J Appl Physiol 124: 364–373, 2018.
First published November 16, 2017; doi:10.1152/japplphysiol.00687.2017.
RESEARCH ARTICLE
Exercise induces muscle fiber type switching via transient receptor potential
melastatin 2-dependent Ca2⫹ signaling
Seo-Ho Lee,1,2 Byung-Ju Kim,1,2 Dae-Ryoung Park,1,2 and Uh-Hyun Kim1,2,3
1
Department of Biochemistry, Chonbuk National University Medical School, Jeon-ju, South Korea; 2National Creative
Research Laboratory for Ca2⫹ Signaling Network, Chonbuk National University Medical School, Jeon-ju, South Korea; and
3
Institute of Cardiovascular Research, Chonbuk National University Medical School, Jeon-ju, South Korea
Submitted 27 July 2017; accepted in final form 2 November 2017
Lee SH, Kim BJ, Park DR, Kim UH. Exercise induces muscle fiber
type switching via transient receptor potential melastatin 2-dependent
Ca2⫹ signaling. J Appl Physiol 124: 364 –373, 2018. First published
November 16, 2017; doi:10.1152/japplphysiol.00687.2017.—The aim of
the present study was to examine whether transient receptor potential
melastatin 2 (TRPM2) plays a role in muscle fiber-type transition during
exercise. Mice were trained at a speed of 12 m/min at a slope of 0° for 60
min for 5 consecutive days/wk for 4 wk. Exhaustion tests were performed
on the treadmill (the speed was set at 6 m/min at a slope of 0° and
increased at a rate of 1 m/min every 6 min). Isolated primary skeletal
muscle cells from TRPM2-knockout (KO) mice showed lower ampli-
tudes of electrical stimuli (ES)-induced Ca2⫹ signals when compared
with wild-type (WT) mice due to a defect in Ca2⫹ influx. Moreover,
TRPM2-KO mice had a higher proportion of fast-twitch skeletal
muscle fibers and a lower proportion of slow-twitch muscle fibers
before exercise than WT mice. After exercise, the expression of
slow-twitch skeletal muscle fibers was increased only in WT mice but
not in TRPM2-KO mice. ES-induced nuclear translocation of the
Ca2⫹-dependent transcription factor NFATc1 was significantly lower
in TRPM2-KO mice than in WT mice. TRPM2-KO mice also showed
decreased mitochondrial Ca2⫹ and membrane potential. Lactate levels
were higher in the skeletal muscle cells of TRPM2-KO mice before
and after ES compared with WT mice. Collectively, these data
indicate that TRPM2-mediated Ca2⫹ signaling plays a critical role in
the regulation of fiber-type switching and mitochondrial function in
skeletal muscle.
NEW & NOTEWORTHY TRPM2 has been shown to play an
important role in a variety of cellular functions. However, the role of
TRPM2 in skeletal muscle remains poorly understood. Here, we
provide evidence that TRPM2-mediated Ca2⫹ signaling is required
for training-induced improvement in skeletal muscle mitochondrial
function and fiber type transition.
Ca2⫹ signaling; exercise; mitochondria; nuclear factor of activated T
cells, cytoplasmic 1; skeletal muscle
INTRODUCTION
Skeletal muscles have a remarkable capacity to undergo
adaptive changes in response to use and disuse, including
changes in fiber size, fiber type, and muscle force (46). Train-
ing for endurance, strength, and power can bring out specific
characteristics of muscle fibers that influence muscle contrac-
tile function and performance (36, 46). The major biochemical
Address for reprint requests and other correspondence: U. H. Kim, Dept. of
Biochemistry, Chonbuk National University, Medical School, Keum-am dong,
Jeonju 561-182, South Korea (e-mail: uhkim@chonbuk.ac.kr).
364 8750-7587/18 Copyright © 2018 the American Physiological Society
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characteristics of muscle fibers, which affect the function of
skeletal muscle, are oxidative capacity, constitutive proteins,
capillary density, myoglobin density, and the type of muscular
myosin heavy chain (MHC) (36). Based on their physiological
and biochemical characteristics, there are three classical fiber
types: type I (oxidative slow-twitch), type IIa (oxidative fast-
twitch), and type IIb (glycolytic fast-twitch) fibers in rodent,
which are equivalent to type IIx (glycolytic fast-twitch) fibers
in humans (36).
Adult skeletal muscle undergoes conversion between these
fiber types in response to exercise (5, 36, 46). Endurance
training induces the transition from fast-twitch muscle fiber to
slow-twitch muscle fiber, whereas strength training results in
slow-twitch to fast-twitch muscle fiber transition. Muscle fiber
transition is regulated by many factors, such as myogenic
regulatory factors (MyoD and myogenin), peroxisome prolif-
erator-activated receptor (PPAR)-␥ coactivator-1␣ (PGC-1␣)
and Ca2⫹-dependent transcription factor, and the nuclear factor
of activated T cells, cytoplasmic 1 (NFATc1) (5, 28, 36, 48).
NFATc1 controls fiber type composition and is required for
fast-to-slow fiber type switching in response to exercise. Re-
cently, it was demonstrated that NFATc1 inhibits MyoD-
dependent fast-twitch muscle fiber gene promoters by physi-
cally interacting with the NH2-terminal activation domain of
MyoD and blocking recruitment of the essential transcriptional
coactivator p300 (5). Together, it is apparent that NFATc1
coordinates the adaptive response of skeletal muscle to phys-
ical activity and exercise. NFATc1 nuclear translocation is
dependent on Ca2⫹ signals (18, 41). Calcineurin is a calcium/
calmodulin-regulated protein phosphatase that acts on
NFATc1, resulting in its nuclear translocation and the expres-
sion of muscle fiber type transition-related genes (2). NFAT
regulates the expression not only of fatty acid oxidation-related
PPARs and mitochondria potential-related ERR and MEF
genes but also of mitochondria biogenesis-related PGC-1␣ in
skeletal muscle cells (9).
Increases in mitochondrial Ca2⫹ and potential play impor-
tant roles in energy metabolism, to maintain the contractile
activity of skeletal muscle as well as muscle fiber type transi-
tion, although the exact mechanism remains to be clarified
(17). Mitochondrial Ca2⫹ is essential for the activities of
Ca2⫹-dependent enzymes, which include pyruvate dehydroge-
nase (PDH), isocitrate dehydrogenase (IDH), and ␣-ketogluta-
rate dehydrogenase (KGDH) of the tricarboxylic acid (TCA)
cycle in the production of adenosine triphosphate (ATP) to
maintain muscle contraction (29). These Ca2⫹ signals result
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 ROLE OF TRPM2 IN EXERCISE-INDUCED MUSCLE FIBER TYPE SHIFT
from the regulation of mitochondrial Ca2⫹ channels, voltage-
dependent anion channel (VDAC), and mitochondrial calcium
uniporters (MCU), which are in turn regulated by the rise in
Ca2⫹ signals released from the endoplasmic reticulum (ER)/
sarcoplasmic reticulum (SR) or through T-type Ca2⫹ channels
(26, 30). Succinate dehydrogenase (SDH) is an indicator of
mitochondrial oxidative potential, the expression of which is
known to be increased by exercise (19, 24).
Transient receptor potential melastatin 2 (TRPM2) is a
Ca2⫹-permeable cation channel activated by oxidant stress,
adenosine diphosphate ribose (ADPR), tumor necrosis factor-␣
(TNF␣), and intracellular calcium (39). TRPM2 is expressed in
many cell types, including the brain, pancreas, heart, and
immune cells (39). TRPM2 has been shown to play an impor-
tant role in a variety of cellular functions, which include cell
proliferation, insulin release, cell motility, and cell death (39,
45). However, the role of TRPM2 in skeletal muscle remains
poorly understood. In the present study, we subjected both
TRPM2 wild-type (WT) and knockout (KO) animals to a 4-wk
running training protocol to investigate whether skeletal mus-
cles of TRPM2-KO mice have defects in the capacity to
exercise. In particular we tested 1) whether skeletal muscle
fiber type transition and mitochondrial function were different
between the two groups following exercise training and 2), if
present, what is the mechanism by which TRPM2 mediates
Ca2⫹ signals in skeletal muscle during exercise.
METHODS
Animals. Pathogen-free wild-type (WT) C57BL/6 male mice,
12-wk of age, were obtained from Da-mul Science. TRPM2-KO male
mice were obtained from Prof. Mori Y (Department of Synthetic
Chemistry and Biological Chemistry, Kyoto University, Kyoto, Ja-
pan) (45). Although TRPM2 has been shown to play a role in insulin
secretion (43), the TRPM2-KO mice were viable and were born at the
expected Mendelian frequency. From there, they remained healthy
and showed no signs of defects in their locomotor activities in the
cages. Genotyping was performed with TRPM2 (genomic DNA)
primers (forward, 5=-CTT GGG TTG CAG TCA TAT GCA GGC-3=;
reverse, 5=-GCC CTC ACC ATC CGC TTC ACG ATG-3=; Pneo5,
5=-GCC ACA CGC GTC ACC TTA ATA TGC G-3=), and real-time
PCR analysis was performed with TRPM2 (mRNA) primers (for-
ward,5=-AGT GAC TTC TGG AAC AAA CT-3=; reverse, 5=-ATC
ATC CGC TTC ACG ATG AT-3=). All animal studies were per-
formed according to protocols approved by the Institutional Animal
Care and Use Committee at Chonbuk National University Medical
School (CBNU 2017-0029).
Mouse training. Mice were designated to either a nontrained group
or a trained group. Mice in the trained group were subjected to
treadmill training. Mice were trained at a speed of 12 m/min at a slope
of 0° for 60 min 5 consecutive days/wk for 4 wk. An air stream and
dark chamber in front of the treadmill were used as motivation for
running (34, 35). Mouse skeletal muscle samples were collected 24 h
after the last bout of exercise (4, 22).
Exhaustion test. The exhaustion test was performed on the tread-
mill in a darkroom. The treadmill was set at a speed of 6 m/min at a
slope of 0°, and speeds were increased at a rate of 1 m/min every 6
min. When the mouse was exhausted, running time and distance were
measured. Exhaustion was defined by ⬎10 falls/min into the motiva-
tional grid (3).
Western blotting analysis. Gastrocnemius muscle samples were
obtained from mice and lysed with NP-40 lysis buffer [50 mM
Tris·HCl, pH 7.4, 250 mM NaCl, 1% NP-40, 10 mM EDTA, 50 mM
NaF, 1 mM Na3VO4, and protease inhibitor cocktail (Roche)] for fast
MHC and RIPA buffer [50 mM Tris·HCl, pH 7.4, 140 mM NaCl, 1%
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365
Triton X-100, 1% deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM
Na3VO4, and protease inhibitor cocktail (Roche)] and slow MHC.
After lysis, the lysate was centrifuged at 16,000 g for 10 min and the
supernatant harvested. Protein concentrations were determined by
BCA protein assay kit (Thermo Scientific). Proteins were loaded at 10
␮g/well in SDS-PAGE gels with the sample buffer. After electropho-
resis, proteins were transferred to a PVDF membrane (Bio-Rad),
blocked in 3% BSA in 50 mM Tris·HCl, pH 7.5, 100 mM NaCl, and
0.3% Tween-20, and incubated with fast MHC antibodies (1:1,000
dilution; Abcam), slow MHC antibodies (1:1,000 dilution; Abcam),
␤-tubulin antibodies (1:1,000 dilution; Santa Cruz Biotechnology),
histone H3 antibodies (1:1,000 dilution; Cell Signaling Technology),
SDH antibodies (1:1,000 dilution; Cell Signaling Technology), PDH
antibodies (1:1,000 dilution, Cell Signaling Technology), and
NFATc1 antibodies (1:1,000 dilution; Millipore). Anti-mouse anti-
bodies (1:1,000 dilution; Santa Cruz Biotechnology) or anti-rabbit
antibodies (1:1,000 dilution; Santa Cruz Biotechnology) were used as
secondary antibodies. The blots were developed in ImageQuant LAS
4000 (GE Healthcare Life Science), and their densities were quanti-
fied by ImageJ.
Nucleus isolation. After electrical stimulation (ES; 55 V, 20 Hz,
10 s every 30 s, 1 h), skeletal muscle tissue was immediately obtained
and homogenized in a homogenization buffer [25 mM Tris·HCl (pH
7.5), 0.5 mM EDTA, 0.5 mM EGTA, protease inhibitor cocktail
(Roche), 25 mM NaF, 1 mM Na3VO4, and 1 mM DTT] at 4°C and
incubated in ice for 10 min with continuous vortexing. After centrif-
ugation at 22,000 g for 15 min at 4°C, the supernatant was collected
as a cytosolic fraction. Insoluble pellets containing nuclei were
washed with a wash buffer [10 mM HEPES, 1.5 mM MgCl2, 10 mM
KCl, protease inhibitor cocktail (Roche), 25 mM NaF, 1 mM Na3VO4,
and 1 mM DTT]. After centrifugation at 3,000 g for 10 min at 4°C, the
supernatant was removed, and the remaining pellets were resuspended
in a solubilizing buffer [20 mM HEPES, 20% glycerol, 0.42 M NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 1% triton X-100, protease inhibitor
cocktail (Roche), 25 mM NaF, 1 mM Na3VO4, and 1 mM DTT]. After
vortexing for 15 s every 10 min for 40 min and centrifugation at
22,000 g for 20 min at 4°C, the supernatant was collected as a nuclear
fraction (8). Fractions were analyzed by immunoblotting to identify
nucleus and cytosolic fractions and identified using antibodies against
each marker: nucleus (histone) and cytosol (␤-tubulin).
Electrical stimulation. ES (55 V, 20 Hz, 10 s every 30 s, 1 h) of the
gastrocnemius muscle was performed as described, with modifications
(2, 20). ES was applied with carbon electrodes using stimulators
(Digitimer DG2A and MK II) (25). The maximum current was limited
to 0.2 mA.
Immunohistochemistry. Gastrocnemius muscle tissue was fixed in
20% paraformaldehyde-PBS overnight at RT, embedded in paraffin,
and sectioned at 7-␮m thickness. The sections were stained with
hematoxylin and eosin (HE) for general histological analyses. Images
of slides were acquired using a ScanScope digital scanner (Aperio
ePathology Solutions). Fast MHC antibodies (1:1,000 dilution; Ab-
cam) and slow MHC antibodies (1:1,000 dilution; Abcam) were used,
and anti-mouse biotin (Dako E0354) or anti-rabbit-biotin (Dako
Eisolation of 0432) was used as a secondary antibody. Dako K3461
was used for the chromogen reaction.
Isolation of primary skeletal muscle cells. Primary skeletal muscle
cells were isolated from the gastrocnemius muscle of 12-wk WT and
TRPM2-KO male mice and placed in 15-ml Falcon tubes containing
9 ml of Dulbecco’s modified Eagle’s medium (DMEM; Welgene) and
2% type I collagenase for 200 min in a shaking water bath (50 rpm)
at 37°C. After incubation, the skeletal muscle cells were resuspended
by shaking. Resuspended skeletal muscle cells were centrifuged at
1,000 g for 1 min, and the supernatant was discarded. Skeletal muscle
cells were resuspended with DMEM containing 30% of horse serum
and incubated in CO2 incubator for 1 day. Skeletal muscle cells were
isolated and incubated in a CO2 incubator for 1 additional day (25).
366 ROLE OF TRPM2 IN EXERCISE-INDUCED MUSCLE FIBER TYPE SHIFT

Fig. 1. Transient receptor potential melastatin 2-knockout (TRPM2-KO) mice have less exercise capacity than wild-type mice in exhaustion tests. A–C:
TRPM2-KO mice and wild-type mice performed exhaustion test following 4 wk of treadmill training. The treadmill speed was gradually increased from 6 m/min,
1 m/min increased every 6 min: running time (A), running distance (B), and %running mice (C). Blue, nontrained wild-type mice (n ⫽ 8); red, nontrained
TRPM2-KO mice (n ⫽ 8); green, trained wild-type mice (n ⫽ 6); purple, trained TRPM2-KO mice (n ⫽ 6). Statistical analysis was performed using the t-test.
Data are represented as means ⫾ SD. *P ⬍ 0.05.

Calcium analysis. Primary skeletal muscle cells were attached to
Matrigel Matrix-coated dishes (BD Bioscience). Skeletal muscle cells
were stained with DMEM (without phenol red) containing 5 ␮M
Fluo-4 AM (Invitrogen) for cytosolic calcium (green fluorescence was
determined at 488-nm excitation/530-nm emission) or 10 ␮M Rhod-
2-AM (Invitrogen) for mitochondrial calcium (red fluorescence were
determined at 543 nm excitation/560 nm emission) at 37°C for 40
min. Calcium was measured with confocal microscopy (Nikon Eclipse
C1). Calculations were performed by using an equation developed by
Tsien et al. (42): [Ca2⫹]i ⫽ Kd (F ⫺ Fmin)/(Fmax ⫺ F).
Measurement of cADPR and NAADP. After ES (55 V, 1 Hz, 1
min), primary skeletal muscle cells were treated with 0.2 ml of 0.6 M
perchloric acid (PCA) under sonication, and precipitates were re-
moved by centrifugation at 20,000 g for 10 min. PCA was removed by
mixing in three volumes of 2 M KHCO3. After centrifugation at
16,000 g for 10 min, the supernatant was collected and neutralized
with 20 mM sodium phosphate (pH 8). [cADPR]i and [NAADP]i
were measured using a cyclic enzymatic assay, as described previ-
ously (7, 23, 25). Resorufin fluorescence was determined at 544-nm
excitation/590-nm emission.
Measurement of mitochondrial potential. Primary skeletal muscle
cells were attached to Matrigel Matrix-coated dishes (BD Bioscience).
Skeletal muscle cells were stained with DMEM containing 1 ␮M
tetramethylrhodamine methyl ester (TMRM; Invitrogen) for mito-
chondrial potential (red fluorescence was determined at 543-nm ex-
citation/560-nm emission). Ten micromolars carbonyl cyanide m-
chlorophenyl hydrazone (CCCP; C2759; Sigma,) was used to inhibit
oxidative phosphorylation (27). Mitochondrial potential was mea-
sured with confocal microscopy (Nikon Eclipse C1).
Measurement of lactate level in tissue. After ES (55 V, 20 Hz,
10 s every 30 s, 1 h), skeletal muscle tissue was immediately obtained
and homogenized with 1 ml of 0.6 M PCA, and precipitates were
removed by centrifugation at 20,000 g for 10 min. PCA was removed
by mixing in three volumes of 2 M KHCO3. After centrifugation at
16,000 g for 10 min, the supernatant was collected and neutralized
with 20 mM sodium phosphate (pH 8). One unit per milliliter of LDH
(L2500; Sigma) was added to 100 ␮l of sample and incubated in a
37°C water bath for 30 min. After incubation, 100 ␮l of cycling
reagent mixture (20 ␮M resazurin, 10 ␮g/ml diaphorase, 10 mM
nicotinamide, 0.1 mg/ml BSA, 100 mM NaPO4, pH 8, and 10 ␮M
FMN) was added to the sample, and Resorufin fluorescence was
determined at 544-nm excitation/590-nm emission (10, 23).
Measurement of ATP level in tissue. After ES (55 V, 20 Hz, 10 s
every 30 s, 1 h), skeletal muscle tissue was immediately obtained and
homogenized with 1 ml of 0.6 M PCA, and precipitates were removed
by centrifugation at 20,000 g for 10 min. PCA was removed by
mixing in three volumes of 2 M KHCO3. After centrifugation at
16,000 g for 10 min, the supernatant was collected and neutralized
with 20 mM sodium phosphate (pH 8). ATP concentrations were
measured with an ATP Determination Kit (A22066; Thermo). Lumi-
nescence were determined at 400-nm excitation/650-nm emission and
analyzed by the Multimode Microplate Reader System (Perkin-Elmer)
installed at the Center for University-Wide Research Facilities
(CURF) at Chonbuk National University.

Table 1. Comparison of the weights of the skeletal muscles and body and heart weight between wild-type and
TRPM2-KO mice
Body weight, g GM weight, mg SOL Weight, mg TA Weight, mg EDL Weight, mg Heart Weight, mg

Nontrained
Wild type 26.13 ⫾ 0.86 144.96 ⫾ 3.15 10.28 ⫾ 0.43 45.60 ⫾ 2.60 10.63 ⫾ 0.48 152.06 ⫾ 3.70
TRPM2-KO 26.73 ⫾ 1.45 145.76 ⫾ 9.68 10.40 ⫾ 0.64 45.70 ⫾ 2.98 11.05 ⫾ 0.58 140.20 ⫾ 9.90
Trained
Wild type 26.15 ⫾ 0.90 137.35 ⫾ 6.74 10.73 ⫾ 0.87 45.23 ⫾ 2.02 9.78 ⫾ 1.02 134.02 ⫾ 14.01
TRPM2-KO 25.86 ⫾ 3.27 147.70 ⫾ 11.78 10.68 ⫾ 1.45 45.48 ⫾ 4.36 10.65 ⫾ 1.00 133.36 ⫾ 15.15
Values are means ⫾ SD. Transient receptor potential melastatin 2-knockout, TRPM2-KO, SOL, soleus; GM, gastrocnemius; EDL, extensor digitorum longus;
TA, tibialis anterior. Whole body and skeletal muscle and heart of mice were weighted after 4 wk of treadmill training. Nontraining, n ⫽ 8; training, n ⫽ 6).
Statistical analysis was performed using 1-way ANOVA.

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 ROLE OF TRPM2 IN EXERCISE-INDUCED MUSCLE FIBER TYPE SHIFT
Measurement of pyruvate dehydrogenase activity. Isolated primary
skeletal muscle cells were used to measure PDH activity. After ES
(55V 3 Hz), primary skeletal muscle cells were homogenized in a
PDH assay buffer (component included in the assay kit) and kept on
ice for 10 min. After centrifugation at 16,000 g for 10 min, the
supernatant was collected and PDH activity measured with a PDH
assay kit (K679-100; BioVision). Absorbance was measured at 450
nm for 60 min at 37°C.
Statistical analysis. Data are represented as the mean ⫾ SD of at
least three independent experiments. Statistical analysis was per-
formed by one-way ANOVA and t-test as appropriate. P ⬍ 0.05 was
considered significant.
RESULTS
TRPM2 KO mice have fewer exercise capabilities than WT
mice in exhaustion tests. Given that TRPM2 is a Ca2⫹-perme-
able cation channel, we speculated that the exercise abilities of
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367
TRPM2-KO mice would be impaired. Thus, we performed an
exhaustion test to determine exercise capability and measured
running time and distance. TRPM2-KO mice recorded signif-
icantly inferior results in both running time/distance compared
with WT mice (Fig. 1, A–C). TRPM2-KO mice lagged behind
WT mice after ⬃40 min, and the frequency of this occurrence
was higher than in TRPM2-KO mice, and they were also
observed resting more often than WT mice (See Supplemental
Video S1; Supplemental Material for this article can be found
on the Journal of Applied Physiology web site). To check
whether this issue was resolved through training, each group
underwent 4-wk treadmill training. Consequently, exercise
capability was significantly elevated in trained WT mice, but
not in trained TRPM2-KO mice, in comparison with the
nontrained group (Fig. 1, A–C). This result indicates that
exercise capability was not improved through training in
Fig. 2. TRPM2 is involved in muscle fiber
type transition. A: nuclear factor of activated
T cells, cytoplasmic 1 (NFATc1) nuclear
translocation was determined by Western
blotting before and after electrical stimula-
tion (ES; 55 V, 20 Hz, 10 s every 30 s, 1 h).
Blue, control wild-type; red, control
TRPM2-KO; green, ES wild type; purple,
ES TRPM2-KO. Inset: confirmation of nu-
clear fraction in skeletal muscle; Western
blotting was performed for a nuclear marker
protein (histone) and a cytosolic marker pro-
tein (␤-tubulin), and whole lysate (left), cy-
tosolic fraction (middle), and nuclear frac-
tion (right) were loaded. B: Western blotting
was performed to measure levels of fast
myosin heavy chain (MHC), slow MHC,
pyruvate dehydrogenase (PDH), and succi-
nate dehydrogenase (SDH) in skeletal mus-
cle tissue following 4 wk of treadmill train-
ing. Blue, nontrained wild-type mice; red,
nontrained TRPM2-KO mice; green, trained
wild-type mice; purple, trained TRPM2-KO
mice. Statistical analysis was performed us-
ing the t-test. Data are represented as
means ⫾ SD. *P ⬍ 0.05.
368 ROLE OF TRPM2 IN EXERCISE-INDUCED MUSCLE FIBER TYPE SHIFT

TRPM2-KO mice. To determine whether there were any
differences that affect exercise capability in the muscle
tissue of WT and TRPM2-KO mice, we measured the
weights of the skeletal muscles, as well as body and heart
weight, but there were no significant differences in any of
the parameters (Table 1). Thus, we hypothesized two pos-
sibilities underlying the reduced exercise capabilities of
TRPM2-KO mice: muscle fiber type transition and down-
regulation of mitochondrial function.
TRPM2 is involved in muscle fiber type transition. Because
NFATc1 nuclear translocation is induced by calcineurin, a
Ca2⫹-dependent phosphatase (11), we examined NFATc1 nu-
clear translocation in TRPM2-KO mice. Western blot analysis
of NFATc1 nuclear translocation following 1-h ES revealed
significantly lower NFATc1 translocation levels in
TRPM2-KO mice than in WT mice (Fig. 2A). Next, we
compared the expression levels of slow MHC (slow-twitch
muscle fiber marker) and fast MHC (fast-twitch muscle fiber
marker) in the skeletal muscle of WT and TRPM2-KO mice
before and after 4 wk of treadmill training. TRPM2-KO mice
showed significantly higher expression levels of fast MHC than
WT mice regardless of the 4-wk treadmill training (Figs. 2B
and 3). By contrast, WT mice showed a significant increase in
slow MHC muscle after the 4 wk of treadmill training, whereas
TRPM2-KO mice showed no change after the 4 wk of tread-
mill training, maintaining the low expression levels of slow
MHC muscle. This result indicates that muscle fiber type
transition is affected by TRPM2 function.
Ca2⫹ amplitudes are lower in TRPM2-KO skeletal muscle
cells. We compared the ES-induced Ca2⫹ signals of isolated
primary skeletal muscle cells from WT and TRPM2-KO mice,
and found that the Ca2⫹ amplitude of TRPM2-KO mice was
relatively low compared with WT mice (Figs. 3 and 4A).
Previously, we observed that the cytoplasmic Ca2⫹ in skeletal
muscle is increased by exercise in an NAADP-dependent
manner (25). Thus, we compared the Ca2⫹ signals of primary
skeletal muscle cells from WT and TRPM2-KO mice follow-
ing treatment with the cell-permeable NAADP analog
NAADP-AM. Ca2⫹ levels were significantly lower in skeletal
muscle cells from TRPM2-KO mice than those from WT mice,
especially during the later phases of the Ca2⫹ signals following
NAADP-AM treatment (⬃300 s). To find out whether these
differences were due to Ca2⫹ influx, we compared the
NAADP-AM-induced Ca2⫹ signals of the two groups under
the Ca2⫹-free condition. In the absence of extracellular Ca2⫹,
there were no Ca2⫹ signals in the later phase (Fig. 4, B and C),
indicating that the differences in the latter part of the Ca2⫹
signal was due to Ca2⫹ influx. Previously, we found that
among the three well-known Ca2⫹ signaling messengers, only
cyclic ADPR (cADPR) and NAADP, but not inositol trispho-
sphate (IP3), were produced in ES-treated skeletal muscle cells
(25). We examined whether the skeletal muscle cells of
TRPM2-KO mice could generate these Ca2⫹ signaling mes-
sengers in response to ES. NAADP and cADPR were produced
to a similar extent in both WT and TRPM2-KO mice upon ES
(Fig. 4D). These results indicate that TRPM2-KO mice have a

Fig. 3. Fast MHC levels were increased in TRPM2-KO mice. Representative immunohistochemistry images of gastrocnemius section after 4 wk of training.
Sections were stained with fast MHC (top) and slow MHC (bottom).

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 ROLE OF TRPM2 IN EXERCISE-INDUCED MUSCLE FIBER TYPE SHIFT 369

Fig. 4. Ca2⫹ signals and Ca2⫹ signaling messenger formation in primary skeletal muscle cells from wild-type and TRPM2-KO mice. A: the ES-induced Ca2⫹
oscillation was measured in primary skeletal muscle cells of wild-type mice (left) and TRPM2-KO mice (right). ES was performed with 55 V, 1 Hz. B and C:
NAADP-AM (50 nM) was treated in primary skeletal muscle cells in the presence and absence of extracellular Ca2⫹. D: levels of NAADP and cyclic adenosine
diphosphate ribose (cADPR) were measured in primary skeletal muscle cells of wild-type mice and TRPM2-KO mice before and after ES (55 V, 1 Hz, 1 min).
Blue, control wild-type mice; red, control TRPM2-KO mice; green, ES wild-type mice; purple, ES TRPM2-KO mice. Statistical analysis was performed using
the t-test. Data are represented as means ⫾ SD. *P ⬍ 0.05.

defect in the Ca2⫹ influx of skeletal muscle cells during muscle
contraction, which does not seem to be directly caused by
intracellular Ca2⫹ signaling messengers.
Mitochondrial function is decreased in TRPM2-KO mice.
Because Ca2⫹ signals also play an important role in mitochon-
dria, we looked for any defects in mitochondrial function in
TRPM2-KO mice. Ca2⫹ levels in the matrix of the mitochon-
dria regulate the activity of enzymes involved in the TCA cycle
(29). The Ca2⫹ channels responsible for regulating Ca2⫹ entry
into the mitochondria are VDAC and MCU. Ca2⫹ can move
freely through VDAC but not MCU (26, 29). It is generally
accepted that the resting Ca2⫹ levels of the mitochondrial
matrix almost match that of the cytosol (⬃100 nM). ES-
induced Ca2⫹ levels in the cytosol were lower in TRPM2-KO
mice than in WT mice (Fig. 4A), and we speculated that the
mitochondrial Ca2⫹ levels induced by ES would be reduced in
TRPM2-KO mice as well. We compared ES-induced mito-
chondrial Ca2⫹ signals in the skeletal muscle cells of WT and
TRPM2-KO mice using Rhod-2. As expected, the mitochon-
drial Ca2⫹ levels induced by ES in TRPM2-KO mice was
about one-half that of WT mice (Fig. 5A). A functioning TCA
cycle, which is regulated by mitochondrial Ca2⫹, is required
for mitochondrial ATP synthesis potential. Thus, a reduction
in the mitochondrial Ca2⫹ in TRPM2-KO mice could be
expected to result in a reduction of mitochondrial potential. To
measure the potential of mitochondria, skeletal muscle cells
were treated with TMRM, the potential-sensitive dye, and
CCCP, an oxidative phosphorylation inhibitor. The mitochon-
drial potential of TRPM2-KO mice was significantly decreased
compared with that of WT mice (Fig. 5B). Given that a
decrease in mitochondrial potential results in a defect in ATP
production, we measured ATP levels in WT and TRPM2-KO
mice before and after ES. There were no differences in ATP
levels between wild and TRPM2-KO mice before ES, but the
reduction in ATP levels after ES was much more severe in
TRPM2-KO mice (Fig. 5D), suggesting that the decreased
mitochondrial function in TRPM2-KO mice is significant.
Thus, we postulated that anaerobic glycolysis might be utilized
to maintain ATP homeostasis in the skeletal muscle cells of
TRPM2-KO mice. Because glycolysis induces the accumula-
tion of lactate (32), we measured lactate levels in skeletal
muscle before and after ES. Even in the resting state, the lactate
levels of TRPM2-KO mice were higher than those in WT mice
and were similar to levels found in WT mice after ES. The

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370 ROLE OF TRPM2 IN EXERCISE-INDUCED MUSCLE FIBER TYPE SHIFT

Fig. 5. Comparison of the mitochondrial func-

tion of primary skeletal muscle cells from be-
tween wild-type mice and TRPM2-KO mice. A:
ES (55 V 1 Hz)-induced mitochondrial Ca2⫹
was measured with Rhod-2 in primary skeletal
muscle cells. B: mitochondrial potential was
measured with tetramethylrhodamine methyl
ester (TMRM) in primary skeletal muscle cells;
10 ␮M CCCP was treated. C: lactate level was
measured in skeletal muscle tissue before and
after ES (55 V, 20 Hz, 10 s every 30 s, 1 h). Blue,
control wild-type mice (n ⫽ 4); red, control
TRPM2-KO mice (n ⫽ 4); green, ES wild-type
mice (n ⫽ 4); purple, ES TRPM2-KO mice (n ⫽
4). D: ATP was measured in skeletal muscle
tissue before and after ES (55 V, 20 Hz, 10 s
every 30 s, 1 h). Blue, control wild-type mice
(n ⫽ 3); red, control TRPM2-KO mice (n ⫽ 3);
green, ES wild-type mice (n ⫽ 4); purple, ES
TRPM2-KO mice (n ⫽ 4). E: cytosolic and mi-
tochondrial Ca2⫹ were measured by staining
Fluo-4 and Rhod-2 in primary skeletal muscle
cells. Blue, wild-type mice (n ⫽ 32); red, TRPM2
KO mice (n ⫽ 35). F: representative fluorescent
microscope images for cytosolic and mitochon-
drial Ca2⫹ levels. Statistical analysis was per-
formed using the t-test. Data are represented as
means ⫾ SD. *P ⬍ 0.05.

lactate levels of TRPM2-KO mice after ES were further in- enzymes involved in the TCA cycle, which is important for
creased and were significantly higher than those found in ATP production, between WT and TRPM2-KO mice. To test
ES-induced WT mice (Fig. 5C). Therefore, we speculated that this hypothesis, we compared the expression levels of PDH and
a difference would exist between the expression levels of SDH. (Fig. 2B). Interestingly, both PDH and SDH were de-

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 ROLE OF TRPM2 IN EXERCISE-INDUCED MUSCLE FIBER TYPE SHIFT
creased in trained TRPM2-KO mice when compared with WT
mice. PDH did not show any significant changes in WT mice
before and after training, but it was decreased in trained
TRPM2-KO mice. Expression levels of SDH in WT mice were
increased after 4 wk of training, whereas levels were decreased
in TRPM2-KO mice (Fig. 2B). These results suggest that
TRPM2-KO mice have defects in exercise-induced oxidative
energy metabolism, and the decrement in mitochondrial func-
tion was due to the decreased Ca2⫹ signals in the cytosol and
mitochondria. Indeed, the Ca2⫹ levels in the cytosol and
mitochondria were reduced during the normal resting state,
when the Ca2⫹ signals were measured by simultaneous stain-
ing with Fluo-4 and Rhod-2 (Figs. 4 and 5, E and F), confirm-
ing that both cytosolic and mitochondrial Ca2⫹ were decreased
in TRPM2-KO mice in the normal state.
PDH activity was decreased in TRPM2-KO mice. PDH is a
mitochondrial enzyme that catalyzes the transformation of
pyruvate into acetyl-CoA (pyruvate decarboxylation). PDH
phoshpatase is activated by Ca2⫹ to activate PDH by dephos-
phorylation (15). Our data showed that the ES-induced mito-
chondrial Ca2⫹ levels in the skeletal muscle of TRPM2-KO
mice were lower than that of WT mice. These findings suggest
that the PDH activity of TRPM2-KO mice is decreased. To test
this notion, we measured PDH activity in isolated primary
skeletal muscle cells after ES. The PDH activity of
TRPM2-KO mice was significantly lower than that of WT
mice (Fig. 6). These findings suggest that TRPM2-KO mice are
less reliant on oxidative sources of energy, resulting in an
increased dependency on glycolytic metabolism.
DISCUSSION
In the present study, we show that the TRPM2 channel is
responsible for Ca2⫹ influx in ES-induced skeletal muscle,
which determines the characteristics and function of skeletal
muscle. Thus, we have uncovered a novel phenomenon by
which the TRPM2 channel plays an essential role not only in
cytoplasmic Ca2⫹ signals but also in mitochondrial Ca2⫹
signals. The former is essential for NFATc1 nuclear translo-
cation during exercise. The nuclear translocation of NFATc1
resulted in muscle fibers switching from fast-twitch to slow-
Fig. 6. Comparison of the PDH activity of primary skeletal muscle cells
between wild-type mice and TRPM2-KO mice. PDH activity was measured
with primary skeletal muscle cells. ES was performed with 55 V, 3 Hz, 0, 30,
60, 90, 120, and 150 s (n ⫽ 3). Statistical analysis was performed using the
t-test. Data are represented as means ⫾ SD. *P ⬍ 0.05.
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371
twitch fiber types after 4 wk of treadmill training in a TRPM2-
dependent manner, as seen in WT mice. By contrast, the
skeletal muscle of TRPM2-KO mice exhibited a predominance
of fast-twitch muscle fiber regardless of 4-wk treadmill
training. With respect to mitochondrial Ca2⫹ signals, it
plays important roles in mitochondrial ATP generation. We
found that the skeletal muscle of TRPM2-KO mice had
significantly decreased ATP and increased lactate levels
upon ES due to the compensatory increase in glycolysis in
response to the decreased mitochondria ATP output. In
overview, the TRPM2 channel plays critical roles in the
Ca2⫹ signaling, exercise capability, and the determination
of phenotype of skeletal muscle.
Although our results suggest that the role of the TRPM2
channel revolves mainly around Ca2⫹ influx in ES-induced
skeletal muscle, the mechanism by which TRPM2 is activated
remains unclear. Many metabolites are known to be activators
for TRPM2, including cADPR and H2O2 (13). Because
cADPR was generated in the ES-induced skeletal muscle cells,
TRPM2 might be directly activated by cADPR. Alternatively,
TRPM2 could be activated via the mechanism of store-opera-
tive Ca2⫹ entry following Ca2⫹ store depletion by cADPR
and/or NAADP (6). Regardless of the guesswork, our results
indicate that the TRPM2 channel seems to be responsible for
the ES-induced Ca2⫹ influx in the skeletal muscle (Fig. 4, B
and C).
Muscle fiber transformations as the basis of muscular plas-
ticity occur in response to a variety of stimuli in humans and
animals. All muscles use Ca2⫹ as their main regulatory and
signaling molecule. Therefore, muscle plasticity is closely
linked with and highly dependent upon the Ca2⫹ signaling
system, including the regulation of calcineurin and Ca2⫹-
calmodulin kinase (CaMK), which changes the skeletal muscle
MHC isoform (1). ES has been used mainly for fast-to-slow
transition via chronic low-frequency stimulation (continuous at
10 Hz) (14). On the other hand, Jorquera et al. (12) showed that
slow to fast transition was also induced by low-frequency
stimulation (20 Hz) through dihydropyridine receptor (DHPR)
and ATP. It is known that endurance training with repetitive
muscle contractions leads to muscular plasticity in response to
metabolic/functional demands, resulting in an increase of slow-
twitch muscle fibers. CaMKII is activated by endurance train-
ing (38), and the suppression of MEF2-HDAC4/5 association
induces an increase in slow-twitch muscle fibers (37). Consis-
tent with this background knowledge, our data showed that ES
or physical exercise induced an increase of slow-twitch muscle
fibers, which was correlated with NFAT1c nuclear transloca-
tion, in a TRPM2-dependent manner.
Mitochondria provide a major source of ATP for contraction
and also participate in Ca2⫹ buffering. Furthermore, the spatial
and functional organization of mitochondria in close proximity
with the SR promotes the local delivery of Ca2⫹ transients that
mediate the excitation-contraction coupling to the mitochon-
drial matrix (33). The mitochondrial uptake of Ca2⫹ released
from the SR via IP3 receptors or ryanodine receptors ultimately
activates aerobic respiration at the level of both the Ca2⫹-
dependent matrix dehydrogenases and the F0/F1-ATPase in the
mitochondria (40). Also, mitochondrial membrane potential is
required for ATP synthesis by ATP synthase (16). Consistent
with these reports, TRPM2-KO mice showed decreased mem-
372 ROLE OF TRPM2 IN EXERCISE-INDUCED MUSCLE FIBER TYPE SHIFT
brane potential and mitochondrial Ca2⫹ levels, which resulted
in defective ATP production upon ES.
Under limited mitochondrial function, lactate formation is
essential for muscle to produce the cytosolic NAD in muscle
required to support the continued regeneration of ATP from
glycolysis, which ultimately results in metabolic acidosis. Aci-
dosis can affect cellular enzyme activity and exercise capabilities
(31). TRPM2-KO mice showed lactate accumulation even in the
resting state at levels which were comparable with those of
ES-induced WT mice. Naturally, ES-induced TRPM2-KO mice
had very high lactate levels, which can be expected to induce
acidosis. These findings suggest that TRPM2-KO mice have
reduced exercise capacities.
Fast-twitch muscle fibers are prone to diminishing in size
with age, which results in sarcopenia and muscle atrophy (5).
Our findings indicate that TRPM2-KO mice express high
levels of fast-twitch muscle fibers regardless of training (Fig.
2B), which suggests that TRPM2 might play a role in main-
taining slow-twitch muscle fibers in aging muscles. It would be
interesting to see whether TRPM2-KO mice are susceptible to
age-related sarcopenia. Thus, TRPM2 and its related regulatory
pathways, including NFATc1 signaling, could be potential
therapeutic targets in the prevention of muscle atrophy and
sarcopenia.
In the present study, we attempted to focus on the differ-
ences in skeletal muscle fiber switching in WT and
TRPM2-KO mice and could not look at other tissues or
variables, except body weights and heart weights, which
showed no significant differences between the two groups
(Table 1). We also observed that there are no particular
differences in the level of physical activity in their cages and
reproductive capacities. Given that TRPM2-KO mice showed
various phenotypes, including a resistance to high-fat diet-
induced obesity and inflammation (39, 45, 47), resistance to
ischemia-reperfusion injury to the heart (21), and abnormal
insulin secretion in pancreatic islets (43), the possibility that
the skeletal muscle phenotypes observed in the current study
could be attributable to the systemic effects of the KO could
not be fully excluded. Therefore, in the future, it would be
important to conduct studies with skeletal muscle-specific KO
mice to avoid any confounding effects of the KO on the system
as a whole.
In summary, the ES-induced Ca2⫹ increase in skeletal mus-
cle was dependent on Ca2⫹ entry through the TRPM2 channel.
TRPM2-mediated Ca2⫹ entry was essential for the regulation
of cytosolic and mitochondrial Ca2⫹ levels, which then facil-
itate skeletal muscle fiber type switching to slow-twitch fibers
and mitochondrial ATP production, both of which are required
for optimal skeletal muscle function and response during and
after exercise.
ACKNOWLEDGMENTS
We thank Chansu Park for critical reading of the manuscript.
GRANTS
This work was supported by a Korean National Research Foundation Grant
2012R1A3A2026453 (to U.-H. Kim).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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AUTHOR CONTRIBUTIONS
S.-H.L., B.-J.K., and D.-R.P. performed experiments; S.-H.L., B.-J.K.,
D.-R.P., and U.-H.K. analyzed data; S.-H.L., B.-J.K., D.-R.P., and U.-H.K.
interpreted results of experiments; S.-H.L. and B.-J.K. prepared figures;
S.-H.L., B.-J.K., D.-R.P., and U.-H.K. drafted manuscript; U.-H.K. conceived
and designed research; U.-H.K. edited and revised manuscript; U.-H.K. ap-
proved final version of manuscript.
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