Skeletal muscle adaptation in response to voluntary
running in Ca2+/calmodulin-dependent protein kinase
IV-deficient mice
Takayuki Akimoto, Thomas J. Ribar, R. Sanders Williams and Zhen Yan
Am J Physiol Cell Physiol 287:1311-1319, 2004. First published Jun 30, 2004;
doi:10.1152/ajpcell.00248.2004
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Am J Physiol Cell Physiol 287: C1311–C1319, 2004.
First published June 30, 2004; doi:10.1152/ajpcell.00248.2004.
Skeletal muscle adaptation in response to voluntary running in Ca2⫹/
calmodulin-dependent protein kinase IV-deficient mice
Takayuki Akimoto,1,3 Thomas J. Ribar,2 R. Sanders Williams,1 and Zhen Yan1
Departments of 1Medicine and 2Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710,
3
Department of Life Science, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Tokyo 305-8902, Japan
Submitted 21 May 2004; accepted in final form 23 June 2004
several subtypes of myofibers
that differ with regard to metabolic profile, contractile property, and susceptibility to fatigue (4, 29). The fiber type
composition is established during embryonic and postnatal
development (15, 25). Adult skeletal muscles are capable of
adaptation in response to altered functional demands, such as
endurance exercise, involving orchestrated signal transduction
from neuromuscular activity to the genetic regulatory machinery (23). However, the molecular mechanisms responsible for
the maintenance of slow muscle phenotype and exerciseinduced skeletal muscle adaptation remain to be fully elucidated.
Previous studies showed that Ca2⫹ signaling plays an important function linking different patterns of motor nerve
activity to distinct programs of gene expression that establish
phenotypic diversity among skeletal myofibers (5). Investigators at our laboratory (34) hypothesized that sustained increases in regulatory pools of intercellular Ca2⫹ as a result of
tonic patterns of motor neuron activity stimulate the Ca2⫹/
calmodulin-dependent protein phosphatase calcineurin, leading
to increased transcription of genes expressed selectively in
fatigue-resistant skeletal myofiber types (types I and IIa).
Subsequent investigations provided evidence that calcineurindependent signals are transduced by transcription factors that
include nuclear factor of activated T cells (NF-AT) and myocyte enhancer factor 2 (MEF2) to relevant target genes and are
amplified by concomitant activation of other Ca2⫹-regulated
signaling cascades that can be initiated by Ca2⫹/calmodulindependent kinase (CaMK) activities (13). The identity of the
CaMK involved in this process has not been identified, however, and it could be any of the multifunctional CaMK, such as
CaMKI, CaMKII, or CaMKIV.
Along this line of investigation, investigators at our laboratory (32) have reported that forced expression of a constitutively active form of CaMKIV in murine fast-twitch skeletal
muscle results in enhanced mitochondrial biogenesis and increased types I and IIa fibers. These phenotypic changes are
associated with increased mRNA and protein expression of
proliferator-activated receptor ␥-coactivator 1␣ (PGC-1␣), a
pivotal regulatory protein in skeletal muscle fiber type specialization and mitochondrial biogenesis (10, 19). This finding
suggests a possible link between CaMKIV activity and
PGC-1␣ gene regulation, which is supported by a later finding
that Ca2⫹-regulated PGC-1␣ protein expression in differentiated myotubes is dependent on CaMK activity (12). Other
studies have demonstrated that muscle contractile activity induces PGC-1␣ mRNA and protein expression (2, 18, 26) and
that overexpression of PGC-1␣ in fast-twitch skeletal muscle is
sufficient to increase mitochondrial density and percentage of
types I and IIa myofibers in a transgenic mouse model (10).
Collectively, these findings support the notion that a CaMK,
possibly CaMKIV, plays an important role in fiber type specialization and maintenance and in exercise-induced genetic
reprogramming in skeletal muscles.
In light of the above findings, it is important to establish
which CaMK proteins are expressed in mammalian skeletal
muscle. The published findings for CaMKIV are inconsistent.
In two separate reports, CaMKIV protein was reported not to
be readily detected using immunoblot analysis in human and
murine skeletal muscle (22, 32), while in another study,
Address for reprint requests and other correspondence: Z. Yan, Division of
Cardiology, Dept. of Medicine, Duke Univ. Medical Center, 4321 Medical
Park Drive, Suite 200, Durham, NC 27704 (E-mail: zhen.yan@duke.edu).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
cellular signaling; proliferator-activated receptor ␥-coactivator 1␣;
fiber type switching; mitochondrial biogenesis
ADULT SKELETAL MUSCLES CONTAIN
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Akimoto, Takayuki, Thomas J. Ribar, R. Sanders Williams,
and Zhen Yan. Skeletal muscle adaptation in response to voluntary
running in Ca2⫹/calmodulin-dependent protein kinase IV-deficient
mice. Am J Physiol Cell Physiol 287: C1311–C1319, 2004. First
published June 30, 2004; doi:10.1152/ajpcell.00248.2004.—Mammalian skeletal muscles undergo adaptation in response to alteration in
functional demands by means of a variety of cellular signaling events.
Previous experiments in transgenic mice showed that an active form
of Ca2⫹/calmodulin-dependent protein kinase IV (CaMKIV) is capable of stimulating peroxisome proliferator-activated receptor ␥-coactivator 1␣ (PGC-1␣) gene expression, promoting fast-to-slow fiber
type switching and augmenting mitochondrial biogenesis in skeletal
muscle. However, a role for endogenous CaMKIV in skeletal muscle
has not been investigated rigorously. We report that genetically
modified mice devoid of CaMKIV have normal fiber type composition and mitochondrial enzyme expression in fast-twitch skeletal
muscles and responded to long-term (4 wk) voluntary running with
increased expression of myosin heavy chain type IIa, myoglobin,
PGC-1␣, and cytochrome c oxidase IV proteins in plantaris muscle in
a manner similar to that of wild-type mice. Short-term motor nerve
stimulation (2 h at 10 Hz) likewise increased PGC-1␣ mRNA expression in tibialis anterior muscles in both Camk4⫺/⫺ and wild-type
mice. In addition, we have confirmed that no detectable CaMKIV
protein is expressed in murine skeletal muscle. Thus CaMKIV is not
required for the maintenance of slow-twitch muscle phenotype and
endurance training-induced mitochondrial biogenesis and IIb-to-IIa
fiber type switching in murine skeletal muscle. Other protein kinases
sharing substrates with constitutively active CaMKIV may function as
endogenous mediators of activity-dependent changes in myofiber
phenotype.
C1312
CAMKIV AND SKELETAL MUSCLE ADAPTATION
MATERIALS AND METHODS
Animals. Mice carrying a null mutation of the Camk4 gene were
generated by using homologous recombination in embryonic stem
cells as described previously (35). To characterize skeletal muscle
phenotype in sedentary mice, soleus, plantaris, and white vastus
lateralis muscles were harvested from the Camk4⫺/⫺ mice and their
wild-type littermates (C57BL/6J/129/Sv; n ⫽ 5 for each group) after
the mice were killed with an overdose of sodium pentobarbital (250
mg/kg body wt) delivered by intraperitoneal (ip) injection. The muscle
samples were processed for Western immunoblot and indirect immunofluorescence analyses as described below. To determine the effects
of long-term voluntary running on skeletal muscle, wild-type (C57BL/
6J) and Camk4⫺/⫺ mice (n ⫽ 6 for each group) were individually
housed in cages (13 ⫻ 13 ⫻ 30 cm) equipped with running wheels (11
cm in diameter). Animals were maintained on a 12:12-h light-dark
cycle, and wheel-running activity was monitored continuously with
the Dataquest Acquisition and Analysis System (Data Sciences International, St. Paul, MN). After 4 wk of voluntary running, plantaris
muscles were harvested from each mouse as described above for
AJP-Cell Physiol • VOL
Western immunoblot analysis. To induce muscle contraction by nerve
stimulation, a separate group of Camk4⫺/⫺ mice and their wild-type
littermates (n ⫽ 5 for each genotype) were used. Electrode implantation was performed as described previously in rats (38). Briefly,
while the animals were under anesthesia (50 mg/kg sodium pentobarbital ip), their hindlimbs were shaved with an electric hair clipper and
treated with iodine applied twice, followed by 70% ethanol applied
twice. An incision of ⬃1 cm was made on the left lateral side of the
thigh to expose the deep peroneal nerve. Two stainless steel electrodes
were secured under the deep peroneal nerve, with two sutures used to
fasten each electrode. To accommodate the change in the size of the
animal, the two electrodes were kept ⬃3 mm apart. Motor nerve
stimulation was initiated within 30 min after surgery and lasted for
2 h, during which time the mouse was kept under anesthesia. The
stimulation parameters were 0.25-ms duration and 10-Hz frequency as
previously described (38), except that the amplitude was adjusted
between 1 and 3 V to maintain maximal contractions and minimize
possible damage. The contralateral tibialis anterior (TA) muscle was
used as a control after performing a sham operation without stimulation. Immediately after motor nerve stimulation, both the stimulated
and the contralateral control TA muscles were harvested and processed for total RNA preparation. All experimental protocols were
approved by the Duke University Institutional Animal Care and Use
Committee.
Western immunoblot analysis. Immediately after tissue procurement, skeletal muscles were homogenized in glass homogenizers in
0.3 ml of complete protein loading buffer containing 50 mM
Tris 䡠 HCl, pH 6.8, 1% sodium dodecyl sulfate (SDS), 10% glycerol,
20 mM dithiothreitol, 127 mM 2-mercaptoethanol, and 0.01% bromophenol blue, supplemented with protease inhibitors (Roche) and
phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). The muscle
homogenates were transferred to microfuge tubes, heated for 5 min at
100°C, and centrifuged in a microfuge for 5 min at 13,000 rpm at
room temperature. Protein concentration of each sample was determined by amido black protein assay (24), and 10 ␮g (for contractile
proteins) or 40 ␮g (for other proteins) of total protein was resolved on
8 –12% (depending on the molecular weight of the protein of interest)
SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a
nitrocellulose membrane, and immunodetected by using an enhanced
chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway,
NJ). The following antibodies were used for immunoblot analysis:
PGC-1␣ polyclonal antibody (catalog no. SC-13067; Santa Cruz
Biotechnology, Santa Cruz, CA), ␣-tubulin antibody (catalog no.
13-8000; Zymed Laboratories, South San Francisco, CA), myosin
heavy chain (MHC) mouse monoclonal antibodies (catalog no. BF-F8
for MHC type I, catalog no. SC-71 for MHC type IIa, and catalog no.
BF-F3 for MHC type IIb; German Collection of Microorganisms and
Cell Cultures, Braunschweig, Germany), myoglobin polyclonal antibody (catalog no. A0324; Dako, Carpinteria, CA), CaMKIV (catalog no. 610275; BD Transduction Laboratories, San Diego, CA),
cytochrome c oxidase IV (COXIV) antibody (catalog no. A-21348;
Molecular Probes, Eugene, OR), CaMKII␦ (catalog no. SC-1542;
Santa Cruz Biotechnology), CaMKII␥ antibody (catalog no. SC1541; Santa Cruz Biotechnology), and CaMKI antibody (generous
gift from A. R. Means, Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC). The intensities of the bands were quantified by using Scion Image software
(Scion, Frederick, MD).
Indirect immunofluorescence. For fiber type composition determination, soleus muscles were harvested and frozen in isopentane cooled
in liquid nitrogen. Frozen cross sections (5 ␮m) were immunostained
using monoclonal antibodies against types I, IIa, and IIb. Briefly,
muscle sections were fixed in 4% paraformaldehyde/PBS for 10 min
at 4°C and permeabilized with 0.3% Triton X-100-PBS for 10 min at
4°C. The sections were blocked in 5% normal goat serum (NGS)-PBS
for 30 min at room temperature, followed by incubation with MHC
type IIb antibody (BF-F3) diluted 1:100 in 5% NGS-PBS at 4°C
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CaMKIV was reported to be expressed in murine skeletal
muscle and further induced by energy deprivation (40). On the
other hand, CaMKII autonomous activity (independent of
Ca2⫹ and calmodulin) has been shown to be increased in
contracting skeletal muscles (6, 22), suggesting a potential
functional role for CaMKII in mediating the genetic events in
skeletal muscle adaptation. Thus it is necessary to determine
unambiguously the dependence of skeletal muscle adaptation
on specific CaMK activities in response to acute and long-term
exercise training.
An intriguing finding from a recent study brought additional
attention to the possible dependence of mitochondrial biogenesis and fiber type switching on CaMKIV activity. Zong et al.
(40) reported that energy deprivation increased CaMKIV and
PGC-1␣ protein expression as well as activation of the endogenous AMP-activated protein kinase (AMPK) and enhanced
mitochondrial biogenesis in murine skeletal muscle; all of
these were blocked by forced expression of a dominant-negative form of AMPK. Previous studies clearly linked increased
AMPK activity in skeletal muscle to mitochondrial biogenesis
(3, 20, 31, 39), at least partially through the activation of
transcription factors such as nuclear respiratory factor 1 (3).
Zong et al.’s (40) findings suggest that CaMKIV is expressed
in skeletal muscle and that elevated expression of CaMKIV
upon AMPK activation in skeletal muscle, which stimulates
PGC-1␣ expression, mediates mitochondrial biogenesis in response to energy deprivation. An important question is whether
CaMKIV activity is required for maintenance of slow muscle
phenotype and exercise-induced skeletal muscle adaptation.
In this study, we have used mice with targeted deletion of
the Camk4 gene (Camk4⫺/⫺) to characterize different groups
of muscle comprehensively and to assess muscle adaptation
after long-term voluntary running for the expression of contractile and mitochondrial proteins. Our results show that
CaMKIV activity is not required for the maintenance of the
slow muscle phenotype or for exercise-induced adaptation in
skeletal muscle. We have also obtained unambiguous results to
indicate that CaMKIV protein is not expressed in murine
skeletal muscle. Thus other protein kinases sharing substrates
with constitutively active CaMKIV may function as endogenous mediators of activity-dependent changes in myofiber
phenotype.
CAMKIV AND SKELETAL MUSCLE ADAPTATION
overnight. The muscle sections were washed three times with PBS for
5 min each, followed by incubation with fluorescein-conjugated goat
anti-mouse IgM secondary antibody (1:50 dilution) at room temperature for 30 min. The muscle sections were then washed three times
C1313
with PBS and fixed in 4% paraformaldehyde for 2 min at 4°C and
blocked with 5% NGS-PBS for 30 min. The sections were then
sequentially stained as described above with MHC type I antibody
(BA-F8, 1:100 dilution), followed by rhodamine red-X-conjugated
AJP-Cell Physiol • VOL
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Fig. 1. Expression of contractile proteins and proteins involved in mitochondrial biogenesis in skeletal muscles of
mice with targeted deletion of the Camk4 gene (Camk4⫺/⫺)
and their wild-type littermates. A: Western immunoblot
analysis of myosin heavy chain (MHC) types I, IIa, and IIb,
myoglobin (Mb), peroxisome proliferator-activated receptor ␥-coactivator 1␣ (PGC-1␣), and cytochrome c oxidase
IV (COXIV) proteins in soleus (SO), plantaris (PL), and
white vastus lateralis (WV) skeletal muscles in Camk4⫺/⫺
mice (KO) and the wild-type littermates (WT). ␣-Tubulin
level was measured as a control for the loading and quality
of the protein samples. B: quantification of the relative
abundance of the proteins after normalization to the abundance of ␣-tubulin protein. (n ⫽ 5). **P ⬍ 0.01 vs.
wild-type littermates.
C1314
CAMKIV AND SKELETAL MUSCLE ADAPTATION
Fig. 2. Camk4⫺/⫺ mice have a higher percentage of type I fibers in SO muscle. Indirect
immunofluorescence staining of type I (rhodamine red-X, red), type IIa (cyanine Cy5, blue),
type IIb (fluorescein, green), and other (no staining) myofibers in SO muscles in sedentary KO
and WT. There are no type IIb fibers in these
images, and an increased percentage of type I
myofibers with a concurrent decrease in type IIa
myofibers is evident in Camk4⫺/⫺ mice.
than that in the wild-type littermates (Fig. 1, A and B). Evidently, the change is specific to the soleus muscle, because no
significant differences were observed in plantaris and white
vastus lateralis muscles. To further confirm the finding in
soleus muscle, we used indirect immunofluorescence to determine fiber type composition. Consistent with the Western
immunoblot data, we observed a significantly higher percentage of type I fibers with a concurrent decrease in the percentage
of type IIa fibers in the soleus muscles of Camk4⫺/⫺ mice (Fig.
2 and Table 1).
These morphological and biochemical differences in soleus
muscle were associated with a 20% decrease in muscle mass
(0.23 ⫾ 0.01 mg/g body wt in Camk4⫺/⫺ mice vs. 0.29 ⫾ 0.01
mg/g body wt in wild-type littermates, P ⬍ 0.05) (Table 1). No
significant differences in muscle mass were observed in plantaris and TA muscles (predominantly type IIb fibers) between
Camk4⫺/⫺ mice and their wild-type littermates (not shown).
The mean cross-sectional areas of the two predominant fiber
types (types I and IIa) in soleus muscle were measured, and
there was a trend toward decreased cross-sectional area for
both types I and IIa fibers in Camk4⫺/⫺ mice relative to
wild-type littermates, but the difference was not statistically
significant (Table 1).
RESULTS
Muscle weight, mg/g body wt
Fiber type composition, %
Type I
Type IIa
Type IIb
Other
Cross-sectional area, ␮m2
Type I
Type IIa
Camk4⫺/⫺ mice have normal fiber type composition in
fast-twitch muscles and an increased percentage of type I
myofibers in slow-twitch soleus muscle. To comprehensively
characterize skeletal muscle phenotype in sedentary mice,
Western immunoblot analysis was performed to measure the
expression of MHC proteins in three muscles of distinct fiber
type composition: soleus (predominantly types I and IIa fibers),
plantaris (predominantly types IIa and IIb fibers), and white
vastus lateralis muscles (predominantly type IIb fibers). To our
surprise, MHC type I protein concentration in soleus muscle
was found to be ⬃100% higher (P ⬍ 0.01) in Camk4⫺/⫺ mice
AJP-Cell Physiol • VOL
Table 1. Fiber type analysis of soleus muscle in Camk4⫺/⫺
mice and wild-type littermates
WT (n ⫽ 5)
KO (n ⫽ 4)
0.29⫾0.02
0.23⫾0.01*
37.7⫾1.5
51.9⫾1.5
0.8⫾0.4
9.6⫾1.6
57.5⫾4.1*
39.9⫾3.9†
0.3⫾0.1
2.3⫾0.3†
1,636⫾99
1,245⫾141
1,542⫾16
1,178⫾86
KO, Camk4⫺/⫺ mice; WT, wild-type littermate mice; Type I, myofibers
stained positive for myosin heavy chain (MHC) type I with antibody BA-F8;
Type IIa, myofibers stained positive for MHC type IIa with antibody SC-71;
Type IIb, myofibers stained positive for MHC type IIb with antibody BF-F3;
Other, myofibers with negative staining. Values are means ⫾ SE. *P ⬍ 0.05
vs. WT. †P ⬍ 0.01 vs. WT.
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goat anti-mouse IgG and MHC type IIa antibody (SC-71, 1:100
dilution), followed by cyanine Cy5-conjugated goat anti-mouse IgG.
Images were captured under an Olympus confocal microscope, and
total myofibers of each type were counted to calculate fiber type
composition for each muscle section. Myofiber diameter for types I
and IIa (n ⬎ 300 for each section and each fiber type) was measured
by using Scion Image software. The staining, photography, and image
analysis were performed by a single individual who had no knowledge
of the coding system.
Semiquantitative RT-PCR. Total RNA preparation and semiquantitative RT-PCR analysis were performed as described previously (37)
to measure endogenous PGC-1␣ mRNA expression in TA muscle in
response to increased contractile activity. PGC-1␣ mRNA data were
normalized by the abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA for each sample and expressed as the
relative change compared with the contralateral control muscle. The
PCR primers used were as follows: GAPDH forward primer, 5⬘GTGGCAAAGTGGAGATTGTTGCC-3⬘; GAPDH reverse primer,
5⬘-GATGATGACCCGTTTGGCTCC-3⬘; PGC-1␣ forward primer,
5⬘-AAACTTGCTAGCGGTCCTCA-3⬘; and PGC-1␣ reverse primer,
5⬘-TTTCTGTGGGTTTGGTGTGA-3⬘. Sequential denaturing (94°C
for 30 s), annealing (60°C for 30 s), and extension (72°C for 45 s)
reactions were repeated 26 and 16 times for PGC-1␣ mRNA and
GAPDH mRNA, respectively. The linearity of the PCR reactions was
ensured by preliminary tests in which the most and least abundant
samples were used with multiple reactions of different cycle numbers.
Statistics. Data are presented as means ⫾ SE. For comparisons
between the Camk4⫺/⫺ and wild-type mice and between the sedentary
and trained mice, a two-tailed Student’s t-test was used. For differences between the motor nerve-stimulated TA muscle and the contralateral control, a two-tailed, paired t-test was used. P ⬍ 0.05 was
accepted as statistically significant.
CAMKIV AND SKELETAL MUSCLE ADAPTATION
activity is required for skeletal muscle adaptation in response
to endurance exercise, Camk4⫺/⫺ mice were subjected to
long-term voluntary running (4 wk), and myoglobin, PGC-1␣,
COXIV, and MHC proteins were measured in the active
plantaris muscles. Camk4⫺/⫺ mice ran an average of 3.9 ⫾ 0.7
km/day at the beginning of the training, and the running
distance increased to 5.7 ⫾ 0.7 km/day after 28 days of
voluntary running (Fig. 4A). The average running distance of
the wild-type C57BL/6J mice was about twice that of the
Camk4⫺/⫺ mice before and after the long-term voluntary
running (8.3 ⫾ 1.1 km/day in sedentary mice and 10.7 ⫾ 0.9
km/day after training). C57BL/6J mice are known to run the
longest daily distance among different mouse strains on the
voluntary running wheel (1) and were used as a wild-type
control in this study for comparison. Consistent increases in
myoglobin, PGC-1␣, COXIV, and MHC type IIa protein
expression similar to that observed in the C57BL/6J wild-type
mice were noted in the plantaris muscle of Camk4⫺/⫺ mice
(Fig. 4, B and C).
CaMKIV protein is not detectable in murine skeletal muscle.
To determine without ambiguity whether CaMKIV protein is
expressed in murine skeletal muscles, proteins in the total cell
lysates from the brain and soleus muscle were resolved using
SDS-PAGE and immunoblotted with an anti-CaMKIV antibody (catalog no. 610275; BD Transduction Laboratories) with
or without preincubation with recombinant CaMKIV protein.
Consistent with the previous report (40), this antibody recognized CaMKIV protein as a dominant band at a molecular mass
of ⬃61 kDa only in the brains of wild-type mice and not in the
brains of Camk4⫺/⫺ mice (Fig. 5). Interestingly, a protein with
apparent molecular mass similar to that of CaMKIV was
detected in the soleus muscles of both the wild-type and
Camk4⫺/⫺ mice. However, when the anti-CaMKIV antibody
was preincubated with recombinant glutathione S-transferaseCaMKIV protein to titrate the active antibody, the ability of
this antibody to recognize CaMKIV in the brain was completely abolished, while the recognition of the 61-kDa protein
in the soleus muscle on the same membrane was not affected.
We conclude that the antibody mixture detects a protein distinct from CaMKIV in skeletal muscle.
To rule out possible compensation by enhanced expression
of other CaMK, we also performed Western blot analysis for
CaMKI, CaMKII␥, and CaMKII␦ in the soleus muscles of
wild-type and Camk4⫺/⫺ mice. There were no significant
differences in the expression of these CaMK proteins (Fig. 6),
confirming that the normal muscle phenotype in fast-twitch
muscles in Camk4⫺/⫺ mice is not due to compensatory mechanisms involving these CaMK proteins in skeletal muscle as a
result of genetic disruption of the Camk4 gene.
DISCUSSION
Fig. 3. Contractile activity induces PGC-1␣ mRNA expression in tibialis
anterior (TA) muscles of Camk4⫺/⫺ mice and WT littermates. A: agarose gel
image of semiquantitative RT-PCR analysis of PGC-1␣ mRNA in the stimulated (Stim) and contralateral control (Con) TA muscles. Samples from the
same mouse are underlined. B: quantification of the relative abundance of
PGC-1␣ mRNA in the stimulated and contralateral control TA muscles after
normalization to the abundance of glyceraldehyde-3-phosphate dehydrogenase
mRNA. **P ⬍ 0.01 compared with contralateral control muscle (n ⫽ 6).
AJP-Cell Physiol • VOL
The mechanisms of adult skeletal muscle adaptation have
been the subject of investigation for more than four decades,
and compelling evidence supports an important role for neuromuscular activity, particularly the motor neuron firing pattern, in the determination and maintenance of muscle mitochondrial biogenesis and fiber type composition (17, 27, 30).
Accumulating evidence supports the view that Ca2⫹ signaling
has a pivotal function in linking different patterns of motor
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PGC-1␣ mRNA expression is induced in Camk4⫺/⫺ mice in
response to increased contractile activity. Several laboratories
have reported that a single bout of contractile exercise is
sufficient to induce a transient increase in PGC-1␣ mRNA
expression in fast-twitch skeletal muscle in rats and humans (2,
18, 26). Investigators at our laboratory have observed similar
changes in mice responding to a single bout of voluntary
running or motor nerve stimulation (Pohnert SC, Akimoto T,
Rosenburg PB, Williams RS, and Yan Z, unpublished observation). This induced expression of PGC-1␣ mRNA in skeletal
muscle may play an important functional role in mediating the
skeletal muscle adaptation. To determine whether CaMKIV
activity is required for activity-dependent regulation of
PGC-1␣ expression in skeletal muscle, we stimulated the TA
muscle via the common peroneal nerve. Semiquantitative RTPCR showed that short-term (2 h), low-frequency (10 Hz)
motor nerve stimulation resulted in a reproducible 150% (P ⬍
0.01) and 100% increase (P ⬍ 0.01) in PGC-1␣ mRNA
compared with the contralateral control TA muscle in the
wild-type littermates and the Camk4⫺/⫺ mice, respectively
(Fig. 3, A and B).
Camk4⫺/⫺ mice undergo normal fiber type switching and
enhanced mitochondrial biogenesis in plantaris muscle after
long-term voluntary running. To determine whether CaMKIV
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Fig. 4. Voluntary running induces skeletal
muscle adaptation in KO and WT mice. A:
average daily running distance before and
after 4 wk of training increases in KO mice
(C57BL/6J/129/Sv) and C57BL/6J WT mice
(n ⫽ 5). ⫹⫹⫹P ⬍ 0.001 compared with
WT mice. B: Western immunoblot analysis
of MHC I, IIa, and IIb, Mb, PGC-1␣, and
COXIV proteins in PL muscle in sedentary
(S) and trained (T) KO mice and their WT
littermates. SO muscle lysate was loaded as
a control for MHC I protein expression.
␣-Tubulin level was measured for the loading and quality of the protein samples. C:
quantification of relative abundance of the
proteins after normalization to the abundance of ␣-tubulin protein (n ⫽ 5). *P ⬍
0.05 vs. sedentary mice of the same genotype. **P ⬍ 0.01 vs. sedentary mice of the
same genotype.
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CAMKIV AND SKELETAL MUSCLE ADAPTATION
nerve activity to distinctive programs of gene expression that
establish phenotypic diversity among skeletal myofibers (14).
Gain-of-function and loss-of-function studies have suggested that stimulation of the calcineurin pathway, leading to
activated NF-AT and MEF2 transcription factors, plays an
essential role in slow muscle fiber specialization (5, 11, 16).
More recent findings have suggested the functional role for a
parallel CaMK pathway in supporting slow muscle gene expression (33). For example, investigators at our laboratory have
demonstrated that overexpression of a constitutively active
form of CaMKIV in the skeletal muscles of transgenic mice
triggers significant mitochondrial biogenesis and fast-to-slow
fiber type transformation, along with enhanced expression of
PGC-1␣ (32). Other groups also have observed activation of
PGC-1␣ gene expression and promoter activity by ectopic
expression of this active form of CaMKIV (7, 40). In addition,
Ca2⫹ signaling induces PGC-1␣ protein and mRNA expression
in cultured myotubes in a CaMK activity-dependent manner
(12). Furthermore, enhanced mitochondrial biogenesis in skeletal muscle in response to energy starvation has been suggested
to be associated with increased CaMKIV protein expression in
a AMPK-dependent manner (40). An important remaining
question is whether CaMKIV is part of the physiological
mechanism by which skeletal muscle undergoes adaptation in
response to stimuli such as endurance exercise in vivo. Mice
with deletion of the Camk4 gene (36) provided an opportunity
to address this important question in a loss-of-function manner.
If endogenous CaMKIV participates in signaling mechanisms that establish and maintain the slow muscle fiber type,
one would expect targeted mutation of the Camk4 gene to
result in a decreased percentage of types I and IIa myofibers
and a concurrent increase in the percentage of type IIb fibers,
as well as decreased expression of mitochondrial proteins, in
skeletal muscle. The results of this study show clearly that the
maintenance of slow muscle fibers and basal level mitochondrial biogenesis are not dependent on CaMKIV activity in
skeletal muscle. Thus we conclude that CaMKIV activity is not
required for the maintenance of slow muscle fiber type and
mitochondrial biogenesis in skeletal muscle.
AJP-Cell Physiol • VOL
Camk4⫺/⫺ mice showed an increased percentage of type I
fibers and a concurrent decrease in type IIa fibers and muscle
mass in soleus muscles. This is an unexpected finding that
argues against our initial hypothesis. The decrease in muscle
mass is likely due to a decrease in muscle fiber size, because
we noticed a trend of decreased cross-sectional area of both
types I and IIa fibers in the soleus muscles of Camk4⫺/⫺ mice.
The subtle, but not statistically significant, difference (⬃5%) in
the mean cross-sectional area between the Camk4⫺/⫺ mice and
their wild-type littermates appears to be consistent with a
volume reduction of 20%. It is known that CaMKIV protein is
highly expressed in developing spinal cord, including both
dorsal root and sympathetic ganglia (9, 28), and plays a
functional role (8, 21) in the central and peripheral nervous
systems. The changes in fiber type composition and muscle
mass in the soleus muscles of Camk4⫺/⫺ mice may result from
subtle neuronal defects that alter neural input to certain skeletal
muscles in the absence of CaMKIV activity. Further experimentation is required to address the functional role of this
potential function of CaMKIV in motor nerve.
The role for CaMKIV within skeletal muscles in signal
transduction arising from changes in neuromuscular activity
was rigorously tested by subjecting Camk4⫺/⫺ mice to 4 wk of
voluntary running followed by a comprehensive phenotypic
Fig. 6. Expression of other CaMK proteins in SO muscles of KO mice and
their WT littermates. A: total tissue homogenates from the SO muscles of KO
mice and their WT littermates were resolved on SDS-PAGE gel, transferred to
nitrocellulose membrane, and probed with anti-CaMKI, anti-CaMKII␥, and
anti-CaMKII␦ antibodies. ␣-Tubulin level was measured as a control for the
loading and quality of the protein samples. B: quantification of the relative
abundance of the proteins after normalization to the abundance of ␣-tubulin
protein. (n ⫽ 5).
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Fig. 5. Western immunoblot analysis of CaMKIV in skeletal muscle. Total
tissue homogenates from brains and SO muscles of KO mice and their WT
littermates were resolved on SDS-PAGE gel, transferred to nitrocellulose
membrane, and probed with anti-CaMKIV antibody (top) or anti-CaMKIV
antibody preincubated with 10 ␮g of glutathione S-transferase-CaMKIV recombinant protein. Arrows indicate position of the endogenous CaMKIV
protein.
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munoblots representing endogenous CaMKIV protein in the
wild-type mice with great certainty, because the CaMKIV
protein band was not detectable in the brain extract of
Camk4⫺/⫺ mice. Preincubation with recombinant CaMKIV
protein to titrate the antibody abolished the detection of this
band in the brain extract of wild-type mice, further proving its
identity. A protein band detected at approximately the molecular size predicted for CaMKIV was evident in the soleus
muscles of both Camk4⫺/⫺ and their wild-type littermates,
suggesting that this is not CaMKIV, because targeted mutation
did not affect its expression. Furthermore, detection of this
protein was unaffected by preincubation with recombinant
CaMKIV protein, providing strong evidence that this protein is
not CaMKIV but a cross-reacting protein that is not a product
of the Camk4 gene. The expression of this cross-reacting
protein is markedly induced by motor nerve stimulation
(data not shown), consistent with the previous finding that
its expression is sensitive to changes in energy metabolism
(40).
The confirmation that CaMKIV is not expressed in skeletal
muscle is valuable to understanding of our findings that
Camk4⫺/⫺ mice are normal in muscle fiber type composition
and with regard to activity-dependent changes in myofiber
specialization. These findings suggest that future research
should focus on the potential functional roles of CaMKI,
CaMKII, CaMKK, or other protein kinases that act on substrates shared with the constitutively active form of CaMKIV
as mediators of skeletal muscle plasticity.
ACKNOWLEDGMENTS
We thank Drs. A. R. Means and J. M. Colomer Font for careful review of
the manuscript. We appreciate the excellent technical support of Mei Zhang, C.
Ireland, and C. Gumbs.
GRANTS
This work was supported by American Heart Association Grant 0130261N
(to Z. Yan) and National Institute of Arthritis and Musculoskeletal and Skin
Diseases Grant AR-40849 (to R. S. Williams). T. Akimoto is a recipient of a
Grant-in-Aid for Overseas Research Scholars from the Ministry of Education,
Science, and Culture of Japan. The Camk4⫺/⫺ mice were generated previously
with the support of National Institute of Child Health and Human Development
Grant HD-07503 (to A. R. Means).
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