The Journal of Experimental Medicine
JEM © The Rockefeller University Press $30.00
Vol. 204, No. 13, December 24, 2007 3173-3181 www.jem.org/cgi/doi/10.1084/jem.20070956
Asthma and chronic obstructive pulmonary dis-
ease (COPD) are infl ammatory airway diseases
that are characterized by diff erent patterns of air-
way remodeling ( 1 ). Nevertheless, the decrease
in lung function that characterizes both diseases
is associated with an increased mass of bronchial
smooth muscle (BSM) ( 2, 3 ), which is likely to
be the most important abnormality responsible
for the airway narrowing observed in response
to bronchoconstricting stimuli ( 4 ). The mecha-
nisms underlying such remodeling of smooth
muscle remain largely unknown. On the one
hand, in smooth muscle from asthmatic patients,
excessive in vitro proliferation of BSM cells has
been demonstrated ( 5 – 7 ). In addition, decreased
apoptosis of BSM cells has also been demon-
strated, although this was in a rat model of ex-
perimental asthma ( 7 ). On the other hand, in
COPD, smooth muscle remodeling appears lim-
ited to airways distal to the fourth generation
( 3, 8 ). Although an increase in TGF- ? 1 pro-
duction by BSM cells has been proposed, such
localization and the complete mechanism remain
unexplained ( 9 ). Whatever its cause, BSM re-
modeling is poorly sensitive to current therapeu-
tics in both asthma and COPD.
Mitochondria play a major role in both cell
proliferation and apoptosis ( 10, 11 ). In cancer,
for instance, targeting of mitochondrial function
and mitochondrial antiapoptotic protein bcl-2 has
been used to either suppress the proliferation of
tumor cells ( 10 ) or induce cell apoptosis in solid
tu mors ( 12 ). Mitochondria are also involved
in other diseases, such as neuron-degenerative
Abbreviations used: ANOVA,
analysis of variance; BSM,
bronchial smooth muscle;
dependent protein kinase IV;
COPD, chronic obstructive
pulmonary disease; mtTFA,
factor A; NRF, nuclear respira-
tory factor; PGC, peroxisome
The online version of this article contains supplemental material.
Bronchial smooth muscle remodeling
involves calcium-dependent enhanced
mitochondrial biogenesis in asthma
Thomas Trian, 1,2 Giovanni Benard, 3 Hugues Begueret, 1,2,4
Rodrigue Rossignol, 3 Pierre-Olivier Girodet, 1,2,4 Debajyoti Ghosh, 1,2
Olga Ousova, 1,2 Jean-Marc Vernejoux, 4 Roger Marthan, 1,2,4
José-Manuel Tunon-de-Lara, 1,2,4 and Patrick Berger 1,2,4
1 Universite Bordeaux 2, Laboratoire de Physiologie Cellulaire Respiratoire, F-33076 Bordeaux, France
Institut National de la Sant é et de la Recherche M é dicale (INSERM), 2 U885 and 3 U688, F-33076 Bordeaux, France
4 Centre Hospitalier Universitaire de Bordeaux, F-33076 Bordeaux, France
Asthma and chronic obstructive pulmonary disease (COPD) are characterized by different
patterns of airway remodeling, which all include an increased mass of bronchial smooth
muscle (BSM). A remaining major question concerns the mechanisms underlying such a
remodeling of BSM. Because mitochondria play a major role in both cell proliferation and
apoptosis, we hypothesized that mitochondrial activation in BSM could play a role in this
remodeling. We describe that both the mitochondrial mass and oxygen consumption were
higher in the BSM from asthmatic subjects than in that from both COPD and controls. This
feature, which is specifi c to asthma, was related to an enhanced mitochondrial biogenesis
through up-regulation of peroxisome proliferator-activated receptor ? coactivator (PGC) –
1 ? , nuclear respiratory factor-1, and mitochondrial transcription factor A. The priming
event of such activation was an alteration in BSM calcium homeostasis. BSM cell apoptosis
was not different in the three groups of subjects. Asthmatic BSM was, however, character-
ized by increased cell growth and proliferation. Both characteristics were completely abro-
gated in mitochondria-defi cient asthmatic BSM cells. Conversely, in both COPD and control
BSM cells, induction of mitochondrial biogenesis reproduced these characteristics. Thus,
BSM in asthmatic patients is characterized by an altered calcium homeostasis that in-
creases mitochondrial biogenesis, which, in turn, enhances cell proliferation, leading to
SMOOTH MUSCLE MITOCHONDRIAL BIOGENESIS IN ASTHMA | Trian et al.
and 18 ± 2.8 yr, respectively. Of the 19 control subjects who
received no treatment, 8 of them were lifelong nonsmokers,
whereas 11 were former smokers.
diseases ( 11 ). However, their role in asthma or COPD remains
to be investigated. We hypothesized that mitochondrial
activation in BSM from asthmatic or COPD patients could
contribute to smooth muscle remodeling. To investigate this
hypothesis, we have compared mitochondrial mass, activity,
and biogenesis in BSM obtained from asthmatics, COPD
patients, and normal controls. We describe that both the
mitochondrial mass and oxygen consumption were higher
in the BSM from asthmatic subjects than in that from both
COPD and controls. This feature, which is specifi c to asthma,
is related to an enhanced mitochondrial biogenesis as a conse-
quence of an increase in extracellular calcium infl ux upon ac-
tivation of asthmatic BSM cells. We also demonstrate a
specifi c mitochondria-dependent pathway for asthmatic BSM
cell proliferation. Targeting such a pathway may thus rep-
resent a new approach for the treatment of airway remodel-
ing in asthma.
The clinical characteristics of all subjects are shown in Table I .
All of the 14 severe persistent asthmatics were lifelong non-
smokers and received stable treatments, including oral or
inhaled corticosteroids and ? 2 agonists. 10 of them were atopic.
The 17 moderate to severe COPD patients were either current
or former smokers, and 9 of them received stable treatments,
including oral or inhaled corticosteroids and/or ? 2 agonists.
None of the asthmatic or COPD patients experienced a
recent ( < 3 mo) exacerbation of the disease. The mean duration
of the disease in asthmatic and COPD patients was 26 ± 4.6
Table I. Clinical and functional characteristics of subjects
Characteristic Patients with asthmaPatients with COPDControls
No. of patients
BMI (kg/m 2 )
Current (no. of patients)
Former (no. of patients)
Years since quitting
LABA (no. of patients)
ICS (no. of patients)
OCS (no. of patients)
Percentage of predicted value
FEV 1 : FVC ratio (% of FVC)
Liters sec ? 1
Percentage of predicted value
14 17 19
42.3 ± 5.7
26.9 ± 1.1
59.8 ± 3.0
24.0 ± 0.9
60.4 ± 2.5
27.4 ± 1.1
0 ± 0
46.3 ± 4.8
11.4 ± 4.8
19.5 ± 5.7
13.1 ± 3.8
2.2 ± 0.2
83.1 ± 6.3
71.9 ± 3.7
1.8 ± 0.1
60.2 ± 2.5
56.3 ± 2.2
2.9 ± 0.1
101.7 ± 3.2
81.8 ± 1.7
1.9 ± 0.3
54.6 ± 7.8
0.9 ± 0.1
28.6 ± 3.1
2.9 ± 0.1
90.2 ± 4.0
Data are the mean ± the SEM. BMI, body mass index; LABA, long-acting ? 2 agonist; ICS, inhaled corticosteroid; OCS, oral corticosteroid; FEV 1 , forced expiratory volume in one
second; FVC, forced vital capacity; FEF 25-75, forced expiratory fl ow between 25 and 75% of FVC.
Figure 1. BSM remodeling in both asthma and COPD. Representative
optic microscopic images from bronchial sections stained with HES were
obtained from an asthmatic (A), a COPD (B), or a control subject (C)
and observed at 200 × magnifi cation. Smooth muscles were visualized
(SM). Bars, 50 μ m. (D) The normalized smooth muscle area was assessed
from microscopic images. Bronchial specimens were obtained from asth-
matic (black column; n = 10), COPD (gray column; n = 7), and control
subjects (white column; n = 6). Data are the mean ± the SEM. *, P < 0.05
between populations using ANOVA with the use of Bonferroni ’ s test.
JEM VOL. 204, December 24, 2007
.20070956/DC1; P = 0.02). There was a signifi cant increase
in the mitochondrial density in asthmatic BSM cells both
ex vivo ( Fig. 2 E ; P = 0.01) and in vitro (Fig. S1 E; P = 0.01)
as compared with both COPD and controls. Based on elec-
tronic microscopy, the mitochondrial density has been shown
to refl ect the mitochondrial activity ( 13, 14 ). To assess the
smooth muscle specifi city of these results, we also analyzed
ultrastructural mitochondrial parameters in other cell types
from the same bronchial specimens. There was no diff erence
between the three groups, in terms of both number (P = 0.35
and P = 0.89) and density of mitochondria (P = 0.47 and P =
0.87) in endothelial and epithelial cells, respectively (unpub-
lished data). The increased mass of asthmatic BSM mitochon-
dria was further confi rmed, in vitro, by an increase in the porin
content compared with that of both COPD and controls
( Fig. 2 F ; P = 0.001). We also found within the asthmatic
population that both duration of the disease and forced expi-
ratory volume in one second (FEV 1 )/ forced vital capacity
Mitochondrial mass and activity are increased only
in the BSM of asthmatics
A morphological analysis of BSM was performed in the three
groups of subjects ( Fig. 1, A – C ). BSM mass was increased in
both asthmatic and COPD patients as compared with controls
( Fig. 1 D ; P = 0.01). Ultrastructure of BSM mitochondria
was then compared in asthma and COPD to control subjects
( Fig. 2, A-C ). The number of mitochondrial sections was
higher in the BSM of asthmatics than in that of both COPD
and controls ( Fig. 2 D ; P < 0.001). The mean area per section
was, however, unchanged (0.1 ± 0.007, 0.1 ± 0.016, and 0.1 ±
0.009 μ m 2 for asthmatics, COPD, and controls, respectively;
analysis of variance [ANOVA] P = 0.87). Collectively, these
results favor the hypothesis that the increase in mitochondrial
mass in the BSM of asthmatics is related to an increase in
number rather than in individual size. Similar results were
obtained with cultured growth-arrested BSM cells (Fig. S1,
A – D, available at http://www.jem.org/cgi/content/full/jem
Figure 2. Increased mitochondrial mass and activity in asthmatic BSM. Representative electronic microscopic images from bronchial sections
were obtained from an asthmatic (A), a COPD (B), or a control subject (C) and observed at 26,000 × magnifi cation. Some smooth muscle mitochondria
were visualized (arrows). Bars, 0.2 μ m. The number (D) and the density (E) of mitochondria were assessed from electronic microscopic images ( n = 4 for
each population). Mitochondrial mass was assessed by the porin content using Western blot (F; n = 8 for asthmatics, n = 5 for COPD, and n = 7 for con-
trols). Endogenous cellular oxygen consumption was evaluated by oxygraphy (G; n = 5 for asthmatics, n = 4 for COPD, and n = 4 for controls). BSM cells
(BSMC) were obtained from asthmatic (black columns), COPD (gray columns), and control subjects (white columns). Data are the mean ± the SEM.
*, P < 0.05 between populations using ANOVA with the use of Bonferroni ’ s test.
SMOOTH MUSCLE MITOCHONDRIAL BIOGENESIS IN ASTHMA | Trian et al.
D and E ). Upstream mechanisms that would explain such an
increased mitochondrial biogenesis were then examined. The
two transcription factors nuclear respiratory factor-1 (NRF-1)
and peroxisome proliferator – activated receptor ? coactivator –
1 ? (PGC-1 ? ) were both elevated in asthmatic BSM cells at
both the protein and the transcription levels ( Fig. 3, D and E ).
Among the various factors that could activate PGC-1 ? , we
observed that the calcium/calmodulin-dependent protein
kinase IV (CaMK-IV) was phosphorylated and thus activated in
asthmatic BSM cells compared with controls and COPD ( Fig.
4 A ). We thus investigated whether calcium homeostasis was
deregulated in asthmatic BSM cells using microspectrofl uo-
rimetry. The resting calcium concentration was consistent
in asthmatic (125 ± 8 nM), COPD (127 ± 7 nM), or control
BSM cells (133 ± 4 nM; P = 0.60). However, the calcium re-
sponse to acetylcholine was signifi cantly altered in asthmatic
BSM cells ( Fig. 4, B – D ). Whereas the amplitude of the calcium
rise was unchanged (275 ± 20, 284 ± 43, and 272 ± 27 nM in
asthmatics, COPD, and controls, respectively; P = 0.95), the
area under the curve was signifi cantly increased in asthmatic
BSM cells ( Fig. 4 E ). A similar result was obtained in asthmatic
BSM cells stimulated with histamine, confi rming that it was
not agonist-specifi c (Fig. S3, A – D, available at http://www
.jem.org/cgi/content/full/jem.20070956/DC1). An enhanced
calcium infl ux in asthmatic BSM cells accounts for this result,
as both removal of extracellular calcium using EGTA and
blockade of calcium channels using methoxyverapamil (D600)
abolished such abnormal calcium responses in asthmatic BSM
cells ( Fig. 4 E and Fig. S3 D). Blockade of calcium infl ux using
D600 also inhibited the activation of CaMK-IV ( Fig. 4 A ) and
the subsequent activation of mitochondrial biogenesis through
(FVC) ratio were correlated with the porin content (r = 0.77,
P = 0.03 and r = ? 0.84, P = 0.01, respectively), whereas no
correlation was found within the COPD or the control pop-
ulation. Similarly, the oxygen consumption of asthmatic BSM
cells was specifi cally enhanced compared with COPD or control
BSM cells ( Fig. 2 G ; P = 0.01). Such an increase in the mito-
chondrial respiratory rate was not associated with diff erences
in the coupling degree, as demonstrated by the effects of
inhibitors of the phosphorylation system. Similarly, oxygen
consumption of COPD and control BSM, although lower, also
refl ected coupled mitochondrial respiration (Fig. S2; P <
0.04, paired t tests). These inhibitors decreased the mito-
chondrial respiration of asthmatics, COPD, and controls by
18.6, 21.2, and 24.7%, respectively, whereas cyanide com-
pletely inhibited oxygen consumption of all BSM cells. The
increased mitochondrial respiration in asthma thus appears to
result from the increased organelle content and subsequent
enhancement in the overall oxidative capacity in BSM.
Mitochondrial biogenesis is increased in asthmatic BSM
cells through a calcium-dependent pathway
We next analyzed the mitochondrial network by confocal
microscopy ( Fig. 3, A – C ). Surprisingly, asthmatic BSM cells
presented a typical aspect of intense mitochondrial biogenesis,
as shown by the presence of several budding areas with intense
dots, and a wider network with increased ramifi cation ( Fig. 3 A ).
Because the mitochondrial transcription factor A (mtTFA) is
the main factor involved in mitochondrial biogenesis, we
measured its protein content and transcription level in the
three groups of BSM. As compared with both controls and
COPD, mtTFA was increased in asthmatic BSM cells ( Fig. 3,
Figure 3. Increased mitochondrial biogenesis in asthmatic BSM. Representative confocal images of the mitochondrial network after three dimen-
sional reconstruction were obtained from asthmatic (A), COPD (B), or control (C) BSM cells. Bars, 10 μ m. mtTFA, NRF-1, and PGC-1 ? levels were assessed
by both Western blot (D) and quantitative RT-PCR (E). BSM cells were obtained from asthmatic (black columns; n = 6), COPD (gray columns; n = 7), and
control subjects (white columns; n = 6). Data are the mean ± the SEM. *, P < 0.05 between populations using ANOVA with the use of Bonferroni ’ s test.
JEM VOL. 204, December 24, 2007
of anaerobic glycolysis. For this purpose, BSM cell proliferation
curves were plotted in the three groups of subjects using
either glucose or galactose in the culture medium ( Fig. 6,
A and B ). In the presence of glucose, which allows ATP to be
produced by both aerobic and anaerobic glycolysis, asthmatic
BSM cell growth was signifi cantly increased compared with
that of COPD and control subjects ( Fig. 6 A ; P = 0.02), with
a concomitant decrease in the doubling time ( Fig. 6 C ; P =
0.02). When galactose, which only allows cells to produce
ATP by mitochondrial oxidative phosphorylations, was substi-
tuted for glucose, the doubling time of the cell growth from
both COPD (P = 0.02) and control subjects (P = 0.02) sig-
nifi cantly increased, whereas that of asthmatic cells remained
constant ( Fig. 6, B and C ). Because an increase in cell growth
can be related to a decreased apoptosis and/or an increased
proliferation, we analyzed Annexin V binding and BrdU in-
corporation, respectively. Taking into account the percent-
age of Annexin V – positive cells, spontaneous apoptosis was
not altered in asthmatic BSM cells as compared with that of
COPD and controls (Fig. S4, available at http://www.jem
.org/cgi/content/full/jem.20070956/DC1). However, BrdU
incorporation increased in BSM cells from asthmatics as com-
pared with that in both COPD and controls in the presence
of glucose ( Fig. 6 D ; P = 0.01). Incubation for 11 d in the
absence of glucose signifi cantly inhibited BrdU incorporation
in BSM cells from controls (P = 0.02) and COPD patients
(P = 0.02), but not in that from asthmatics ( Fig. 6 D ; P = 0.99).
Collectively, these results demonstrate that the proliferation
of asthmatic BSM cells mainly uses mitochondrial-dependent
PGC-1 ? , NRF-1, and mtTFA ( Fig. 5, A – C ), leading to the
increase in the mitochondrial mass as assessed by the porin
content ( Fig. 5 D ). Thus, these results indicate that an enhanced
extracellular calcium infl ux, specifi c to asthmatic BSM cells, is
the initial priming event leading to an increased mitochondrial
biogenesis and mass.
Only asthmatic BSM cell proliferation is mitochondria
To further assess the specifi c role of mitochondria in asth-
matic BSM proliferation contributing to airway remodeling,
we next compared BSM cell growth in the presence or absence
Figure 4. Altered cell calcium homeostasis in asthmatic BSM.
(A) Phosphorylated CaMK-IV (P-CaMK-IV) levels were assessed by Western
blot. Representative intracellular calcium responses after stimulation for
30 s by 10 ? 5 M acetylcholine (ACh) are presented in BSM cells from asth-
matic (B), COPD (C), or control subjects (D). As a reference, response from
the control cell (D) is presented as a gray line (B and C). The area under
the curve was assessed from the calcium response (E). BSM cells were
analyzed in the absence (-) or presence (+) of 2 mM extracellular Ca 2 + or
1 μ M methoxyverapamil (D600). Cells were obtained from asthmatic
(black columns; n = 5), COPD (gray columns; n = 4), and control subjects
(white columns; n = 4). Data are the mean ± the SEM. *, P < 0.05 between
populations within an experimental condition using ANOVA with the use
of Bonferroni ’ s test. † , P < 0.05 between experimental conditions versus 2
mM Ca 2+ without D600 within a population using paired Student’s t tests.
Figure 5. Effect of methoxyverapamil (D600) on mitochondrial
biogenesis and content. PGC-1 ? (A), NRF-1 (B), mtTFA (C), and porin (D)
levels were assessed by Western blot in BSM cells cultured in the absence
(-) or presence (+) of 1 μ M D600 for 48 h. Cells were obtained from asth-
matic (black columns; n = 5), COPD (gray columns; n = 4), and control
subjects (white columns; n = 4). Data are the mean ± the SEM. *, P < 0.05
between populations within an experimental condition using ANOVA with
the use of Bonferroni ’ s test. † , P < 0.05 between the absence and the
presence of D600 within a population using paired Student’s t tests.
SMOOTH MUSCLE MITOCHONDRIAL BIOGENESIS IN ASTHMA | Trian et al.
porin in the three groups of subjects ( Fig. 7 ). Fig. 6 C dem-
onstrates that ethidium bromide signifi cantly increased the
doubling time of asthmatic BSM cells (P < 0.001), but did
not change that of both COPD (P = 0.15) and control cells
(P = 0.09). Similarly, proliferation of mitochondria-defi cient
asthmatic BSM cells decreased (P < 0.001), whereas that of
COPD and controls remained unchanged (Fig. S5, available
However, ethidium bromide also decreased BrdU incorpor-
ation of BSM cells from the three groups of subjects ( Fig. 6 D ).
In a second alternative approach, we stimulated mitochon-
drial biogenesis using cyclic GMP for 6 d, as previously de-
scribed ( 16 ). The amount of mitochondria increased in such
stimulated BSM cells from both COPD and controls ( Fig. 7 ;
P = 0.02 for both). Because the amount of mitochondria
was already up-regulated in BSM cells from asthmatics, cy-
clic GMP failed to additionally increase the porin content
(P = 0.2). Cyclic GMP signifi cantly decreased the doubling
time (P = 0.01), and increased the BrdU incorporation
(P = 0.02) and the proliferation of BSM cells from COPD
patients (P = 0.01) ( Fig. 6, C and D, and Fig. S5). Similarly,
cyclic GMP also decreased the doubling time (P < 0.001)
and increased the BrdU incorporation (P < 0.001) and the
proliferation of BSM cells from controls (P < 0.001; Fig. 6,
C and D, and Fig. S5). However, cyclic GMP had no eff ect
in asthmatic BSM cell growth and proliferation. Finally, in
a last approach, we analyzed the eff ect of altering calcium
homeostasis on the proliferation of BSM cells. D600, which
blocks calcium infl ux ( Fig. 4 E ) and the resulting increase in
mitochondrial biogenesis and content only in asthmatic BSM
cells ( Fig. 5 ), also signifi cantly inhibited the increased BrdU
oxidative phosphorylations, whereas that of COPD and con-
trol subjects mainly uses mitochondrial-independent anaero-
Increased mitochondrial mass explains increased asthmatic
BSM cell proliferation
To determine whether the increased mitochondrial biogen-
esis found in asthmatic BSM cells is a cause or a consequence
of the asthmatic BSM cell increased proliferation, three alter-
native approaches were used. First, mitochondria-defi cient
BSM cells were generated using culture with ethidium bro-
mide, as previously described ( 10, 15 ). After 16 d of culture,
all of the asthmatic BSM cells died, whereas those from both
COPD patients and controls were still alive up to 30 d in the
ethidium bromide medium (unpublished data). 6 d of incubation
with ethidium bromide signifi cantly decreased the amount of
Figure 6. Asthmatic BSM cell proliferation is mitochondria depen-
dent. BSM cell proliferation curves were obtained using either glucose (A)
or galactose (B) in the culture medium. The doubling times of cell growth
(C) were obtained from the proliferation curves. (D) BrdU incorporations
were measured. BSM cells were cultured in various experimental condi-
tions, i.e., glucose, galactose, glucose + ethidium bromide (Et Br), or glu-
cose + cyclic GMP (cGMP). BSM cells were obtained from asthmatic (black
symbols and columns; n = 4), COPD (gray symbols and columns; n = 4),
and control subjects (white symbols and columns; n = 4). Data are the
mean ± the SEM. *, P < 0.05 between populations within an experimental
condition using ANOVA with the use of Bonferroni ’ s test. † , P < 0.05 be-
tween experimental conditions versus glucose within a population using
paired Student’s t tests.
Figure 7. Effect of ethidium bromide and cyclic GMP on the porin
content. Mitochondrial mass was assessed by the porin content using
Western blot. BSM cells were obtained from asthmatic (black columns;
n = 4), COPD (gray columns; n = 4), and control subjects (white columns;
n = 4) and were cultured in the absence (-) or presence (+) of ethidium
bromide (Et Br) or cyclic GMP (cGMP) for 6 d before the experiments. Data
are the mean ± the SEM. *, P < 0.05 between populations within an ex-
perimental condition using ANOVA with the use of Bonferroni ’ s test. † ,
P < 0.05 between experimental conditions versus glucose within a popu-
lation using paired Student’s t tests.
JEM VOL. 204, December 24, 2007
quantitative RT-PCR and Western blot to assess mitochon-
drial respiratory chain content, as mitochondrial overall con-
tent and respiratory chain content are not always linked ( 19 ).
The increased number of mitochondria was limited to
asthmatic BSM cells compared with endothelial and epithe-
lial cells from asthmatic, COPD, or control subjects. However,
two characteristics of such asthmatic populations deserve fur-
ther comment. First, it is unlikely that asthma treatments in-
terfered with the observed changes because, on the one hand,
9/17 COPD patients took treatments similar to that of asth-
matics, and, on the other hand, no diff erence has been found
between controls and COPD. Second, the younger mean age
of the asthmatic population is unlikely to contribute to this in-
crease in mitochondrial content because (a) this increase per-
sisted when comparing asthmatics to a subgroup of nonsmoking
controls whose mean age was similar, and (b) in skeletal muscle,
if not any other muscle, age does not infl uence mitochondrial
content ( 20 ). To the best of our knowledge, this is the fi rst de-
scription of such mitochondrial characteristics in any type of
smooth muscle cells with potential pathophysiological impli-
cations. It has been previously reported that in a variety of
diff erentiated tissues, a mitochondrial dysfunction increases
mitochondrial biogenesis, suggesting a cellular compensatory
mechanism ( 21 ). However, there was no mitochondrial dys-
function in our study because the respiration of asthmatic BSM
mitochondria was effi ciently coupled. Similarly, artifi cial activa-
tion of mitochondrial biogenesis of mouse neonatal cardiac my-
ocytes largely induced a coupled respiration ( 22 ).
Mitochondrial biogenesis is controlled by many factors.
PGC-1 ? is a well-known master activator of mitochondrial
biogenesis through the production of both NRF-1 and mtTFA
in various cell types, including myoblast ( 23 ), fi broblast, or adi-
pocytes ( 24 ). In this study, we demonstrated that such a cascade
is activated in asthmatic BSM cells, as shown by the concomi-
tant up-regulation of PGC-1 ? , NRF-1, and mtTFA. More-
over, cyclic GMP has been shown to activate PGC-1 ? and
mitochondrial biogenesis in various cell lines, including U937,
L6, and PC12 ( 16, 25 ). We have observed that cyclic GMP
induces mitochondrial biogenesis in both control and COPD
BSM cells, but not in asthmatic BSM cells. These fi ndings sug-
gest that mitochondrial biogenesis in asthmatic BSM cells may
already be up-regulated. In this connection, cyclic GMP also
improved cell proliferation of control and COPD BSM cells,
but not those of asthmatics. It could be argued that cyclic GMP
regulates many diff erent genes in smooth muscle, such as vas-
cular smooth muscle ( 26 ). However, cyclic GMP has been
shown to inhibit, rather than enhance, the proliferation of vas-
cular smooth muscle cells, as well as that of mesangial cells and
various fi broblasts ( 26 ). In addition, direct improvement of
mitochondrial biogenesis by transgenic overexpression of
PGC-1 ? activates skeletal muscle atrophy ( 27 ) and cardiac
muscle dysfunction ( 22 ). Conversely, cyclic GMP increases the
proliferation of endothelial cells, but the role of mitochondria
in this phenomenon has not been investigated so far ( 28 ).
PGC-1 ? activation can be calcium-dependent ( 29 ). In this
study, we found that CaMK-IV was more phosphorylated in
incorporation in BSM cells from asthmatics, thus confi rming
that this enhanced cellular calcium infl ux represents the ini-
tial priming event ( Fig. 8 ).
This study indicates that whereas both asthma and COPD
are characterized by BSM remodeling, a specifi c mitochon-
dria-dependent pathway is required for BSM proliferation
only in asthma. This pathway is initiated by an altered cal-
cium homeostasis, upon the activation of asthmatic BSM cells
(Fig. S6, available at http://www.jem.org/cgi/content/full/
jem.20070956/DC1). Proliferation of BSM in both health and
COPD is, at the very least, less mitochondria dependent. Thus,
these results suggest that mitochondria may represent a specifi c
new therapeutic target in airway remodeling in asthma.
In this study, we paid special attention when comparing
data from severe asthmatics to that of COPD patients because
both diseases have been shown to present smooth muscle re-
modeling ( 2, 3, 17 ). Using a variety of diff erent experimental
approaches, we provide evidence that asthmatic BSM express
a higher number of active mitochondria and a clear aspect of
intense mitochondrial biogenesis. In our study, we assessed
mitochondrial mass using various parameters, including the
number of mitochondria by electron microscopy both ex vivo
and in vitro, the mitochondrial network by confocal micros-
copy, and the porin content by Western blot. All of these
methods provided consistent results. We also found signifi cant
correlations between in vitro BSM porin content, which is a
relevant quantitative estimate of mitochondrial mass, and both
the duration of the disease and the FEV 1 /FVC ratio within the
asthmatic population. These correlations further support our
hypothesis because, on the one hand, the longer the duration
of the disease that is known to favor airway remodeling ( 18 ),
the higher the mitochondrial mass, and on the other hand, the
lower the FEV 1 /FVC ratio, which also refl ects airway remod-
eling, the higher the mitochondrial mass. Moreover, to obtain
a comprehensive assessment of mitochondrial content, we ad-
ditionally measured the expression level of mtTFA by both
Figure 8. Effect of methoxyverapamil (D600) on BSM cell prolif-
eration. BSM cell proliferation was measured using BrdU incorporations.
Cells were cultured in the absence (-) or presence (+) of 1 μ M D600 for
48 h. BSM cells were obtained from asthmatic (black columns; n = 4), COPD
(gray columns; n = 4), and control subjects (white columns; n = 4). Data
are the mean ± the SEM. *, P < 0.05 between populations within an ex-
perimental condition using ANOVA with the use of Bonferroni ’ s test. † ,
P < 0.05 between the absence and the presence of D600 within a popula-
tion using paired Student’s t tests.
SMOOTH MUSCLE MITOCHONDRIAL BIOGENESIS IN ASTHMA | Trian et al.
software (Soft Imaging System) at a magnifi cation of 200 × . This smooth
muscle area was normalized by the whole area of the corresponding tissues
and presented as percentages of whole area.
Bronchial specimens were also embedded in epon for electron micros-
copy (Tecnai 12; Philips), as previously described ( 39 ). 10 60-nm-thick
ultrathin serial sections per specimen were cut and examined by a pathologist
to locate whole nucleated BSM cells and endothelial and epithelial cells. The
number of mitochondrial sections normalized by the whole-cell area and the
density of mitochondria normalized by the cytoplasmic density were assessed
manually in these 3 cell types from 10 to 25 measurements per section in a
blinded fashion using ScanView and ImageJ (National Institutes of Health)
softwares at a magnifi cation of 6000 × .
Primary cultures of BSM cells were established from bronchial specimens,
as previously described ( 37, 38, 40 ). All experiments were performed on phe-
notypically confi rmed smooth muscle cells between passages 2 and 4. Cells
were seeded on glass coverslips for confocal microscopy and microspectrofl u-
orimetry, on culture fl asks for electron microscopy and protein extraction, or
on culture plates for RNA extraction. We used cells transfected with the plas-
mid mitochondrion-targeted GFP (mito-GFP) ( 14 ) to obtain confocal images
of the mitochondrial network. Such images were acquired with FluoView laser
scanning microscope (Nikon) and reconstituted in three-dimensional images
using Imaris Software (Bitplane) ( 38 ). Immunoblotting was performed on cell
protein extracts ( 38, 40 ) using primary antibodies directed against porin,
mtTFA, NRF-1, and PGC-1 ? . After reverse transcription, real-time quanti-
tative PCR was performed on a Rotor-Gene 2000 (Corbett Research)
( 37, 38, 40 ), using appropriate primers designed to target mtTFA, NRF-1, or
PGC-1 ? . Endogenous cell oxygen consumption, as well as coupling degree,
were assessed in a thermostatically controlled chamber equipped with a Clark
oxygen electrode (Oxygraph System; Hansatech), as previously described ( 14 ).
Cell calcium was assessed by microspectrofl uorimetry using Indo-1 probe, as
previously described ( 40, 41 ). Cell proliferation was evaluated using both
BrdU incorporation and cell counting. Cell apoptosis was fi nally studied using
FITC – Annexin V and fl ow cytometry. A complete description of all methods
is available in the Supplemental materials and methods.
Statistical analysis. The statistical analysis was performed with NCSS 2001
software. Comparison between the three groups was performed by means
of ANOVA, with the use of Bonferroni ’ s test for multiple comparisons or
paired Student’s t tests. Values are presented as the mean ± the SEM. A
Pearson correlation matrix was built between in vitro and in vivo measure-
ments. A P value < 0.05 was considered statistically signifi cant.
Online supplemental material. Fig. S1 provides ultrastructural character-
istics of mitochondria from pellets of BSM cells. Fig. S2 demonstrates cou-
pled endogenous BSM cell respiration. Fig. S3 provides BSM cell intracellular
calcium responses to histamine. Fig. S4 shows spontaneous BSM cell apop-
tosis. Fig. S5 shows the eff ect of ethidium bromide and cyclic GMP on BSM
cell proliferation. Fig. S6 illustrates the mechanisms of BSM proliferation in
asthma. The online version of this article is available at http://www.jem
We would like to thank Liliane Dubuisson (SERCOMI, Universit é Bordeaux2) for
This study was supported by grants from the Fondation pour la Recherche
M é dicale, France (DAL 2005120574); the Agence Nationale de la Recherche, France
(0591/ANR05 SEST 042-01); and the Programme Hospitalier de Recherche Clinique,
Centre Hospitalier Universitaire de Bordeaux, France. D. Ghosh was funded by the
Fondation pour la Recherche M é dicale.
The authors have no confl icting fi nancial interests.
Submitted: 11 May 2007
Accepted: 31 October 2007
1 . Busse , W.W. , and R.F. Lemanske Jr . 2001 . Asthma. N. Engl. J. Med.
344 : 350 – 362 .
asthmatic BSM cells than in both controls and COPD. Rises in
calcium concentration have been previously shown to activate
CaMK-IV in other cell types, including skeletal muscle cells
( 29 ) or osteoclasts ( 30 ). Interestingly, we also demonstrated that
calcium homeostasis in asthmatic BSM cells was altered, thus
providing a mechanistic explanation for the increased activation
of calcium-dependent signaling enzymes such as CaMK-IV
( 31 ). A D600-sensitive calcium infl ux accounts for such asthma-
induced alteration in calcium homeostasis. We also provide ev-
idence that this calcium infl ux was the initial priming event
because, when blocked, mitochondrial biogenesis and subse-
quent asthmatic BSM cell – increased proliferation was inhibited.
Whereas blockade of such infl ux may be benefi cial, clinical re-
sponses remain to be examined because previous studies using
methoxyverapamil were focused on short-term eff ects for up to
4 wk and did not assess airway remodeling ( 32, 33 ).
Finally, we generated mitochondria-defi cient BSM cells
by depletion of mitochondrial DNA with ethidium bromide,
which is a potent inhibitor of mitochondrial DNA replication
and transcription ( 10, 15 ). Mitochondria-defi cient BSM cells
from asthmatics were unable to proliferate, thereby confi rming
the importance of mitochondria in asthmatic BSM cell prolif-
eration. Thus, the increased mitochondrial biogenesis observed
in asthmatic BSM cells appears to be a cause rather than a con-
sequence of the asthmatic BSM cell increased proliferation. How-
ever, it is well known that training can increase mitochondrial
biogenesis in skeletal muscle ( 34 ). It is unlikely that a similar
phenomenon appears to the BSM from asthmatics. In this study,
none of the asthmatics presented recent exacerbations, and all
of these patients were treated by relaxant ? 2 agonists.
In conclusion, this study reveals that asthmatic BSM is
characterized by an increased mitochondrial biogenesis that, in
turn, enhances cell proliferation. Mitochondria may thus repre-
sent a new target for the treatment of asthmatic smooth mus-
cle remodeling. Further studies are required to assess whether
drugs interacting with mitochondrial biogenesis, including drugs
acting at the site of calcium homeostasis, can prevent and/or
reverse BSM remodeling in asthma.
MATERIALS AND METHODS
Study populations. A total of 14 patients with severe persistent asthma, 17
moderate to severe COPD patients, and 19 normal controls were prospectively
recruited from the Centre Hospitalier Universitaire of Bordeaux according to
both the Global Initiative for Asthma ( 35 ) and the Global Initiative for Chronic
Obstructive Lung Disease guidelines ( 36 ). All subjects gave their written in-
formed consent to participate in the study, after the nature of the procedure
had been fully explained. The study followed recommendations outlined in
the Helsinki Declaration and received approval from the local ethics commit-
tee. Bronchial specimens from all subjects were obtained by either fi beroptic
bronchoscopy or lobectomy, as previously described ( 37, 38 ). A complete
description of subjects is available in the Supplemental materials and methods
(available at http://www.jem.org/cgi/content/full/jem.20070956/DC1).
Study procedures. Bronchial specimens were embedded in paraffi n. As-
sessable BSM was identifi ed by a pathologist in a blinded fashion using both
morphological characteristics and ? -smooth muscle actin staining, as previ-
ously described ( 39 ). There was assessable BSM in the bronchial specimens
from all 14 asthmatics, 17 COPD, and 19 controls. The total area of smooth
muscle layer was calculated manually in a blinded fashion using ScanView
JEM VOL. 204, December 24, 2007 Download full-text
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