIH) Grants
AR048816 and HL098945 to J.-P. Jin and the Intramural Research Program of
the National Institute of Diabetes and Digestive and Kidney Diseases, NIH, US
Department of Health and Human Services.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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in the soleus muscle of Gs␣-deficient mice is unlikely an
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It is worth noting that, although the mass of MGsKO
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MGsKO enhances fast-to-slow-fiber-type switch selectively
in the slow- and mixed-fiber muscles. It was interesting to find
that Gs␣ deficiency specifically affected slow and mixed-fibertype muscles, but not fast-fiber-type muscle. The fast-toslow-fiber-type switch was highly prominent in soleus muscle. Diaphragm muscle showed a detectable increase in slow
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These observations suggest a hypothesis that the fiber-type
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