Photochemistry and Photobiology, 20**, **: *–*
Low-level Laser (Light) Therapy Increases Mitochondrial Membrane
Potential and ATP Synthesis in C2C12 Myotubes with a Peak Response
, Beatriz Kaippert
, Pinar Avci
, Ying-Ying Huang
, Marcelo V. P. de Sousa
Vanderlei S. Bagnato
, Nivaldo A. Parizotto
and Michael R. Hamblin
Laboratory of Electrothermophototherapy, Department of Physical Therapy, Federal University of Sao Carlos, Sao Carlos,
Post-Graduation Program in Biotechnology, Federal University of Sao Carlos, Sao Carlos, SP, Brazil
Optics Group, Physics Institute of Sao Carlos, University of S~
ao Paulo, Sao Carlos, SP, Brazil
Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA
Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Department of Dermatology, Harvard Medical School, Boston, MA
Laboratory of Radiation Dosimetry and Medical Physics, Institute of Physics, Sao Paulo University, Sao Carlos, SP, Brazil
Harvard-MIT Division of Health Science and Technology, Cambridge, MA
Received 2 September 2014, accepted 25 November 2014, DOI: 10.1111/php.12397
Low-level laser (light) therapy has been used before exercise
to increase muscle performance in both experimental animals
and in humans. However, uncertainty exists concerning the
optimum time to apply the light before exercise. The mecha-
nism of action is thought to be stimulation of mitochondrial
respiration in muscles, and to increase adenosine triphosphate
(ATP) needed to perform exercise. The goal of this study was
to investigate the time course of the increases in mitochondrial
membrane potential (MMP) and ATP in myotubes formed
from C2C12 mouse muscle cells and exposed to light-emitting
diode therapy (LEDT). LEDT employed a cluster of LEDs
with 20 red (630 10 nm, 25 mW) and 20 near-infrared
(850 10 nm, 50 mW) delivering 28 mW cm
for 90 s
(2.5 J cm
) with analysis at 5 min, 3 h, 6 h and 24 h post-
LEDT. LEDT-6 h had the highest MMP, followed by LEDT-
3 h, LEDT-24 h, LEDT-5 min and Control with signiﬁcant
differences. The same order (6 h >3h>24 h >5 min >
Control) was found for ATP with signiﬁcant differences. A
good correlation was found (r=0.89) between MMP and
ATP. These data suggest an optimum time window of 3–6h
for LEDT stimulate muscle cells.
Mitochondria are the organelles responsible for energy produc-
tion in cells and for this reason have a very important role in cel-
lular function and maintenance of homeostasis. This organelle
has an intriguing and well-designed architecture to generate
adenosine triphosphate (ATP) that is the basic energy supply for
all cellular activity (1,2).
Mitochondria contain a respiratory electron transport chain
(ETC.) able to transfer electrons through complexes I, II, III and
IV by carrying out various redox reactions in conjunction with
pumping hydrogen ions (H
) from the mitochondrial matrix to
the intermembrane space. These processes generate water as the
metabolic end-product, as oxygen is the ﬁnal acceptor of elec-
trons from the ETC., that is coupled with synthesis of ATP when
ions return back into mitochondrial matrix through complex
V (ATP synthase), thus completing the ETC. Changes in the
ﬂow of electrons through the ETC. and consequently in H
pumping produce signiﬁcant modulations in the total proton
motive force and ATP synthesis. These changes can be measured
by mitochondrial membrane potential (MMP) and content of
Since the earliest evidence that low-level laser (light) therapy
(LLLT) can increase ATP synthesis (3,4), several mechanisms of
action have been proposed to explain LLLT effects on mitochon-
dria. One of the ﬁrst studies reported increased MMP and ATP
synthesis measured at an interval of 3 min after LLLT (3). Years
later, other authors extended the measurement of this “extra”
ATP-induced by LLLT in HeLa (human cervical cancer) cells
(4). With intervals of 5 to 45 min, these authors found no
change in ATP synthesis during the ﬁrst 15 min after LLLT, but
after 20–25 min ATP levels increased sharply and then came
back to control levels at 45 min (4).
More recent studies have reported LLLT effects on mitochon-
dria in different types of cells (5–9). In neural cells LLLT seems
to also increase MMP, protect against oxidative stress (5) and
increase ATP synthesis in intact cells (without stressor agents)
(6). In mitochondria from ﬁbroblast cells without stressor agents,
LLLT also increased ATP synthesis and mitochondrial complex
IV activity in a dose-dependent manner (7). In myotubes from
C2C12 cells, LLLT could modulate the production of reactive
oxygen species (ROS) and mitochondrial function in a dose-
dependent manner in intact cells or in cells stressed by electrical
Increases in mitochondrial metabolism and ATP synthesis
have been proposed by several authors as a hypothesis to explain
*Corresponding author email: email@example.com (Michael R.
©2014 The American Society of Photobiology
LLLT effects on muscle performance when used for muscular
preconditioning or muscle recovery postexercise (10–12). How-
ever, there is a lack in the literature to identify immediate and
long-term effects of LLLT on mitochondrial metabolism and
ATP synthesis in skeletal muscle cells that in turn could conﬁrm
This study aimed to identify the time-response for LLLT by
light-emitting diode therapy (LEDT) in modulation of MMP and
ATP content in myotubes from C2C12 intact cells (mouse mus-
cle cells) only under the stress of the culture. Moreover, the sec-
ond objective was to correlate MMP with ATP content within a
time range of 5 min to 24 h after LLLT. Our goal was to ﬁnd
the best time-response for LLLT which could be useful in future
experimental and clinical studies investigating muscular precon-
ditioning, muscle recovery postexercise or any other photobio-
modulation in muscle tissue.
MATERIALS AND METHODS
Cell culture. C2C12 cells were kindly provided by the Cardiovascular
Division of the Beth Israel Deaconess Medical Center, Harvard Medical
School, USA. Cells were grown in culture medium (DMEM, Dulbecco’s
Modiﬁed Eagle’s Medium - Sigma-Aldrich) with fetal bovine serum
(20% FBS - Sigma-Aldrich) and 1% antibiotic (penicillin and streptomy-
cin) in humidiﬁed incubator at 37°C and 5% CO
C2C12 cells were cultured and a total of 1.71 910
mately were counted in a Neubauer chamber. Next, these cells were dis-
tributed equally into 30 wells (approximately 5.7 910
cells per well)
into two different plates:
1 15 wells in black plate (Costar
96-Well Black Clear-Bottom Plates)
for analysis of MMP.
2 15 wells in white plate (Costar
96-Well White Clear-Bottom Plates)
for analysis of ATP synthesis.
Moreover, both plates were subdivided into ﬁve columns with three
wells per column (triplicate):
1 LEDT-Control: no LEDT applied to the cells.
2 LEDT-5 min: LEDT applied to the cells and assessments of ATP and
MMP after 5 min.
3 LEDT-3 h: LEDT applied to the cells and assessments of ATP and
MMP after 3 h.
4 LEDT-6 h: LEDT applied to the cells and assessments of ATP and
MMP after 6 h.
5 LEDT-24 h: LEDT applied to the cells and assessments of ATP and
MMP after 24 h.
After plating C2C12 cells were cultured for 9 days in culture medium
(DMEM) containing 2% heat-inactivated horse serum (Sigma-Aldrich) in
a humidiﬁed incubator at 37°C and 5% CO
to induce cell differentiation
into myotubes, as described in a previous study (9). At the 10
LEDT-24 h group received LEDT. At 11
day all remaining groups
received LEDT and were assessed for ATP and MMP at speciﬁc times in
accordance with each group.
Light-emitting diode therapy (LEDT). A cluster of 40 LEDs (20 red –
630 10 nm; 20 infrared –850 20 nm) with a diameter of 76 mm
was used in this study. The cluster was positioned at a distance of
156 mm from the top of each plate and irradiation lasted 90 s with ﬁxed
parameters as described in Table 1. Each group of wells received LEDT
individually, and all others wells of each plate (groups) were covered
with aluminum foil to avoid light irradiation (Fig. 1). LEDT parameters
were measured and calibrated using an optical energy meter PM100D
and sensor S142C (area of 1.13 cm
). In addition, we chose
use red and near-infrared light therapy at the same time to promote a
double band of absorption by cytochrome c oxidase (Cox) based on spe-
ciﬁc bands of absorption reported previously (2,13–16). The room tem-
perature was controlled (22–23°C) during LEDT irradiation, which did
not increase temperature on the top of plates more than 0.5°C. This
increase of 0.5°C was dissipated to room within 2 min after LEDT.
Mitochondrial membrane potential (TMRM) assay. This analysis was
performed using cells placed into black plate. MMP was assessed using
tetramethyl rhodamine methyl ester (TMRM –Invitrogen/Molecular
Probes) at a ﬁnal concentration of 25 nM. Nuclei of myotubes from
C2C12 cells were labeled using Hoechst (Sigma-Aldrich) at a concentra-
tion of 1 mg mL
. Each well was incubated for 30 min, 37°C and 5%
with 100 lL of solution containing TMRM and Hoechst. Next, this
solution was carefully removed from each well and added 100 lL of buf-
fer solution containing HBSS (Hank’s Balanced Salt Solution –Life
Technologies Corporation) and 15 mM HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid –Life Technologies Corporation). The
myotubes were imaged in a confocal microscope (Olympus America Inc.
Center Valley, PA) using an excitation at 559 nm and emission at
610 nm. Three random ﬁelds per well were imaged with a magniﬁcation
of 409water immersion lens. Images were exported and TMRM ﬂuores-
cence incorporation into mitochondrial matrix was measured using soft-
ware Image J (NIH, Bethesda, MD).
Adenosine triphosphate (ATP) assay. This analysis was performed
using cells placed into white plate. First, the medium was carefully
removed from each well followed by addition of 50 lL per well of
CellTiter Glo Luminescent Cell Viability Assay reagent (Promega). After
10 min of incubation at room temperature (25°C), luminescence signals
were measured in a SpectraMax M5 Multi-Mode Microplate Reader
(Molecular Devices, Sunnyvale, CA) with integration time of 5 s to
increase low signals (17). A standard curve was prepared using ATP
standard (Sigma) according to manufacturer’s guideline and then ATP
concentration was calculated in nanomol (nmol) per well.
Pearson product-moment correlation coefﬁcient (Pearson’s r). The
correlation between TMRM and ATP content in myotubes from C2C12
cells was calculated using Pearson’s r. The rvalues were interpreted as
recommended previously (18): 0.00–0.19 =none to slight; 0.20–0.39 =
low; 0.40–0.69 =modest; 0.70–0.89 =high; and 0.90–1.00 =very high.
Sample size calculation. The sample size was calculated based on that
necessary to obtain signiﬁcant differences among all groups with ATP
Table 1. All parameters of light-emitting diode therapy (LEDT). Control
did not receive LEDT.
Number of LEDs (cluster): 40 (20 infrared-IR and 20 red-RED)
Wavelength: 850 20 nm (IR) and 630 10 nm (RED)
LED spot size: 0.2 cm
Pulse frequency: continuous
Optical output of each LED: 50 mW (IR) and 25 mW (RED)
Optical output (cluster): 1000 mW (IR) and 500 mW (RED)
LED cluster size: 45 cm
Power density (at the top of plate): 28 mW cm
Treatment time: 90 s
Cluster energy density applied on the top plate: 2.5 J cm
Application mode: without contact
Distance from plate or power meter: 156 mm
Figure 1. Myotubes from C2C12 cells. Experimental setup for irradia-
tion of the white and black plates containing myotubes from C2C12 cells
using light-emitting diode therapy (LEDT) without contact.
2 C. Ferraresi et al.
content. The statistical power of 80% and the effect size (greater than
0.75) were found to be satisfactory.
Statistical analysis. Shapiro–Wilk’s W test veriﬁed the normality of
the data distribution. ATP and TMRM were compared among all groups
using one-way analysis of variance (ANOVA) with Tukey HSD post hoc
test. Pearson product-moment correlation coefﬁcient (Pearson’s r) was
conducted between TMRM and ATP. Signiﬁcance was set at P<0.05.
Mitochondrial membrane potential (TMRM)
LEDT-6 h group increased MMP (10.77 AU, SEM 0.88) com-
pared to: Control (3.79 AU, SEM 0.46): P<0.001; LEDT-5 min
(4.11 AU, SEM 0.52): P<0.001; LEDT-24 h (4.91 AU, SEM
0.47): P=0.001. LEDT-3 h (7.87 AU, SEM 0.59) increased
MMP compared to Control (P=0.019) and LEDT-5 min
(P=0.031). These results are graphically presented in Fig. 2. All
nonsigniﬁcant results were Control versus LEDT-5 min
(P=0.997) and versus LEDT-24 h (P=0.816); LEDT-5 min
versus LEDT-24 h (P=0.935); LEDT-3 h versus LEDT-6 h
(P=0.113) and versus LEDT-24 h (P=0.103).
LEDT-6 h increased ATP contents (4.53 nmol per well, SEM
0.19) compared to: Control (1.28 nmol per well, SEM 0.05):
P<0.001; LEDT-5 min (2.01 nmol per well, SEM 0.16):
P<0.001; LEDT-24 h (2.77 nmol per well, SEM 0.16): P=
0.007. LEDT-3 h increased ATP contents (3.73 nmol per well,
SEM 0.17) compared to Control (P<0.001) and LEDT-5 min
(P=0.008). LEDT-24 h increased ATP contents compared to
Control (P=0.020). These results are graphically presented in
Fig. 3A. All nonsigniﬁcant results were Control versus LEDT-
5 min (P=0.385); LEDT-3 h versus LEDT-6 h (P=0.299)
and versus LEDT-24 h (P=0.169); LEDT-24 h versus LEDT-
5 min (P=0.338).
The statistical power and the effect size regarding ATP content
in all groups were calculated to ensure the minimal power of
80% and large effect size (>0.75). We used the mean ATP con-
tent of each group and the highest value of standard deviation
among all groups, which was observed in LEDT-6 h. Our results
demonstrate a difference between groups with a statistical power
of 99%, effect size of 3.42 (very large effect) and total sample
size of 10, i.e. 2 wells per group (ﬁve groups). These calcula-
tions demonstrate that our sample size was small, but adequate
(3 wells per group).
Pearson product-moment correlation coefﬁcient (Pearson’sr)
TMRM incorporation into mitochondrial matrix of myotubes
from C2C12 cells showed a high correlation (r=0.89) with
ATP content (P<0.001). This result is presented in Fig. 3B.
This study identiﬁed a well-deﬁned time-response for the LEDT-
mediated increase in MMP and ATP synthesis in myotubes from
C2C12 cells under the stress of the cell culture. The light dose
used was based on previous study that already reported beneﬁts
of LLLT on mitochondria of myotubes (9). In addition, we found
a strong correlation between MMP and ATP content measured
Figure 2. TMRM. Analysis of mitochondrial membrane potential using tetramethyl rhodamine methyl ester (TMRM) stained in red. Images with a
magniﬁcation of 409. Abbreviations: LEDT=light-emitting diode therapy; AU =arbitrary units; C =control group; 5 min =LEDT-5 min group;
24 h =LEDT-24 h group; *=statistical signiﬁcance (P<0.05) using one-way analysis of variance (ANOVA).
Photochemistry and Photobiology 3
during a wide range from 5 min (immediate effect) to 24 h
(prolonged effect). To our knowledge this is the ﬁrst study inves-
tigating the time-response for light therapy modulation of mito-
chondrial metabolism in conjunction with ATP synthesis in
C2C12 is a cell line originally isolated from dystrophic mus-
cles of C3H mice by Yaffe and Saxel (19). In culture it rapidly
differentiates into contractile myotubes (muscle ﬁbers) especially
when treated with horse serum instead of fetal bovine serum.
These myotubes contain multinucleated cells that express pro-
teins characteristic of skeletal muscle such as myosin heavy
chain and creatine kinase (20).
One of ﬁrst effects of LLLT reported in literature was a mod-
ulation on MMP and ATP synthesis in mitochondria isolated
from rat liver (3) and in HeLa cells (4). Our results are in accor-
dance with these previous studies, showing an increased MMP
and ATP synthesis in myotubes from C2C12 cells. However,
light therapy seems to produce a different time-response of
MMP and ATP synthesis among different cell types. While HeLa
cells showed a peak of ATP synthesis around 20 min after light
therapy (4), mitochondria from liver showed an immediate
increase in MMP and ATP synthesis (3). In this study, we found
that muscle cells need a longer time in the range of 3 h to 6 h to
show the maximum effect of light therapy and convert it into a
signiﬁcant increase in MMP and ATP synthesis, comprising an
increase around 200% to 350% over the control values. In addi-
tion, we found that 24 h after irradiation, myotubes could still
produce signiﬁcantly more ATP compared to LEDT-Control
while LEDT-5 min showed no signiﬁcant difference.
Cytochrome c oxidase (Cox) has been reported to be the main
chromophore in cells exposed to red and near-infrared light
(2,15,16,21). However, although Cox activity is important in the
immediate effects of photon absorption, the measurement of its
activity may be insufﬁcient to conﬁrm whether light therapy can
induce “extra”ATP synthesis. For this reason, the measurement
of MMP in conjunction with ATP synthesis can provide informa-
tion on how fast changes occur in the electron transport chain
(ETC.), and H
pumping from the mitochondrial matrix to the
intermembrane space, as well as how much H
ions are returning
to the mitochondrial matrix (1). In this perspective, our results
are consistent with Xu et al. (9) who reported no immediate
effects of light therapy on MMP. Moreover, although Xu et al.
Figure 3. ATP and Pearson’sr. (A) Analysis of adenosine triphosphate (ATP) content between groups. (B) Pearson product-moment correlation coefﬁ-
cient (Pearson’sr) between ATP and mitochondrial membrane potential using TMRM. Abbreviations: LEDT =light-emitting diode therapy;
TMRM =tetramethyl rhodamine methyl ester; nmol =nanomol; AU =arbitrary units; C =control group; 5 min =LEDT-5 min group; 24 h =LEDT-
24 h group; *=statistical signiﬁcance (P<0.05) using one-way analysis of variance (ANOVA).
Figure 4. Mechanism of action of LEDT on mitochondria. (A) Mito-
chondria of myotubes from C2C12 cells without low-level laser therapy
(LLLT) or light-emitting diode therapy (LEDT). There is a normal ﬂux
of electrons (red arrow) through all complexes of electron transport chain,
normal pumping of H
, normal synthesis of ATP and modest take up of
TMRM by the mitochondrial matrix. (B) Mitochondria of myotubes from
C2C12 cells 3–6 h after LEDT. There is an increased ﬂux of electrons
(ticker red arrow), increased pumping of H
, increased synthesis of ATP
and increased take up of TMRM by the mitochondrial matrix. Abbrevia-
tions: I, II, III, IV and V =complexes of the mitochondrial electron
transport chain; H
=proton of hydrogen; - =electron of hydrogen;
O=metabolic water; Q =quinone; Cox =cytochrome
c oxidase; ATP =adenosine triphosphate;TMRM =tetramethyl rhoda-
mine methyl ester.
4 C. Ferraresi et al.
(9) did not assess ATP content, our results showed no signiﬁcant
responses for ATP increment immediately after light therapy
compared to control group.
Our results for MMP in conjunction with ATP content had a
high correlation (Pearson’sr=0.89) during the time range of
5 min to 24 h, suggesting a linear and positive dependence of
ATP synthesis on the value of MMP (ETC. and H
muscle cells, suggesting a new and more efﬁcient time-response
or time window for LEDT stimulate muscle cells (see Fig. 4A,
B). These results are very important for muscle recovery postex-
ercise (10,11) because they suggest a prolonged effect of light
therapy on ATP synthesis necessary to repair muscle damage. In
addition, muscular preconditioning using light therapy for
improvement of performance before a bout of exercise (12) could
possibly be optimized by application at the appropriate time.
However, more studies in vivo and clinical trials are needed to
conﬁrm our hypotheses.
Muscular preconditioning using LLLT or LEDT have been
reported as therapeutic approaches to improve muscle perfor-
mance in both experimental models (22–24) and in clinical trials
(12). However, although this improvement reported in the litera-
ture has been signiﬁcant, some studies have not found positive
results (25). Furthermore, differences between groups treated
with light therapy or placebo seem to be not so large. These dif-
ferences reported in experimental models varied between 80%
and 150% of the values found for control groups for fatigue test
induced by electrical stimulation (22–24). In clinical trials these
differences varied between 5% and 57% increases in number of
repetitions and maximal voluntary contraction (12). Possibly
these relatively modest increases could be due to allowing insuf-
ﬁcient time necessary for the muscle cells to convert light ther-
apy into biological responses as identiﬁed in our study for MMP
and ATP synthesis. Consequently, protocols for muscular pre-
conditioning that have been done up to now (12,22–24), i.e. gen-
erally applying light 5 min before the exercise, may not possibly
achieve the best result. On the basis of our results, we suggest to
wait 3–6 h after light therapy irradiation to obtain the best
increase in muscle performance in muscular preconditioning regi-
men, as MMP and ATP availability are important for muscle
performance (26,27). Once more time, we would like to remark
the needed of more studies in vivo and clinical trials to conﬁrm
our hypotheses. At this point, it is valuable to reference two
previous studies that had a similar initiative (28,29). Hayworth
et al. (28) found increments in Cox activity 24 h after apply
LEDT over rats muscles; Albuquerque-Pontes et al. (29) found a
time window, wavelength-dependence and dose response for
Cox activity increase also after LLLT in rats muscles. Both stud-
ies used animals without any kind of stress, such as this study
used cells only under the stress of the cell culture. We believe
that these previous studies combined with our results are extre-
mely valuable for the discovery and understanding of mecha-
nisms of action of LLLT on muscle tissue, and may offer
guidance on the future use of LLLT in clinical practice.
Our study was designed to test one dose of light during a
time-response to show that there is time-dependency for LLLT
to produce secondary responses in muscle cells. For this reason,
this study used a constant dose (ﬂuence) of light as reported in a
previous study (9) as well as a constant power density. As there
is a possible biphasic dose response (30,31), use of different
parameters such as ﬂuence, wavelengths or irradiance could pro-
duce different responses. In addition, red and near-infrared light
therapy was delivered at the same time to take advantage of the
double bands in Cox to absorb the light (2,13–16).
This is the ﬁrst study reporting the beneﬁts of mixed red and
near-infrared light therapy on MMP in conjunction with ATP
synthesis in myotubes from C2C12 cells (muscle cells from
mice). Moreover, a well-deﬁned time-response was found for the
increase in ATP synthesis mediated by MMP increased by light
therapy in myotubes.
Our data suggest that 3–6 h could be the best time-response
for light therapy to improve muscle metabolism. In addition, our
results lead us to think there may be possible cumulative effects
if light therapy is applied at intervals less than 24 h that may
have clinical relevance when LLLT is used for muscle postexer-
cise recovery. Finally, we believe that use of light therapy for
muscular preconditioning could be optimized in future studies
whether the time-response for increases in ATP and MMP found
in this study are taken account.
Acknowledgements—We thank Professor Zoltan Pierre Arany and his
instructor Glenn C. Rowe for the C2C12 cells and Andrea Brissette for
assistance with multiple roles including purchase of reagents. Cleber
Ferraresi thank FAPESP for his PhD scholarships (numbers 2010/07194-
7 and 2012/05919-0). MR Hamblin was supported by US NIH grant
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