Tài liệu Báo cáo khoa học: Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice ppt

Attenuation of cardiac mitochondrial dysfunction
by melatonin in septic mice
Germaine Escames
1
, Luis C. Lo
´
pez
1
, Francisco Ortiz
1
, Ana Lo
´
pez
1
, Jose
´
A. Garcı
´a
1
, Eduardo Ros
2
and Darı
´
o Acun
˜
a-Castroviejo
1,3
1 Instituto de Biotecnologı
´
a, Departamento de Fisiologı
´

a, Universidad de Granada, Spain
2 Servicio de Angiologı
´
a y Cirugı
´
a Vascular, Hospital Universitario San Cecilio, Granada, Spain
3 Servicio de Ana
´
lisis Clı
´
nicos, Hospital Universitario San Cecilio, Granada, Spain
Sepsis-induced multiple organ failure is the major
cause of mortality in critically ill patients, and its inci-
dence is rising [1]. The heart and cardiovascular sys-
tems are seriously affected during sepsis [2]. Although
myocardial impairment in sepsis has been extensively
studied, its etiology remains unclear [3]. Some reports
Keywords
ATP production; mitochondrial failure;
mitochondrial nitric oxide synthase;
oxidative stress; therapy
Correspondence
D. Acun˜ a-Castroviejo, Departamento de
Fisiologı
´
a, Facultad de Medicina, Avenida de
Madrid 11, E-18012, Spain
Fax: +34 958246295
Tel: +34 958246631
E-mail:

(Received 4 December 2006, revised 9
February 2007, accepted 23 February 2007)
doi:10.1111/j.1742-4658.2007.05755.x
The existence of an inducible mitochondrial nitric oxide synthase has been
recently related to the nitrosative ⁄ oxidative damage and mitochondrial dys-
function that occurs during endotoxemia. Melatonin inhibits both inducible
nitric oxide synthase and inducible mitochondrial nitric oxide synthase
activities, a finding related to the antiseptic properties of the indoleamine.
Hence, we examined the changes in inducible nitric oxide synthase ⁄ indu-
cible mitochondrial nitric oxide synthase expression and activity, bioener-
getics and oxidative stress in heart mitochondria following cecal ligation
and puncture-induced sepsis in wild-type (iNOS
+ ⁄ +
) and inducible nitric
oxide synthase-deficient (iNOS
– ⁄ –
) mice. We also evaluated whether melato-
nin reduces the expression of inducible nitric oxide synthase ⁄ inducible
mitochondrial nitric oxide synthase, and whether this inhibition improves
mitochondrial function in this experimental paradigm. The results show
that cecal ligation and puncture induced an increase of inducible mito-
chondrial nitric oxide synthase in iNOS
+ ⁄ +
mice that was accompanied by
oxidative stress, respiratory chain impairment, and reduced ATP produc-
tion, although the ATPase activity remained unchanged. Real-time PCR
analysis showed that induction of inducible nitric oxide synthase during
sepsis was related to the increase of inducible mitochondrial nitric oxide syn-
thase activity, as both inducible nitric oxide synthase and inducible mito-
chondrial nitric oxide synthase were absent in iNOS

– ⁄ –
mice. The induction
of inducible mitochondrial nitric oxide synthase was associated with mito-
chondrial dysfunction, because heart mitochondria from iNOS
– ⁄ –
mice
were unaffected during sepsis. Melatonin treatment blunted sepsis-induced
inducible nitric oxide synthase⁄ inducible mitochondrial nitric oxide syn-
thase isoforms, prevented the impairment of mitochondrial homeostasis
under sepsis, and restored ATP production. These properties of melatonin
should be considered in clinical sepsis.
Abbreviations
CLP, cecal ligation and puncture; ETC, electron transport chain; GPx, glutathione peroxidase; GRd, glutathione reductase; GSH, glutathione;
GSSG, oxidized glutathione; iNOS, inducible nitric oxide synthase; i-mtNOS, inducible mitochondrial nitric oxide synthase; LPO, lipid
peroxidation; mtNOS, constitutive mitochondrial nitric oxide synthase; nNOS, neuronal nitric oxide synthase.
FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2135
have shown that mitochondria are the primary targets
injured in both vital and nonvital organs during
inflammation [4–6]. Besides other factors, mitochond-
rial dysfunction in sepsis is directly associated with an
increase in reactive oxygen species and reactive nitro-
gen species [6–10].
It has been shown that mitochondria from several
organs, such as lungs, liver, diaphragm, and hind leg
skeletal muscle, contain an inducible mitochondrial
nitric oxide synthase (i-mtNOS) [9,11–13], which is
encoded by the same gene as cytosolic inducible nitric
oxide synthase (iNOS) [9,11,12,14,15]. Moreover, mito-
chondria contain a constitutive NOS (mtNOS) derived
from neuronal NOS (nNOS) [16,17]. The expression

and activity of i-mtNOS, but not those of mtNOS,
increase during sepsis [9,11,12]. Other studies have
shown induction of mitochondrial NOS in the dia-
phragm and heart of septic rats, although these reports
did not distinguish between constitutive and inducible
forms [8,18].
Increasing evidence suggests that the nitric oxide
(NO) produced by i-mtNOS plays a role in mitochond-
rial dysfunction during sepsis [9,11,12]. Because iNOS
– ⁄ –
mice do not express i-mtNOS, and the mitochondria
of these mice were unaffected by sepsis, it was sugges-
ted that the overproduction of NO by i-mtNOS is the
main factor responsible for mitochondrial nitrosative ⁄
oxidative stress and impairment during endotoxemia
[9,12]. The induction of i-mtNOS after lipopoly-
saccharide administration leads to an increase in NO
and other reactive species, such as superoxide anion
(O
2
–
), hydrogen peroxide (H
2
O
2
) and peroxynitrite
(ONOO
–
), in heart and diaphragm mitochondria
[8,18]. Cecal ligation and puncture (CLP) also induces

i-mtNOS in these tissues, increasing mitochondrial lipid
peroxidation (LPO) and the oxidized glutathione ⁄
glutathione (GSSG ⁄ GSH) ratio, and reducing the
activity of the electron transport chain (ETC) com-
plexes [9,11,12]. Although NO is a physiologic modula-
tor of mitochondrial respiration [19,20], high levels of
NO may inhibit the ETC, increasing the formation of
O
2
–
and H
2
O
2
[21]. NO reacts with O
2
–
to yield
ONOO
–
, which in turn impairs the ETC and ATP syn-
thase [19]. The parallel failure of the respiratory chain
and oxidative phosphorylation leads to mitochondrial
dysfunction, energy depletion, and cell death. A reduc-
tion in the capacity of the mitochondria to produce
ATP may be related to the organ failure in sepsis
[4,5,22].
There is evidence that antioxidants may be useful in
protecting against mitochondrial damage induced by
oxidative and ⁄ or nitrosative stress [23]. Several reports

have shown that melatonin (aMT) protects against
mitochondrial oxidative stress, due to its antioxidant
properties and its ability to enter mitochondria [24–
28]. In muscular tissues such as skeletal muscle and
diaphragm of septic mice, aMT administration inhi-
bited the activity of i-mtNOS, restoring the mito-
chondrial GSH pool and the ETC activity in these
animals [9,12,29].
Mitochondrial dysfunction is an important patho-
physiologic event related to heart failure during sepsis,
and i-mtNOS may be directly related to it. To address
this question, we induced sepsis by CLP in iNOS
+ ⁄ +
and iNOS
– ⁄ –
mice, and explored in heart mitochon-
dria: (a) the presence and source of i-mtNOS; (b) the
relationship between i-mtNOS induction, ETC dys-
function, and oxidative phosphorylation activity; (c)
the steady-state energy and ATP production; and (d)
the protective effect of aMT against mitochondrial
damage produced during sepsis.
Results
Mitochondrial NOS activities
Figure 1 shows that heart mitochondria from
iNOS
+ ⁄ +
mice contain two mitochondrial NOS iso-
forms: a constitutive, Ca
2+

-dependent form (mtNOS),
and an inducible, Ca
2+
-independent form (i-mtNOS).
In iNOS
+ ⁄ +
mice, sepsis induced a significant increase
in i-mtNOS activity, whereas mtNOS activity remained
unchanged (Fig. 1A). Control iNOS
– ⁄ –
mice exhibited
only the constitutive component of mitochondrial
NOS that was partially inhibited during sepsis
(Fig. 1B). aMT administration counteracted sepsis-
induced i-mtNOS activity in iNOS
+ ⁄ +
mice, without
affecting mtNOS activity (Fig. 1A). aMT also restored
mtNOS activity that had been depressed by sepsis in
mutant mice (Fig. 1B).
Some considerations should be borne in mind
regarding the purity of the mitochondrial preparation
used here. Heart mitochondria were isolated by differ-
ential centrifugation, and purified by Percoll centrifu-
gation [9,11,43]. To remove contaminants, purified
mitochondria were washed with high ionic strength
solution (150 mm KCl). This protocol yields a very
pure mitochondrial fraction without contaminating
organelles and broken mitochondria, as reported else-
where [11,12]. The lack of mitochondrial contamina-

tion with cytosolic NOS was assessed by the absence
of any detectable NOS activity and nitrite levels in the
supernatant of the final centrifugation step (data not
shown). These data confirm the purity of the mito-
chondria used in our experiments, and guarantee the
mitochondrial origin of the NOS activity reported
NOS and heart mitochondrial dysfunction in sepsis G. Escames et al.
2136 FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS
here. Moreover, the method used for NOS measure-
ment specifically detects mtNOS activity, and the addi-
tion of NG-monomethyl- l-arginine (l-NMMA) (300 lm)
to the reaction mixture of mitochondrial samples from
septic mice blocked the transformation of l-arginine to
l-citrulline, due to mtNOS inhibition (14.67 ± 3.09
versus 1.09 ± 0.87 pmol citrullineÆmin
)1
Æmg
)1
protein,
CLP and CLP + l-NMMA, respectively) [9,11,12].
iNOS
+ ⁄ +
mice exhibited a slight increase in nitrite
level after sepsis, which was counteracted by aMT
treatment (Table 1). Interestingly, iNOS
– ⁄ –
mice showed
a significant decrease in nitrite level during sepsis, coin-
ciding with the mtNOS activity inhibition, that was
partially counteracted by aMT.

iNOS mRNA expression
Figure 2 shows the results obtained in quantitative
RT-PCR experiments. Because this is a semiquantita-
tive technique, the data are expressed as the relative
quantity of mRNA in experimental versus control
samples, giving to the control samples a value ¼ 1
after deducting basal and background values. Sepsis
resulted in a significant increase in the transcription of
the mRNA encoding iNOS in heart of iNOS
+ ⁄ +
mice.
The expression of iNOS mRNA was not detected in
iNOS
– ⁄ –
mice. Treatment with aMT absolutely coun-
teracted the transcription of iNOS mRNA induced by
sepsis.
Mitochondrial oxidative stress
Sepsis significantly increased LPO levels in heart mito-
chondria from iNOS
+ ⁄ +
mice, whereas aMT admin-
istration reduced LPO below the control values
(Table 1). Sepsis, however, did not modify the levels of
LPO in iNOS
– ⁄ –
mice, although aMT also reduced
them below control values.
Fig. 1. Total heart mitochondrial NOS activ-
ity comprises constitutive, Ca

2+
-dependent,
and inducible, Ca
2+
-independent, compo-
nents in iNOS
+ ⁄ +
mice (A). Deficient iNOS
mice, however, show only the constitutive,
Ca
2+
-dependent component (B). In both
cases, mice were subjected to CLP to
induce sepsis, and killed 24 h later. Pure
mitochondrial preparations were used to
determine NOS activity with
L-[
3
H]arginine
as substrate. Data represent the means
± SE of six experiments per group. C, con-
trol; S, sepsis; S + aMT, sepsis + aMT.
*P<0.05, **P<0.01, ***P<0.001 versus
C;
#
P<0.05,
###
P<0.001 versus S.
Table 1. Effects of sepsis and aMT treatment on the mitochondrial nitrite and LPO levels, and on the activity of the mitochondrial ATPase in
wild-type and iNOS knockout mice. C, control; S, sepsis; S + aMT, sepsis + aMT. Sepsis was induced by CLP, and the animals were killed

24 h later. Nitrite, nmolÆmg protein
)1
; LPO, nmolÆmg protein
)1
; ATPase, nmol P
i
Æmin
)1
Æmg protein
)1
. Data are means ± SE, n ¼ 6.
iNOS
+ ⁄ +
iNOS
– ⁄ –
C S S + aMT C S S + aMT
Nitrite 3.07 ± 0.27 3.23 ± 0.31 2.63 ± 0.29 3.05 ± 0.24 2.23 ± 0.34* 2.48 ± 0.15
LPO 2.33 ± 0.1 2.94 ± 0.1* 2.05 ± 0.09*
,
 2.53 ± 0.12 2.48 ± 0.9 2.13 ± 0.05
ATPase 2641 ± 169 2753 ± 188 1430 ± 97 2280 ± 201 2452 ± 134 2205 ± 142
*P<0.05 versus C; P<0.05 versus S.
G. Escames et al. NOS and heart mitochondrial dysfunction in sepsis
FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2137
Figure 3A shows that glutathione peroxidase (GPx)
activity increased in heart mitochondria of iNOS
+ ⁄ +
mice after sepsis, and this increase was preserved after
aMT administration. In these animals, mitochondrial
glutathione reductase (GRd) activity decreased during

sepsis, whereas aMT treatment increased it to above
control values (Fig. 3B). Heart mitochondria from
iNOS
– ⁄ –
mice did not show changes in GPx and GRd
activities with any treatment (Fig. 3). Basal GPx activ-
ity was lower in iNOS
– ⁄ –
than in iNOS
+ ⁄ +
mice
(Fig. 3A).
The mitochondrial level of GSH decreased and that
of GSSG increased in hearts from iNOS
+ ⁄ +
mice after
CLP (Fig. 4A,B), raising the GSSG ⁄ GSH ratio
(Fig. 4C). Sepsis also reduced total glutathione levels in
iNOS
+ ⁄ +
mice (Fig. 4D). Treatment with aMT
increased GSH levels and reduced GSSG levels in
iNOS
+ ⁄ +
mice, normalizing the GSSG ⁄ GSH ratio
(Fig. 4A–C). aMT also increased the total glutathione
pool in this mouse strain (Fig. 4D). No changes in glu-
tathione levels were found in heart mitochondria
of iNOS
– ⁄ –

mice under any experimental conditions
(Fig. 4A–D).
ETC complexes and ATPase activities
Figure 5A–D shows that, after CLP, the activity of the
four ECT complexes was significantly reduced in
iNOS
+ ⁄ +
mice. aMT administration increased the
activity of these complexes above the control values
(Fig. 3A–D). The activity of the ETC complexes in
heart mitochondria from iNOS
– ⁄ –
mice was unaffected
by sepsis. The basal activities of complex I and com-
plex II were significantly lower, and those of com-
plex III and IV were significantly higher, in iNOS
– ⁄ –
Fig. 2. Effects of aMT treatment on CLP-induced mRNA levels
of iNOS. Ten nanograms of RNA extracted from mouse heart
was used, and quantification of iNOS mRNA was performed by
real-time RT-PCR. The relative level was calculated as
the ratio of inflammatory mRNA expression to b-actin mRNA
expression. ***P<0.001 versus C;
###
P<0.0001 versus S. Each
value represents the mean ± SE for three independent experi-
ments.
Fig. 3. Changes in heart mitochondrial GPx
(A) and GRd (B) activities after sepsis and
aMT treatment in iNOS

+ ⁄ +
and iNOS
– ⁄ –
mice. Data represent the means ± SE of six
experiments per group. C, control; S, sep-
sis; S + aMT, sepsis + aMT. *P<0.05 and
**P<0.01 versus C;
##
P<0.005 versus S;
+
P<0.05 versus iNOS
+ ⁄ +
mice.
NOS and heart mitochondrial dysfunction in sepsis G. Escames et al.
2138 FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS
than in iNOS
+ ⁄ +
mice (Fig. 5A–D). The activity of
the ETC complexes was also unchanged by aMT treat-
ment in iNOS
– ⁄ –
mice. No changes in ATPase activity
were observed in any mouse strain under sepsis, and
aMT treatment only slightly decreased ATPase activity
in iNOS
+ ⁄ +
mice (Table 1).
Fig. 4. GSH level (A), GSSG level (B),
GSSG ⁄ GSH ratio (C) and GSH + GSSG level
(D) in heart mitochondria of iNOS

+ ⁄ +
and
iNOS
– ⁄ –
mice. Data represent the
mean ± SE of six experiments per group. C,
control; S, sepsis; S + aMT, sepsis + aMT.
**P<0.01 versus C;
#
P<0.05 versus S.
Fig. 5. Complex I (A), II (B), III (C) and IV (D) activities in heart mitochondria of iNOS
+ ⁄ +
and iNOS
– ⁄ –
mice. Data represent the mean ± SE
of six experiments per group. C, control; S, sepsis; S + aMT, sepsis + aMT. *P<0.05 and **P<0.01 versus C;
#
P<0.05,
#
P<0.05,
##
P<0.01 and
###
P<0.001 versus S;
+
P<0.05,
++
P<0.01 and
+++
P<0.001 versus iNOS

+ ⁄ +
mice.
G. Escames et al. NOS and heart mitochondrial dysfunction in sepsis
FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2139
Mitochondrial ATP production
To assess whether sepsis modifies the bioenergetic sta-
tus of heart mitochondria, ATP production was deter-
mined. ATP production was significantly reduced in
iNOS
+ ⁄ +
but not in iNOS
– ⁄ –
mice during sepsis,
whereas aMT administration restored the ability of
mitochondria to produce ATP in the former (Fig. 6).
After the ATP production assay, the amount of AMP
in the samples was less that 3% of the total nucleo-
tides, discounting extramitochondrial ATP production
by adenylate kinase in our assays. The experimental
procedure used here allowed us to detect ATP inside
(pellet, fraction p2) and outside (supernatant, frac-
tion s1) the mitochondria. The results indicated that
92–98% of the ATP produced was detected outside the
mitochondria.
Animal survival
To determine the mortality of CLP-induced sepsis in
our experimental paradigm, and to assess whether the
improvement in mitochondrial function after aMT
treatment was followed by a reduction in mortality,
mice survival was analyzed. Figure 7 shows the survi-

val curves obtained from untreated and aMT-treated
septic mice. The half-life of iNOS
+ ⁄ +
animals with
sepsis was 26.5 h, increasing up to 35 h when they
were treated with aMT. Moreover, there was 100%
mortality at 32.5 h in septic mice, whereas aMT treat-
ment increased survival up to 50 h.
A significant improvement in survival was observed
in iNOS
– ⁄ –
mice (Fig. 7). Untreated animals with sep-
sis showed a half-life of 67.5 h, and aMT treatment
increased it up to 129.5 h. Also, there was 100% mor-
tality at 90 h in septic mice, whereas aMT administra-
tion increased this time up to 150 h.
Discussion
The results of this study demonstrate the presence of
two NOS isoforms with constitutive and inducible kin-
etic properties in heart mitochondria. During inflam-
mation, i-mtNOS activity is increased, but not that of
mtNOS. The induction of i-mtNOS depends on iNOS
expression, because mitochondria from iNOS
– ⁄ –
mice
lack i-mtNOS. These data, and the results obtained
from iNOS mRNA expression, suggest that i-mtNOS
found in heart mitochondria derives from the cytosolic
iNOS and is encoded by the same gene. Sepsis was
also accompanied by increased oxidative stress and

inhibition of the ETC complexes, leading to a reduction
in ATP. Because heart mitochondria from iNOS
– ⁄ –
Fig. 6. Changes in mitochondrial ATP production in heart of
iNOS
+ ⁄ +
and iNOS
– ⁄ –
mice, using succinate as substrate. Data rep-
resent the mean ± SE of six experiments per group. C, control;
S, sepsis; S + aMT, sepsis + aMT. *P < 0.05 and **P<0.01
versus C;
##
P<0.01 versus S.
Fig. 7. Survival curves obtained from untreated and aMT-treated
septic iNOS
+ ⁄ +
and iNOS
– ⁄ –
mice. The total number of animals
used in this study was 20 in each group.
NOS and heart mitochondrial dysfunction in sepsis G. Escames et al.
2140 FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS
mice were unaffected by endotoxemia and they do not
express i-mtNOS, mitochondrial impairment during
sepsis was probably related to i-mtNOS induction in
iNOS
+ ⁄ +
mice. aMT treatment counteracted sepsis-
induced iNOS mRNA expression, a finding related to

the reduction of i-mtNOS. aMT also prevented mito-
chondrial dysfunction, increasing ATP production, and
the survival of septic mice.
Since the discovery of NOS activity in the mitochon-
dria [16,24,30], several reports have shown the presence
of this enzyme in different tissues with properties of
endothelial NOS, nNOS, and ⁄ or iNOS [14,31,32]. In
different models of sepsis and tissues, the existence of
both mtNOS and i-mtNOS isoforms has been reported
[9,11,12]. The constitutive isoform was identified in
liver mitochondria as an nNOS isoform that was
post-translationally modified [11,17,33]. Recent reports
also support the existence of i-mtNOS in mitochondria
from different tissues [9,11–13,15]. Whereas the ab-
sence of mtNOS in nNOS knockout mice suggested
that mtNOS is derived from cytosolic nNOS [16], the
absence of i-mtNOS in iNOS knockout mice supported
its relationship with cytosolic iNOS [9,12].
NO is particularly important in the regulation of
cardiac function [10]. It is involved in vascular and
nonvascular effects, including regulation of cardiomyo-
cyte contractility, in which mitochondrial respiration
and bioenergetics play an important role [15]. Produc-
tion of excessive quantities of NO leads to profound
cellular disturbances and myocardial dysfunction
[8,15,18,34]. Mitochondrial dysfunction is a conse-
quence of inflammation [6], and the induction of
i-mtNOS in heart mitochondria may be responsible for
mitochondrial failure during sepsis [9,11,12]. The exist-
ence of an iNOS isoform was also recently confirmed

in heart mitochondria from rats [13]. Normally, the
induction of i-mtNOS produces a significant increase
in NO and nitrite [9,11,12]. The lack of a significant
increase in nitrite in iNOS
+ ⁄ +
mice after sepsis repor-
ted here could be explained by two main mechanisms.
In mitochondria, the major oxidative decay pathway
of NO is its reaction with O
2
–
to form ONOO
–
[20]. In
turn, ONOO
–
reacts with a variety of biomolecules
[35]. Moreover, ONOO
–
can react with H4-biopterin
(BH), a cofactor necessary for NO synthesis by NOS,
leading to formation of the BH
3
radical [36], and caus-
ing NOS inactivation [37,38]. An alternative explan-
ation for the lack of changes in nitrite under sepsis is
the presence of an NOS-independent NO source in
mitochondria. Alterations in the redox state of the
ETC lead to the formation of reactive nitrogen species,
including NO and ONOO

–
, and thus to nitrite [39]. In
turn, mitochondrial nitrite reductase can recycle NO
from nitrite, masking the nitrite increase during sepsis
[40]. The existence of elevated nitrite levels in mito-
chondria from other tissues under conditions of sepsis
[9,11,12] suggests that the presence of a nitrate reduc-
tase with higher activity in heart mitochondria than in
the other tissues could explain the lack of changes in
nitrite reported here. However, differences in the relat-
ive activities of nitrate reductases in mitochondria
from different tissues have not yet been found.
The reactive species produced as a consequence of
i-mtNOS induction during sepsis are highly toxic, and
they can impair the mitochondrial ETC and oxidative
phosphorylation [19–21]. Our results show a significant
inhibition of the four complexes of the respiratory chain
in septic iNOS
+ ⁄ +
mice. Similar results were reported
for other tissues [5,7–9,11,12]. However, septic iNOS
– ⁄ –
mice did not show alterations in ECT activity during
sepsis, suggesting that the oxidative ⁄ nitrosative stress
derived from i-mtNOS induction is responsible for ETC
dysfunction in inflammation. Unlike the situation with
ETC complexes, our results do not show changes in
ATPase activity in septic mice. It was recently shown
that the ETC complexes, but not ATPase, are damaged
during the early and acute phases of Chagasic cardiomy-

opathy [41]. In these phases, the innate inflammatory
response corresponds with iNOS induction and a subse-
quent increase in NO. These results suggest that ATPase
is more resistant to oxidative ⁄ nitrosative stress than
ETC complexes, probably because ETC complexes,
unlike ATPase, have redox centers such as Fe–S that are
very sensitive to NO [42].
ETC coupled with oxidative phosphorylation is
responsible for the production of 90–95% of the total
ATP synthesized in the cell [26]. Thus, ETC damage
may alter the synthesis of ATP without any effect on
ATPase. Our results show a reduced ability of the
mitochondria to produce ATP during sepsis, which
may reduce cardiomyocyte contractility [43,44]. Be-
cause the activity of the respiratory complexes was not
affected in septic iNOS
– ⁄ –
mice, ATP production by
heart mitochondria was not altered in this mouse
strain. Thus, the reduction in the production of ATP
found in diaphragm and heart after endotoxin admin-
istration [5,22,45] probably reflects mitochondrial
impairment due to i-mtNOS induction by the toxin.
Heart mitochondria from iNOS
– ⁄ –
mice show lower
complex I and II activities and higher complex III and
IV activities than iNOS
+ ⁄ +
mice, a finding also des-

cribed in diaphragmatic and skeletal muscle mitochon-
dria [9,12]. Whereas the latter was attributed to the
lack of the inhibitory effect of the NO derived from
i-mtNOS, which is absent in iNOS
– ⁄ –
mice, the reasons
for the former phenomenon remains unclear [9,12]. In
G. Escames et al. NOS and heart mitochondrial dysfunction in sepsis
FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2141
any case, the data regarding ATP production suggest
that the low activities of complex I and II in iNOS
– ⁄ –
mice are compensated by the higher activities of com-
plex III and IV, allowing normal mitochondrial
homeostasis.
Mitochondrial ETC impairment leads to electron
leakage and formation of O
2
–
through the partial reduc-
tion of oxygen by one electron. Subsequent reduction by
one or two electrons can yield H
2
O
2
, and HO, respect-
ively [46]. However, the main source of mitochondrial
H
2
O

2
is superoxide dismutase activity [47], whereas HO
can be derived from H
2
O
2
and ONOO
–
decomposition,
although the latter is a minor process [35]. The small
effect on complex I compared with the strong inhibition
of complex III produced during sepsis in iNOS
+ ⁄ +
mice suggests that the latter was the most important
source of free radicals in our experimental model. NO
and O
2
–
react to produce ONOO
–
in mitochondria
[7,19,20], increasing ETC damage [19,20] and LPO
activity [48]. Besides causing direct oxidative damage,
ONOO
–
can produce nitration, and to a lesser extent ni-
trosation, of mitochondrial components indirectly [35].
The free radical pathways of ONOO
–
are mainly initi-

ated secondary to the reaction of ONOO
–
with CO
2
,
leading to the rapid formation of carbonate and nitro-
gen dioxide radicals. The ONOO
–
⁄ CO
2
pathway
becomes highly relevant in mitochondria, as these are
the main organelles in which CO
2
is produced, due to
the decarboxylation reactions. Although ONOO
–
can
yield HO
•
, it is a rather minor pathway in mitochondria,
as most ONOO
–
will react directly with either target or
CO
2
. In any case, ONOO
–
promotes, to some degree,
mitochondrial LPO activity; this could be initiated by

nitrogen dioxide and HO
•
radicals. Carbonate radicals,
however, are poor direct initiators of LPO, due to their
negative charge, which limits their diffusion to the
hydrophobic domains of membrane phospholipids [35].
These mechanisms explain the LPO increase found in
heart mitochondria from septic iNOS
+ ⁄ +
mice, a find-
ing related to the i-mtNOS induction, because iNOS
– ⁄ –
mice did not show changes in LPO levels.
Mitochondrial GPx activity increased in iNOS
+ ⁄ +
mice, reflecting a compensatory mechanism to reduce
oxidative stress during sepsis. However, the
GSSG ⁄ GSH ratio remains elevated under these condi-
tions, because the reduced activity of GRd, probably
due to oxidative damage [53], prevents the recovery of
GSH from GSSG. Moreover, total glutathione levels
in these mitochondria were also reduced, probably
reflecting inhibition of GSH transport into the mito-
chondria [49]. The lack of mitochondrial oxidative
stress and the presence of a normal GSH pool in septic
iNOS
– ⁄ –
mice further support the idea that i-mtNOS
induction is the main event related to oxidative stress
during sepsis in heart mitochondria.

Different types of antioxidants with beneficial effects
against mitochondrial oxidative stress have been pro-
posed [23]. One of these molecules is aMT, an
indoleamine with excellent antioxidant and anti-
inflammatory properties in the cell and mitochondria
[24,27,28,50,51]. First, aMT inhibits the expression and
activity of both cytosolic iNOS and mitochondrial
i-mtNOS in septic rats and mice [11,29,37], allowing
the animals to recover from multiorgan failure. Sec-
ond, aMT directly scavenges reactive oxygen species
and reactive nitrogen species [24,26–28,52], induces the
expression of antioxidative enzymes [8], and restores
mitochondrial GSH homeostasis [53]. Third, aMT
increases the activity of the ETC and ATP production
in vitro and in vivo [54,55]. Our results demonstrate
these protective effects of aMT in heart mitochondria
of septic iNOS
+ ⁄ +
mice. aMT treatment counteracted
iNOS expression, reducing the activity of i-mtNOS,
increased the activity of the ETC complexes over the
control values, and normalized the levels of LPO.
Reducing the levels of free radicals with aMT prevents
them from causing oxidative damage to GRd [53], nor-
malizing the GSSG ⁄ GSH ratio. Low oxidative status
also prevents the mitochondrial transition pore open-
ing and uncoupling, a condition associated with ATP
hydrolysis [27,56,57]. In fact, aMT restored the ability
of heart mitochondria to produce ATP. In these
circumstances, cardiomyocytes could have enough

energy for muscle contraction, avoiding myocardial
dysfunction, and probably heart failure, in sepsis.
The significant increase in survival of mice treated
with aMT further supports this observation. It is inte-
resting that aMT had minor effects on heart mito-
chondria from iNOS
– ⁄ –
mice. As reported in other
pathophysiologic conditions, it seems that aMT can
upregulate mitochondrial function when it is impaired
[9,11,12,24,49,53,55], but the indoleamine had minor
effects under normal conditions. The half-life and
maximum survival time of septic iNOS
– ⁄ –
mice were
significantly higher than those of wild-type animals.
Moreover, aMT treatment significantly increased the
half-life and maximum survival time of mutant mice.
Because iNOS
– ⁄ –
mice with sepsis did not show signifi-
cant mitochondrial damage, they probably died by a
mechanism different from iNOS-dependent dysfunc-
tion, such as cyclooxygenase-2 induction. Because
aMT treatment had only minor effects on mitochon-
dria of iNOS
– ⁄ –
mice, the inhibitory effect of aMT on
cyclooxygenase-2 expression may explain the signifi-
cant improvement in survival of iNOS

– ⁄ –
mice. This
hypothesis, however, remains to be studied.
NOS and heart mitochondrial dysfunction in sepsis G. Escames et al.
2142 FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS
In summary, this study demonstrates that, besides
mtNOS, mouse heart mitochondria contain an
i-mtNOS isoform that is induced during sepsis. Among
other consequences, mitochondrial dysfunction, oxida-
tive stress and reduced ability to produce ATP follows
an increase in i-mtNOS. These alterations could con-
tribute to the myocardial dysfunction that often occurs
during sepsis. The parallel increases in iNOS expres-
sion and i-mtNOS activity, the inhibition of both
iNOS mRNA expression and i-mtNOS activity after
aMT treatment, and the lack of i-mtNOS expression in
mitochondria from iNOS
– ⁄ –
mice, suggest that the
enzyme is encoded by the same gene that encodes cyto-
solic iNOS. Moreover, the absence of any sign of mit-
ochondrial dysfunction and the lack of i-mtNOS
expression in iNOS
– ⁄ –
mice further support the role of
i-mtNOS in mitochondrial impairment during sepsis.
Administration of aMT to septic iNOS
+ ⁄ +
mice nor-
malized mitochondrial function, restoring their ability

to produce ATP, and increasing mice survival. These
properties, together with the prevention of endotoxin-
induced circulatory failure in rats [58,59], and the mor-
tality reduction in septic newborns after aMT therapy
[60], suggest that the use of the indoleamine in septic
patients should be seriously considered.
Experimental procedures
Chemicals
l-[2,3,4,5-
3
H]arginine monohydrochloride (58 CiÆmmol
)1
)
was obtained from Amersham Biosciences Europe GmBH
(Barcelona, Spain). Liquid scintillation cocktail (Ecolume)
was purchased from ICN (Madrid, Spain). All other
chemicals, of the purest available grade, were obtained from
Sigma-Aldrich (Madrid, Spain) unless otherwise specified.
Experimental animals
All procedures involving animals were carried out under an
approved protocol and in accordance with the Spanish
Government Guide and the European Community Guide
for animal care. iNOS knockout B6.129P2-Nos2
tm1Lau
mice
(iNOS
– ⁄ –
) and their respective wild-type control C57 ⁄ Bl ⁄ 6
mice (iNOS
+ ⁄ +

) were obtained from Jackson’s Laboratory
through Charles River Laboratories (Barcelona, Spain).
The animals were housed in the university’s facility with a
12 h : 12 h light ⁄ dark cycle (lights on at 07:00 h) at
22±2°C, and given regular chow and tap water. Both
iNOS
+ ⁄ +
and iNOS
– ⁄ –
mice 12–14 weeks of age were
grouped (n ¼ 18 animals ⁄ group) as follows: (a) control
group; (b) sepsis group; and (c) sepsis + aMT group.
Sepsis was induced by CLP [61] under intraperitoneal
equithesin anesthesia (1 mLÆkg
)1
). Four doses of aMT
(30 mgÆkg
)1
) were injected as follows: one dose 30 min
before surgery (intraperitoneal); the second dose just after
surgery (subcutaneous); and the remaining doses 4 h and
8 h after surgery (subcutaneous). Animals were killed 24 h
after CLP, except for the survival studies.
Preparation of cardiac mitochondria
Pure mitochondria were isolated from 12–14-week-old wild-
type and knockout mouse hearts by differential centrifuga-
tion and density gradient centrifugation with Percoll as
follows [9,11]. All procedures were carried out in the cold.
Briefly, cardiac muscle was excised, washed with saline,
treated with proteinase K (1 mgÆmL

)1
) for 30 s, washed
with buffer A (250 mm mannitol, 0.5 mm EGTA, 5 mm He-
pes, 0.1% BSA, pH 7.4, 4 °C), and homogenized (1 : 10,
w ⁄ v) in buffer A at 800 r.p.m. at 4 °C with a Teflon pestle.
The homogenate was aliquoted, and centrifuged at 600 g
for 5 min at 4 °C (twice) (rotor type F34-6-38 Eppendorf
5810R centrifuge), and the supernatants were centrifuged at
10 300 g for 10 min at 4 °C (rotor type F1255 Beckman
TL-100 centrifuge). Then, the mitochondrial pellets were
suspended in 0.5 mL of buffer A, and placed in ultracentri-
fuge tubes containing 1.4 mL of buffer B (225 mm manni-
tol, 1 mm EGTA, 25 mm Hepes, 0.1% BSA, pH 7.4, 4 °C)
and 0.6 mL of Percoll. The mixture was centrifuged at
105 000 g for 30 min at 4 °C (rotor type F1255 Beckman
TL-100 centrifuge). The fraction corresponding to a pure
mitochondrial fraction was collected, washed twice with
buffer A at 10 300 g for 10 min at 4 °C (rotor type F1255
Beckman TL-100 centrifuge) to remove the Percoll, and
washed again with a high ionic strength solution of KCl
(150 mm) to yield a highly pure mitochondrial preparation
without contaminating organelles and broken mitochondria
[28,50]. Aliquots of these pure mitochondrial fractions were
frozen to ) 80 °C. The purity of the mitochondrial prepara-
tions was assessed as described elsewhere [9,11].
Mitochondrial NOS activity measurement
Measurement of constitutive, Ca
2+
-dependent, and indu-
cible, Ca

2+
-independent, mitochondrial NOS activities was
done as previously described [11,62]. Briefly, an aliquot of
frozen mitochondria was thawed and homogenized (0.1
gÆmL
)1
)in25mm Tris buffer (pH 7.6) containing 0.5 mm
dithiothreitol, 10 lgÆmL
)1
pepstatin, 10 lgÆmL
)1
leupeptin,
10 lgÆmL
)1
aprotinin and 1 mm phenylmethanesulfonyl
fluoride at 4 °C (rotor type F34-6-38 Eppendorf 5810R cen-
trifuge). The homogenate was centrifuged at 2500 g for
5 min at 4 °C, and the supernatant was used immediately
for determination of NOS activity; one aliquot was frozen
at ) 80 °C for protein determination [63]. Ten microliters of
the supernatant (2 mgÆmL
)1
protein) were added to the
G. Escames et al. NOS and heart mitochondrial dysfunction in sepsis
FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS 2143
incubation mixture (100 lL, final volume) prewarmed at
37 °C, and containing (final concentration) 25 mm Tris,
1mm dithiothreitol, 30 lm H
4
-biopterin, 10 lm FAD,

0.5 mm inosine, 0.5 mgÆmL
)1
BSA, 0.1 mm CaCl
2
,10lm
l-arginine, and 40 nml-[
3
H]arginine (pH 7.6). The reaction
was started by the addition of 10 l L of NADPH (0.75 mm
final concentration), and continued for 30 min at 37 °C. To
determinate the Ca
2+
-independent activity of NOS
(i-mtNOS), 10 mm EDTA was added to the buffer before
the reaction was started. Control incubations were per-
formed in the absence of NADPH. The reaction was
stopped by adding 400 lL of cold 0.1 m Hepes buffer con-
taining 10 mm EGTA and 1 mml-citrulline (pH 5.5). The
mixture was decanted onto a 2 mL column packed with
Dowex-50W ion exchange resin (Na
+
form), and eluted
with 1.2 mL of water. l-[
3
H]Citrulline was quantified
by liquid scintillation spectroscopy. The retention
of l-[
3
H]arginine by the column was greater than 98%.
Enzyme activity was determined as pmoL l-[

3
H]citrul-
lineÆmin
)1
Æmg protein
)1
.
Real-time quantitative RT-PCR assay of iNOS
mRNA expression
Quantification of the iNOS mRNA levels was done by
SYBR green two-step real-time RT-PCR (Stratagene Mx
3005P; Stratagene, La Jolla, CA, USA). Total cellular
RNA was isolated from the heart using the RNA isolation
kit Real Total RNA Spin Plus (Durviz, S.L., Valencia,
Spain). Ten nanograms of the total RNA extracted was
used. Gene-specific primers for iNOS (forward primer, 5¢-
AGACGGATAGGCAGAGATTGG-3¢, and reverse pri-
mer, 5¢-ACTGACACTTCGCACAAAGC-3¢) and b-actin
(forward primer, 5¢-GCTGTCCCTGTATGCCTCTG-3¢,
and reverse primer, 5¢-CGCTCGTTGCCAATAGTGA
TG-3¢) were designed using the beacon designer software
(Premier Biosoft Int., Palo Alto, CA, USA) and obtained
from Thermo Electron GmbH (Ulm, Germany). Real-time
PCR reactions were carried out in a final volume of 25 lL
of reaction mixture containing 10 ng of RNA, 12.5 lLof
2X SYBR Green Master Mix (Stratagene), 75 nm each spe-
cific gene primer, and H
2
O. The samples were run in tripli-
cate in the amplification program, and the mean value was

used as the final expression value. A negative control with-
out RNA template was run. The PCR program was initi-
ated by 10 min at 95 °C before 40 thermal cycles, each of
30 s at 95 °C and 1 min at 55 °C. Data were analyzed
according to the relative standard curve method, construc-
ted with triplicate serial dilutions (50, 5, 0.5 and 0.05 ng),
and were normalized by b-actin expression.
Nitrite determination
Mitochondrial fractions were thawed and suspended in ice-
cold distilled water, and immediately sonicated to break the
mitochondrial membranes. Aliquots of these samples were
used to calculate nitrite levels following the Griess reaction
[64], and expressed in nmoL nitriteÆmg protein
)1
.
Determination of mitochondrial function
The activities of the four respiratory complexes were deter-
mined as previously described [65,66], with slight modifica-
tions [9,12], and expressed as nmoLÆmin
)1
Æmg protein
)1
.
Complex V (ATP synthase) activity was measured by
following the rate of hydrolysis of ATP to ADP + P
i
.
Ferrous sulfate ⁄ ammonium molybdate reagent was utilized
to determinate P
i

concentration [67]. ATPase activity was
expressed in nmoL P
i
Æmin
)1
Æmg protein
)1
.
Determination of ATP production
For the determination of ATP production, hearts were
excised, washed with saline, treated with proteinase K
(1 mgÆmL
)1
) for 30 s, washed with buffer A (220 mm
mannitol, 70 mm sucrose, 1 mm EGTA, 20 mm Hepes, 1%
BSA, pH 7.2, 4 °C), and homogenized (1 : 10, w ⁄ v) in buf-
fer A at 800 r.p.m. at 4 °C with a Teflon pestle. The homo-
genates were centrifuged at 1500 g for 5 min at 4 °C (rotor
type F1255 Beckman TL-100 centrifuge), and the superna-
tants were centrifuged again at 23 000 g for 5 min at 4 °C
(rotor type F1255 Beckman TL-100 centrifuge). Then, the
mitochondrial pellets were suspended in 1 mL of buffer A,
and centrifuged at 10 300 g for 3 min at 4 °C (rotor type
F1255 Beckman TL-100 centrifuge). The resultant pellets
(p1) were suspended in respiration buffer (225 mm manni-
tol, 75 mm sucrose, 10 mm KCl, 10 mm Tris ⁄ HCl, 5 mm
potassium phosphate, pH 7.2, saturated with O
2
, plus 5 mm
succinate, 30 °C), and ATP production was induced adding

125 nmol of ADP. After 45 s, the sample was centrifuged
at 13 000 g for 3 min at 2 °C (rotor type F1255 Beckman
TL-100 centrifuge) [68,69], and the ATP content in the pel-
let (p2) and supernatant (s1) was measured. Ice-cold 0.5 m
perchloric acid was rapidly added to the p1, p2 and s1 frac-
tions, mixed for 2 min in a vortex mixer, and centrifuged at
25 000 g for 15 min at 2 °C (rotor type F1255 Beckman
TL-100 centrifuge) to precipitate proteins. The pellets were
frozen to ) 80 °C for protein determination [63]; the sup-
ernatants were mixed with 8 lLof5m potassium carbon-
ate to neutralize the pH, and centrifuged at 12 000 g for
10 min at 2 °C (rotor type F1255 Beckman TL-100 centri-
fuge). ATP was measured in the resultant supernatants by
HPLC with a 4 · 250 mm ProPac PA1 column (Dionex,
Barcelona, Spain) [70]. After stabilization of the column
with the mobile phase, samples (20 lL) were injected onto
the HPLC system. The mobile phase consisted of water
(phase A) and 0.3 m ammonium carbonate (pH 8.9) (pha-
se B), and the following time schedule for the binary gradi-
ent (flow rate 1 mLÆmin
)1
) was used: 5 min, 50% A and
50% B; 5 min, 50% to 100% B, and then 100% B for
NOS and heart mitochondrial dysfunction in sepsis G. Escames et al.
2144 FEBS Journal 274 (2007) 2135–2147 ª 2007 The Authors Journal compilation ª 2007 FEBS
25 min; 5 min, 100% to 50% B, and then another 5 min
with 50% B [71]. Water was used for calibration purposes.
A standard curve was constructed with 3.125 lgÆmL
)1
,

6.250 lgÆmL
)1
,12.5lgÆmL
)1
and 25 lgÆmL
)1
of ATP. The
absorbance of the samples was measured with a UV detec-
tor at 254 nm, and the concentration of each nucleotide in
the samples was calculated according to the peak area [70].
ATP production was expressed in lgÆmin
)1
Æmg protein
)1
.
Determination of mitochondrial oxidative stress
For LPO measurement (expressed in nmolÆmg protein
)1
),
mitochondrial fractions were thawed and sonicated in ice-
cold 20 mm Tris ⁄ HCl buffer (pH 7.6) to break the mitoch-
ondrial membranes. Aliquots of these samples were either
stored at ) 80 ° C for total protein determination [63], or
used for malondialdehyde plus 4-hydroxyalkenal determin-
ation as an index of LPO (Bioxytech LPO-568 assay kit;
OxisResearch, Portland, OR, USA) [72]. GSSG and GSH
were measured by fluorescence [73], and expressed in
nmoLÆmg protein
)1
. For GPx and GRd activity determin-

ation, aliquots of the mitochondrial fraction were suspen-
ded in 200 lLof50mm potassium phosphate buffer
containing 1 mm EDTA-K
2
(pH 7.4), and the oxidation of
NADPH was spectrophotometrically measured for 3 min at
340 nm [74]. The activity of GPx and GRd was expressed
in nmolÆmin
)1
Æmg protein
)1
.
Statistical analysis
Data are expressed as means ± SEM. Significance was
determined using two-way ANOVA followed by Dunnet’s
post hoc test, when appropriate. The level of statistical sig-
nificance was taken as P < 0.05.
Acknowledgements
This study was partially supported by grants FIS01⁄
1076, PI03 ⁄ 0817 and G03 ⁄ 137 from the Instituto de
Salud Carlos III, and Consejerı
´
a de Educacio
´
n, Junta
de Andalucı
´
a (CTS-101). L. C. Lo
´
pez is an FPI fellow

from the Ministerio de Educacio
´
n (Spain), and
F. Ortiz and A. Lo
´
pez are predoctoral fellows from
the Instituto de Salud Carlos III (Spain).
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