Download Time course of differential mitochondrial energy metabolism

Cardiovascular Research 66 (2005) 132 – 140
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Time course of differential mitochondrial energy metabolism
adaptation to chronic hypoxia in right and left ventricles
Karine Nouette-Gaulaina,c, Monique Malgata,c, Christophe Rochera,c, Jean-Pierre Savineaub,c,
Roger Marthanb,c, Jean-Pierre Mazata,c, Francois Sztarka,c,*
a
Laboratoire d’anesthésiologie, E.A. Physiologie Mitochondriale, Université Victor Segalen Bordeaux 2, 33076 Bordeaux Cedex, France
b
Laboratoire de Physiologie Cellulaire Respiratoire, Institut National de la Santé et de la Recherche Médicale (Inserm) E 356, France
c
Institut Fédératif de Recherche (IFR) n8 4, Université Victor Segalen Bordeaux 2, 33076 Bordeaux Cedex, France
Received 22 July 2004; received in revised form 8 December 2004; accepted 27 December 2004
Time for primary review 19 days
Abstract
Objective: The present study was designed to characterize mitochondrial adaptation to chronic hypoxia (CH) in the rat heart.
Mitochondrial energy metabolism was differentially examined in both left and right ventricles since CH selectively triggers pulmonary
hypertension and right ventricular hypertrophy.
Methods: Rats were exposed to a hypobaric environment for 2 or 3 weeks and compared with rats maintained in a normoxic environment.
Oxidative capacity (oxygen consumption and ATP synthesis) was measured in saponin-skinned fibers with glutamate or palmitoyl
carnitine as substrates. Enzymatic activities of mitochondrial respiratory chain complexes were measured on tissue homogenates.
Morphometric analysis of mitochondria was performed on electron micrographs. Mitochondrial DNA was quantified using Southern blot
analysis.
Results: Whereas oxidative capacity of both ventricles was decreased following 21 days of CH, oxygen consumption and ATP synthesis
was maintained with the glutamate substrate in the right ventricle following 14 days of CH. As for the oxidative capacity, enzyme
activities were decreased only in the left ventricle following 14 days of CH and in both ventricles following 21 days of CH. These
functional alterations were associated with an increase in numerical density and a decrease in size of mitochondria without a change in
volume density in both ventricles. Finally, 21 days of CH also decreased the ratio of mitochondrial DNA to nuclear DNA in both
ventricles.
Conclusions: CH alters morphometry and function of mitochondria in the heart, but this effect is delayed in the right compared to the left
ventricle, suggesting some adaptive processes at the onset of right ventricular hypertrophy.
D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Chronic hypoxia; Energy metabolism; Mitochondria; Oxidative phosphorylation
1. Introduction
Chronic hypoxia (CH) occurs under either physiologic
(high altitude) or pathologic conditions (e.g., chronic
pulmonary diseases). Previous studies in animals and
* Corresponding author. Laboratoire d’anesthésiologie, E.A. Physiologie
Mitochondriale, Universite Bordeaux 2, 146, Rue Leo Siagnat, 33076
Bordordeaux, France. Tel.: +33 5 56795514; fax: +33 5 56796119.
E-mail address: [email protected] (F. Sztark).
humans have shown complex mechanisms of metabolic
adaptation during exposure to CH [1]. One of these initial
adaptive mechanisms is a marked suppression of adenosine
5V-triphosphate (ATP) demand and supply pathways [2].
This regulation allows ATP level to remain constant, even
while ATP turnover rate greatly declines. Thus, many ATPdependent processes, like ion pumping or protein synthesis,
are down-regulated during exposure to CH [3]. Whereas CH
is characterized by a reduction in oxygen supply, it
selectively induces a chronic functional overload of the
0008-6363/$ - see front matter D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2004.12.023
K. Nouette-Gaulain et al. / Cardiovascular Research 66 (2005) 132–140
right ventricle (RV) due to pulmonary hypertension that
requires additional energy to overcome this increase in
pulmonary vascular resistance.
In the heart, mitochondria provide, through oxidative
phosphorylation, more than 95% of the energy supply in the
form of ATP. In the course of oxidative phosphorylation,
electrons are transferred through the respiratory enzymatic
complexes of the mitochondrial inner membrane, thus
releasing energy used to generate a proton gradient across
the membrane. This protonmotive force allows the synthesis
of ATP from adenosine 5V-diphosphate (ADP) and phosphate by the ATP-synthase. Mitochondrial metabolism is
involved in CH adaptation via energy regulation, generation
of reactive oxygen species, and apoptosis [4–7]. Most of the
studies have examined the global cardiac response to CH
despite the selective effect of CH on the RV. In this
connection, few recent data from our, as well as other,
laboratories have indicated that the adaptive mechanism to
CH was different in left ventricle (LV) and RV. LV appeared
very sensitive to CH and exhibited a rapid decrease in the
oxidative capacity, whereas RV appeared not affected [8,9].
To examine whether ventricular hypertrophy was responsible for the maintained oxidative capacity of RV, we have
compared mitochondrial energy metabolism adaptation to
CH in RV and LV in the rat heart over an extended period of
time of 21 days. Indeed, regarding the time course of CHinduced pulmonary hypertension, we have previously
observed that RV hypertrophy increases linearly with the
duration of exposure to CH from 1 to 3 weeks and then
remains stable up to 4 weeks [10]. Our results indicate that
CH decreases ATP synthesis as a consequence of an
alteration in mitochondrial respiratory chain complexes in
both ventricles but that this effect is delayed in the RV
suggesting some specific, but transient, adaptive processes
at the onset of right ventricle hypertrophy.
2. Materials and method
2.1. Chronic hypoxia
Adult male Wistar rats (aged 8–10 wk, weighing 220–
240 g) were separated into two groups. One group (hypoxic
rats) was exposed to a simulated altitude of 5000 m
(barometric pressure 380 mmHg) in a well-ventilated,
temperature-controlled hypobaric chamber for 14 or 21
days. The chamber was opened twice a week for few
minutes to clean the cages. The other control group
(normoxic rats) was kept in the same room but not in the
hypobaric chamber, with the same 12–12 h light–dark cycle.
Free access to a standard rat diet and water was allowed
throughout the experimental period.
The investigation conforms to the Guide for the Care and
Use of Laboratory Animals published by the US National
Institutes of Health (NIH Publication No.85-23, revised
1996) and European Directives (86/609/CEE).
133
2.2. Heart preparation
Animals were killed by cervical dislocation, and the heart
was quickly removed in a normoxic (i.e., equilibrated with
air) cooled (4 8C) relaxing solution (solution 1: 10 mM
EGTA, 3 mM Mg2+, 20 mM taurine, 0.5 mM dithiothreitol,
5 mM ATP, 15 mM phosphocreatine, 20 mM imidazole, and
0.1 M K+ 2-[N-morpholino]ethane sulfonic acid, pH 7.2);
chemicals were from Sigma Chemical Company (St Louis,
MO). The heart was then dissected and weighed. Pulmonary
hypertension was assessed by measuring the ratio of RV free
wall weight to the sum of septum plus LV free wall (LVS)
weight [10–12].
2.3. Oxidative phosphorylation
Bundles of fibers (non-isolated cardiomyocytes) between
5 and 10 mg were excised from the endocardial surface of
both LV and RV then permeabilized in solution 1 added with
saponin 50 Ag/ml. The bundle was then washed twice for 10
min each time in solution 2 (10 mM EGTA, 3 mM Mg2+, 20
mM taurine, 0.5 mM dithiothreitol, 3 mM phosphate, 1 mg/
ml fatty acid-free bovine serum albumin, 20 mM imidazole,
and 0.1 M K+ 2-[N-morpholino]ethane sulfonic acid, pH
7.2) to remove saponin. All procedures were carried out at 4
8C with extensive stirring. The success of the permeabilization procedure was estimated by determining the activity of
the cytosolic lactate dehydrogenase and the mitochondrial
citrate synthase in the medium. After 15–20 min of
permeabilization, more than 60% of the cytosolic lactate
dehydrogenase was found in the external medium, and the
mitochondrial citrate synthase activity in the medium
remained below 5% [13]. The oxygen consumption rate
was measured polarographically at 30 8C using a Clark-type
electrode (Hansatec, UK) connected to a PC computer that
displayed on-line the respiration rate value (original software developed in the laboratory). Solubility of oxygen in
the medium was considered to be equal to 450 nmol O/ml.
Respiratory rates were determined in a 1-ml oxygraph
cuvette containing one bundle of fibers in solution 2 with 10
mM malate plus 10 mM glutamate or 20 AM palmitoyl
carnitine as substrates; 50 AM di(adenosine 5V)-pentaphosphate, 20 AM EDTA and 1 mM iodoacetate were also added
to the cuvette to inhibit extramitochondrial ATP synthesis
(via the glycolysis or the adenylate kinase) and ATP
hydrolysis [14]. ADP-stimulated respiration, associated with
ATP synthesis, was determined in the presence of 1 mM
ADP. Basal respiration without ATP synthesis was measured
after addition of 70 AM atractyloside and 1 AM oligomycin.
After measurements, fibers were removed, dried on a
precision wipe and weighted. Results were expressed in
nanomoles of atom oxygen consumed per minute and per
milligram wet weight of fiber. Under identical conditions,
the mitochondrial ATP synthesis rate in skinned fibers was
determined by bioluminescence measurement (luciferineluciferase system) of the ATP produced after addition of 1
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K. Nouette-Gaulain et al. / Cardiovascular Research 66 (2005) 132–140
mM ADP [15]. The ATP Bioluminescence Assay Kit HS II
from Roche Diagnostics GmbH (Mannheim, Germany) was
used. At various time intervals after addition of ADP, 10-Al
aliquots were withdrawn from the oxygraph chamber,
quenched in 100 Al DMSO, and diluted in 5 ml ice-cold
distilled water. Standardization was performed with known
quantities of ATP measured under the same conditions. ATP
synthesis rate was expressed in nanomoles ATP produced
per minute and per milligram wet weight of fiber. The
efficiency of oxidative phosphorylation was taken as the
ratio of ATP synthesis rate to oxygen consumption rate
(ATP/O) [14].
2.4. Enzyme activity
About 100 mg of LV or RV were minced and homogenized with a glass Potter homogenizer in a ice-cold medium
(10% w/v) containing 225 mM mannitol, 75 mM sucrose, 10
mM Tris-HCl, 0.10 mM EDTA, pH 7.2. The homogenate
was then centrifuged for 20 min at 650 g. The supernatant
was collected and the protein concentration was determined
[16]. Enzymatic activity was assessed using previously
described spectophotometric procedures (model UVIKON
940, KONTRON) and expressed in nanomoles of substrate
transformed per minute and per milligram of protein. The
enzyme activity of citrate synthase was measured as
described by Srere in the presence of 4% Triton (vol/vol)
by monitoring at 412 nm wavelength at 30 8C the formation
of thionitrobenzoate dianion from the reaction of coenzyme
A and 5,5V-dithiobis(2-nitrobenzoic acid) [17]. The enzyme
activity of complex I, reduced nicotinamide adenine
dinucleotide (NADH) ubiquinone reductase, was measured
as described by Birch-Machin et al. [18]. The oxidation of
NADH by complex I was recorded using the ubiquinone
analog decylubiquinone as the electron acceptor. The
decrease in absorption resulting from NADH oxidation
was measured at 340 nm at 30 8C. Complex I activity was
calculated from the difference in the rate before and after the
addition of rotenone (2 AM), a specific inhibitor of complex
I. The Complex II (succinate dehydrogenase) specific
activity was measured by monitoring the reduction of 2,6dichlorophenol indophenol at 600 nm at 30 8C, in the
presence of phenazine methosulphate [19]. The oxidation of
ubiquinol (UQ1H2) by complex III (ubiquinol cytochrome c
reductase) was determined using cytochrome c(III) as the
electron acceptor [18]. The reduction of cytochrome c(III)
was recorded at 550 nm at 30 8C. Complex IV (cytochrome
c oxidase) was measured by the method described by
Wharton and Tzagoloff using cytochrome c(II) as substrate
[20]. The oxidation of cytochrome c was monitored at 550
nm at 30 8C.
2.5. Morphometric analysis
Three blocks were taken from each heart, and 10–20
electron micrographs of randomly chosen fields were
obtained from each block. Myocardial fibers for electron
microscopic examination were fixed using 2.5% glutaraldehyde and post-fixed in osmium tetroxide; they were then
embedded in epoxy resin. Ultrathin sections were stained
with saturated uranyl acetate and lead citrate. Mitochondrial
volume density (Vv), numerical density (Nv) and mean
volume (V) were determined in final prints of 161 cm2 at
magnification17,250 according to the method of Weibel et
al. [21] as follows.
! Vv is the relative volume fraction of the mitochondria
determined as the relative surface fraction of the unit area
comprised by all mitochondrion slices.
! Nv depends on mitochondria sections (NA) counted per
standard measuring surface, on the size distribution (K)
and, finally on the shape of the mitochondria, writing
Nv=K.NA3/2/b.Vv1/2. The form coefficient b for ellipsoidal structures is a function of the longitudinal and
transverse diameter. On the basis of previous studies it
was assumed to be b=2 in the heart [22]. K is determined
by the size distribution of the objects. According to
previous studies in chronic hypoxic heart, it was assumed
to be K=1.1 in the present study [22].
! V is the mean volume per mitochondria obtained by
simple division of Vv by Nv.
2.6. Southern blot analysis
Total genomic DNA was isolated from homogenates of
muscle tissue according to standard procedures. Approximately 5 Ag of DNA was digested with XhoI (New
England Biolabs, Inc.). Samples were resolved on 0.7%
agarose gels by electrophoresis, transferred to nylon
membranes and hybridized simultaneously with 32P-labeled
probes for mtDNA and the nuclear 18S rRNA gene [23].
Signals were quantified with a phosphorimager using
ImageQuantk software (Molecular Dynamics, Inc.). To
correct for quantitative variations among the samples,
mtDNA signals were normalized relative to nuclear DNA
signals.
Table 1
Effects of chronic hypoxia on physical characteristics
Normoxia
BW (g)
RVW (mg)
LSVW (mg)
RVW/BW (mg/g)
LVSW/BW (mg/g)
RVW/LVSW
364F8
146F4
678F12
0.40F0.01
1.87F0.04
0.22F0.01
Hypoxia
14 days
21 days
312F7*
348F16*
740F24*
1.12F0.05*
2.38F0.08*
0.47F0.02*
259F9*
354F19*
571F17*
1.38F0.09*
2.21F0.08*
0.62F0.02*
Data are meanFSE (n=20 in normoxic group, n=10 in each hypoxic
group). Data were obtained from animals whose hearts were used for
measurement of substrate oxidation. BW=body weight; RVW=right
ventricular free wall weight; LVSW=left ventricular free wall plus septum
weight, *Pb0.05 versus normoxic animals.
K. Nouette-Gaulain et al. / Cardiovascular Research 66 (2005) 132–140
Table 2
Effects of chronic hypoxia on mitochondrial oxidative phosphorylation
Oxygen consumption
ADP
ATP synthesis
ATP/O
14.1F1.2
15.0F1.1
30.1F2.6
31.6F2.8
2.2F0.1
2.1F0.1
11.9F1.1
11.5F0.8*
24.1F3.1
19.7F1.9*,y
2.1F0.3
1.8F0.1
10.4F0.8*
9.3F0.6*
21.7F1.7*
17.6F2.4*
2.1F0.1
1.9F0.3
8.0F0.5
6.8F0.3
17.3F1.3
14.2F1.3
2.1F0.1
2.1F0.2
5.6F0.4*
4.7F0.4*,y
11.6F0.8*
8.4F0.7*
2.1F0.1
1.8F0.1
7.7F0.4
7.5F0.8
12.8F1.1*
11.4F2.2
1.7F0.1
1.6F0.2
135
Student’s t-test was used to compare data of LV and RV in the
same animals. All P values were two-tailed, and a P value of
less than 0.05 was required to reject the null hypothesis.
+ADP
Glutamate/Malate
Normoxia
RV
3.2F0.2
LV
3.5F0.2
14 days Hypoxia
RV
2.7F0.3
LV
2.9F0.1*
21 days Hypoxia
RV
3.2F0.2
LV
2.9F0.3*
3. Results
Palmitoyl carnitine/Malate
Normoxia
RV
3.3F0.3
LV
3.2F0.2
14 days Hypoxia
RV
2.3F0.2*
LV
2.0F0.1*
21 days Hypoxia
RV
3.3F0.2
LV
2.4F0.2*
Data are meanFSE (in each subgroup, n=20 in normoxic group, n=10 in
each hypoxic group). RV=right ventricular free wall; LV=left ventricular
free wall. Experimental conditions are described in bMaterial and MethodsQ.
Basal, without adenosine diphosphate (ADP), and ADP-stimulated oxygen
consumption rates supported by glutamate or palmitoyl carnitine, in the
presence of malate, are expressed in nmol atom oxygen. min1.mg wet
weight1. ATP synthesis rate is expressed in nmol ATP.min1.mg wet
weight1. ATP-to-oxygen ratio (ATP/O) is calculated as the ratio of the rate
of ATP synthesis to the rate of the concomitant respiration in the presence
of ADP. *Pb0.05 versus normoxic animals, yPb0.05 versus right ventricle
under the same conditions.
2.7. Data analysis
Results were expressed as meanFSE. Data were plotted
and analyzed using SigmaPlot 8.0 and Systat 10.0 (SPSS,
Chicago, IL). Differences between groups were tested using
analysis of variance with post hoc Dunnett’s test. Paired
3.1. Physical characteristics
There was little change in body weight and the heart-tobody weight ratio remained constant in normoxic animals
throughout the experiments. In contrast, in rats exposed to
CH for 14 days, cardiac mass increased significantly
(1.65F0.06 g and 1.05F0.04 g in hypoxic and normoxic
animals, respectively, Pb0.05) with also a significant
increase in the heart-to-body weight ratio (0.54F0.09 g/
100 g and 0.29F0.03 g/100 g, respectively, Pb0.05). The
hypertrophy of hypoxic heart was associated with an
increase in the RV mass (348F16 mg and 146F4 mg in
14 days hypoxic and normoxic animals, respectively,
Pb0.05). and in the RV-to-LVS ratio (Table 1). In rats
exposed to hypobaric hypoxia for 21 days, the RV-to-LVS
ratio was almost threefold the control value (0.62F0.02 and
0.22F0.01 in 21 days hypoxic and normoxic animals,
respectively, Pb0.05).
3.2. Oxidative capacity
In normoxic rats, no difference was found in any
respiratory parameter between RV and LV with either
glutamate or palmitoyl carnitine as substrates. On glutamate,
mitochondrial oxygen consumption and ATP synthesis
significantly decreased at 14 days of CH in the LV without
change in ATP/O ratio (Table 2). A similar decrease was still
observed in the LV following 21 days of CH. Likewise,
oxidative capacity of the RV was decreased following 21
days of CH. However, unlike in LV, oxidative phosphorylation on glutamate remained consistent in RV at 14 days of
CH (Table 2). The results look slightly different with
Table 3
Effects of chronic hypoxia on the enzymatic activities of the respiratory chain
Complex I
Complex II
Complex III
Complex IV
Citrate synthase
Normoxia
RV
LV
271F38
236F26
301F45
270F44
964F68
923F81
1984F233
1984F228
1036F73
1004F73
14 days Hypoxia
RV
LV
205F14
136F15*,y
218F19
141F20*,y
806F42
666F49*
1932F177
1550F133y
868F52
766F44*y
21 days Hypoxia
RV
LV
167F29*
205F23y
124F8*
145F6*
658F153*
644F135*
1004F163*
1157F254*
601F70*
662F48*
Data are meanFSE (n=20 in normoxic group, n=10 in each hypoxic group). RV=right ventricular free wall; LV=left ventricular free wall. Experimental
conditions are described in bMaterial and MethodsQ. Enzymatic activity was expressed in nmol substrate.min1.mg protein1. *Pb0.05 versus normoxic
animals, yPb0.05 versus right ventricle under the same conditions.
K. Nouette-Gaulain et al. / Cardiovascular Research 66 (2005) 132–140
0.6
0.6
0.5
0.5
Complex II / CS
Complex I / CS
136
0.4
0.3
0.2
Normoxia
14 days CH
21 days CH
0.4
0.3
0.2
0.1
0.1
0.0
0.0
Right ventricle Left ventricle
Right ventricle Left ventricle
3.0
2.0
Complex IV / CS
Complex III / CS
2.5
1.5
1.0
0.5
2.0
1.5
1.0
0.5
0.0
0.0
Right ventricle Left ventricle
Right ventricle Left ventricle
Fig. 1. Ratio of the enzyme activity of the respiratory chain complexes to the citrate synthase activity. Experimental conditions are described in bMaterial and
MethodsQ. Enzyme activities are reported in Table 3. Values are meansFSE (n=20 in normoxic group, n=10 in each hypoxic group).
palmitoyl carnitine as substrate (Table 2). Mitochondrial
respiration and ATP synthesis were significantly decreased in
both ventricles following 14 days of CH. At 21 days of CH,
oxidative capacities tended to return to normoxic values.
The enzyme activity of the respiratory chain showed
comparable time-dependent changes according to the ventricle. Citrate synthase and the four enzyme complexes of the
respiratory chain decreased following 14 days of CH in the
LV, but not in the RV (Table 3). The amplitude of these
changes represented an averaged reduction of 30% of the
enzymatic activities. Nearly all these activities (except
complex I) were reduced in both ventricles following 21
days of CH (Table 3). However, it is noteworthy that the ratio
of the activity of each respiratory chain complex to citrate
synthase activity were not significantly different, suggesting
a global decrease in mitochondrial enzymes (Fig. 1).
3.3. Morphometric analysis
Following 14 days of CH, mitochondrial Vv, i.e., the
relative mitochondrial fraction of the sarcoplasm, decreased
Table 4
Morphometric analysis of heart mitochondria of rats submitted to chronic
hypoxia
Normoxia
Hypoxia
14 days
21 days
Right ventricle
V
0.93F0.08
Nv
38.68F2.14
Vv
30.24F1.29
0.97F0.06
39.30F1.75
32.55F0.88
0.58F0.04*
57.93F6.16*
29.62F1.19
Left ventricle
V
1.00F0.07
Nv
39.35F2.42
Vv
36.55F1.57y
0.99F0.05
37.89F1.80
33.16F0.83*
0.65F0.06*
57.09F4.89*
32.19F1.73
Data are meanFSE (n=10 in normoxic group, n=5 in each hypoxic group).
Experimental conditions are described in bMaterial and MethodsQ. V: mean
volume of mitochondrion (Am3); Nv: numerical density (102/Am3); Vv:
volume density (%). *Pb0.05 versus normoxic animals, yPb0.05 versus
right ventricle under the same conditions.
Fig. 2. Electron micrographs of mitochondria in rat heart (17,250):
mitochondria in normoxic left ventricle (A), in right ventricle from rat
exposed to 14 days of chronic hypoxia (B) and in left ventricle from rat
exposed to 21 days of chronic hypoxia (C). There was no change at 14 days
of chronic hypoxia. On the contrary, there was a significant increase in
numerical density and a decrease in size of mitochondria after 21 days of
chronic hypoxia.
K. Nouette-Gaulain et al. / Cardiovascular Research 66 (2005) 132–140
mtDNA/nuclear DNA (18S)
2.5
RV
LV
2.0
*
1.5
*
*
1.0
0.5
0.0
Normoxia
14 days
21 days
Hypoxia
Fig. 3. Quantification of mtDNA in right ventricle (RV) and left ventricle
(LV) of normoxic and chronic hypoxic rats. mtDNA/nuclear DNA (18S)
ratio was measured by means of Southern blot analysis from samples of
DNA extracted from hearts of animals. Values are meansFSE (n=6 in
normoxic group, n=3 in each hypoxic group). * Pb0. 05 versus normoxic
animals.
significantly only in LV (36.55%F1.57% and 33.16%F
0.83% in normoxic and 14 days hypoxic group, respectively, Pb0.05) without any change in mitochondrial Nv
(Table 4). Following 21 days of CH, Nv significantly
increased and V significantly decreased in both ventricles
indicating an increase in the number of smaller mitochondria (Fig. 2).
3.4. Mitochondrial DNA
In normoxic animals, the ratio of mtDNA to nuclear
DNA (18S) was similar in RV and LV. Following 14 days of
CH, this ratio was markedly reduced only in LV (Fig. 3) as
was the oxidative capacity in the same ventricle for the same
duration of CH. In contrast, following 21 days of CH,
mtDNA to nuclear DNA ratio was reduced in both
ventricles, without change in the signal of 18S spot.
4. Discussion
The purpose of the present investigation was to
examine the time course of the differential mitochondrial
energy metabolism adaptation to chronic hypoxia in right
and left ventricles in the rat heart taking advantage that,
whereas CH is characterized by a global reduction in
oxygen supply, it selectively induces a functional overload
of the RV due to pulmonary hypertension. Our results
indicate that CH decreases ATP synthesis as a consequence of an alteration in mitochondria function in both
ventricles but that this effect is delayed in the RV.
However, when RV hypertrophy was fully developed,
mitochondrial energy metabolism was decreased as in the
LV, suggesting some specific adaptive processes at the
onset of RV hypertrophy.
CH leads to pulmonary hypertension which induces a
marked RV hypertrophy. In the present study rats were
137
exposed to CH for 2 and 3 weeks on the basis of previous
experiments which have shown that such duration leads to
an alteration of LV and the full development of RV
hypertrophy, respectively [8,10,24]. When RV hypertrophy
reaches its maximal amplitude, following 3 weeks of CH,
mean pulmonary arterial pressure rises from, on average, 10
(control condition) to 32 mmHg and the ratio RVW/LVSW
increases, on average, up to 0.6 [24], a value close to that
found in the present study (Table 1).
Oxidation of glutamate and most enzyme activities of
the respiratory chain decreased in both ventricles following 21 days of CH. At that stage, the change in oxidative
capacity was characterized by a similar decline in ATP
synthesis and in oxygen consumption. The normal ATP/O
ratio indicated that the yield of oxidative phosphorylation
was not modified. Similar findings have been reported in
heart and liver mitochondria of rats acclimatized to a
4400-m simulated altitude. No changes in the oxygen or
ADP dependence of mitochondrial respiration was shown
as a mechanism of adaptation to chronic hypoxia and the
ATP/O ratio was similar in heart mitochondria from
hypoxic or normoxic animals [25–27]. Rumsey et al.
have shown that oxidation of pyruvate and glutamate was
compromised in LV as early as 7 days of hypoxic
exposure in a 10% O2 atmosphere [9]. In terms of
adaptation of enzymes involved in cardiac energetics, total
creatine kinase activity and mitochondrial creatine kinase
were also reduced in LV of rats submitted 28 days to a
simulated altitude of 5500 m [28]. Collectively, these
results suggest that decrease in oxidative capacity is an
early adaptation mechanism to CH, at least in the LV.
The mechanism of the decrease in oxidative phosphorylation can be related to the reduction in the enzyme
activities of the different respiratory chain complexes of the
mitochondrial inner membrane [29,30]. In the present study,
we have observed a global decrease in all enzyme
complexes and in the citrate synthase activity. Normalization of enzyme activities of the respiratory chain
complexes by citrate synthase (a mitochondrial enzyme)
yields no changes during hypoxia. These data suggest that
CH induces a reduction in the functional mitochondrial
mass. The decrease in oxidation observed in skinned fibers
could be explained by the reduction in mitochondrial mass
rather than a decrease in the specific activity of the
complexes This hypothesis is compatible with our morphometric data demonstrating a reduction in the mean volume
of mitochondria following 21 days of CH and is also in
agreement with previous reports regarding the effect on
mitochondria of both hypobaric hypoxia (4400 m) for 9–11
months [25] and normobaric hypoxia (FIO2 at 8%) [22].
The increase in mitochondria number should be considered
as a mechanism of adaptation to hypoxia [25,31]. It favors
the mitochondrial transport of oxygen by increasing the
surface-to-volume ratio of mitochondria.
The reduction of the mitochondrial mass can be
ascribed to a decrease in mitochondrial protein synthesis
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K. Nouette-Gaulain et al. / Cardiovascular Research 66 (2005) 132–140
explained, at least in part, by mtDNA down regulation as
observed in the present study. However, the respiratory
chain complexes depend on both mitochondrial and
nuclear genes. If 13 sub-units of respiratory complexes
are encoded by mtDNA, nuclear genes contribute to the
machinery required for maintenance, replication and
expression of mtDNA. Among the nuclear transcriptional
regulators implicated in mitochondrial biogenesis, the
nuclear respiratory factors (NRF-1 and NRF-2) play an
integrative role [32]. In mice, the loss of function of NRF1 is associated with a lethal phenotype and a dramatic
decrease in the amount of mtDNA [33]. NRF-1 is involved
in the maintenance of the respiratory apparatus. Since NRF
gene expression is down-regulated by hypoxia, it is likely
involved in the alteration of the electron transfer chain and
in the decrease in mtDNA observed during CH [3].
Moreover, protein synthesis is an ATP-consuming process
that is also down-regulated during CH [2]. Therefore, a
global decline in cellular protein synthesis could also, at
least in part, contribute to the decrease in mitochondrial
proteins.
The results concerning the time course of CH-induced
alteration in energy metabolism in the RV deserve further
discussion. As above discussed, we report a decrease in ATP
synthesis in RV at 21 days of CH with all substrates in
agreement with a previous study showing a decrease in
mitochondrial respiration with pyruvate as substrate after 3
weeks of exposure to hypoxic conditions (FiO2 10%) [34].
In contrast, Rumsey et al. have shown an alteration of the
oxidative metabolism in RV only with palmitoyl carnitine as
substrate but not with pyruvate nor glutamate [9]. This
difference could be due to difference in the hypoxic model
and/or the techniques used. In the present study, saponinskinned fibers from each ventricle were used to directly
assess the mitochondrial metabolism. The decrease in food
utilization following long-term exposure to chronic hypoxia
can also in part explain the changes in cardiac energy
metabolism [35]. However, these alterations affect both
ventricles and cannot totally account for the differences
between RV and LV [36].
Oxidation of palmitoyl carnitine was decreased in both
ventricles following 14 days of CH. The mechanism of
the reduction of fatty acid catabolism is not still
elucidated. Recent studies on mechanically overload hearts
revealed that the development of cardiac hypertrophy is
frequently associated with an impaired fatty oxidation
[37]. Therefore, during the development of cardiac
hypertrophy, a shift from fatty acid to glucose utilization
may occur [38]. This alteration could be due to a decrease
in long-chain fatty acid oxidation with alteration in
transporters or decline in fatty acid beta oxidation pathway. Moreover, acclimation to hypoxia might affect the
relative contribution of lipids to energy metabolism with
also a shift toward higher utilisation of carbohydrate [39].
Hypoxia per se induced a rise in hexokinase activity and a
fall of hydroxy-acyl CoA-dehydrogenase activity in both
myocardial ventricles [40]. Probably, the effects of both
overload heart and chronic hypoxia contribute to the
observed decrease in palmitoyl carnitine metabolism in
RV at 14 days of CH. The fact that fatty acid oxidation
tended to return to baseline value by 21 days of hypoxia
in the same time as the activities of all respiratory
complexes decrease remain difficult to explain. Several
hypotheses can be proposed: a specific alteration in fatty
acids transport, a slight uncoupling of oxidative phosphorylation or a differential effect of CH of sub-populations of
heart mitochondria (subsarcolemmal and interfibrilar mitochondria) as shown for aging [41]. Further studies are
necessary to understand fatty acid catabolism during
prolonged hypoxia [9,42].
Interestingly, whereas at 21 days of CH the mitochondrial metabolism was decreased in both ventricles, the
oxidative capacity of the RV was maintained on glutamate
following 14 days of CH, i.e., CH-induced alteration in RV
energy metabolism was delayed. We suggest that the
compensatory increase in RV mass could, in part, explain
that oxidative capacity is maintained at 14 days of CH. In
this connection, it has been shown that increasing energy
demand enhances mitochondrial cytochrome content in
skeletal muscle and the heart [43]. Alternatively, a recent
study in rats submitted to hypobaric hypoxia (450 mmHg
corresponding to an FIO2 of c12%) has indicated that
acclimatizing to a simulated altitude may have a tissuespecific effect on protein expression [44]. Whatever, the
mechanism, it is noteworthy when RV hypertrophy was
fully developed, following 21 days of CH [10,24],
mitochondrial energy metabolism was decreased as in the
LV. The present results suggest that cardiac hypertrophy
only transiently compensates for the hypoxia-induced
alteration in mitochondrial energy metabolism. In this
connection, we have previously observed, at the site of the
pulmonary vasculature, a similar transient adaptive phenomenon at the onset of RV hypertrophy, the occurrence
of rhythmic contractions [10]. Very recent studies indicate
that CH-induced pulmonary hypertension and RV hypertrophy can be prevented, at least in the rat, by administering in vivo, in the course of exposure to hypoxia, a variety
of drugs including serotonin transporter inhibitors [45],
sidenafil [46,47] or DHEA [48]. Whether, under such
conditions, antagonism of RV hypertrophy would accelerate CH-induced alteration in RV energy metabolism
remains to be examined to better understand the differential mitochondrial energy metabolism adaptation to
chronic hypoxia in right and left ventricles.
Acknowledgements
This work was supported by a grant from bConseil
Régional d’AquitaineQ (No 20020301301A) and bMinistère
de l’EnvironnementQ and bADEMEQ (PRIMEQUAL No
0262019).
K. Nouette-Gaulain et al. / Cardiovascular Research 66 (2005) 132–140
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