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Adaptation to hypoxia alters
energy metabolism in rat heart
WILLIAM L. RUMSEY,1 BRIAN ABBOTT,1 DARCI BERTELSEN,1 MICHAEL MALLAMACI,1
KEVIN HAGAN,1 DAVID NELSON,2 AND MARIA ERECINSKA2
1Zeneca Pharmaceuticals, Wilmington, Delaware 19850; and 2School of Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104
oxidative capacity; glycolysis; amino acids; mitochondrial
oxidative phosphorylation; pulmonary hypertension
IT IS WELL ESTABLISHED that chronic exposure to hypoxia
results in pulmonary hypertension. To provide adequate perfusion of the lung, the right ventricle develops much higher pressures under hypoxic conditions
than under normoxia. A response to this chronic functional overload is a compensatory increase in right
ventricular mass. The increased vascular resistance
during hypoxia also imposes a greater burden on the
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energy-producing pathways in matching ATP synthesis
to ATP demand. Furthermore, this increased requirement for energy occurs during conditions of low O2
availability.
In the well-oxygenated heart, .95% of the ATP
supply is generated by oxidative phosphorylation (for
review see Ref. 1) to support cycling of the contractile
proteins, maintain ion gradients, and fuel biosynthetic
reactions along with other ATP-dependent processes.
O2 consumption by heart cells in vitro is limited by PO2
within the physiological range, i.e., ,15 Torr (51).
Measurements of PO2 in animals ventilated with room
air have yielded values of ,20–30 Torr in the epicardial
microvasculature (49, 50), whereas substantially lower
values, down to 4–6 Torr, were found within the cardiac
myocytes (for review see Ref. 64). It might be expected,
therefore, that even a modest lowering of O2 delivery to
the contracting cells would compromise functional capacity of the heart.
Although previous studies have evaluated morphological and biochemical parameters in the chronically
stressed heart, with particular emphasis on the left
ventricle, they have largely focused on events that
occur in the presence of normal O2 delivery (3). Few
investigations (2) have addressed what, if any, alterations in energy metabolism result from a prolonged,
abnormal elevation of cardiac work in the absence of
‘‘normal’’ availability of O2. The present work was
undertaken to characterize more comprehensively the
progressive changes in energy metabolism in right and
left ventricles of rats living in a normobaric atmosphere
of 10% O2. Our data indicate that adaptation to hypoxia
results in significant alterations of substrate utilization. Oxidative capacity was adversely affected, for the
most part, in the musculature of the left ventricle,
although suppression of fatty acid oxidation, the preferred fuel for the heart, was common to both ventricles. Compensatory adjustments, i.e., enhanced capacity for glucose phosphorylation and changes in
amino acid metabolism, were found to support cardiac
work.
METHODS AND MATERIALS
Animals. Eighty 4-day-old, pathogen-free, Sprague-Dawley male rats were housed in standard cages in room air or in
Plexiglas boxes in a 10% O2 atmosphere. In the latter case, no
effort was made to control the level of CO2 (range 0.8–1.8%).
The bedding was changed daily. Animals were permitted free
access to water and standard rat chow.
Physiological measurements. After animals were anesthetized (urethan at 1.5 g/kg body wt im, followed 45 min later
with 0.75 g/kg sc), the left common carotid and right femoral
0363-6135/99 $5.00 Copyright r 1999 the American Physiological Society
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Rumsey, William L., Brian Abbott, Darci Bertelsen,
Michael Mallamaci, Kevin Hagan, David Nelson, and
Maria Erecinska. Adaptation to hypoxia alters energy
metabolism in rat heart. Am. J. Physiol. 276 (Heart Circ.
Physiol. 45): H71–H80, 1999.—The present study characterized metabolic changes in the heart associated with long-term
exposure to hypoxia, a potent stimulus for pulmonary hypertension and right ventricular hypertrophy. When anesthetized rats adapted to chronic hypoxia spontaneously respired
room air, their mean right intraventricular peak systolic
pressure (RVSP) was twice that in normal control animals
with the same arterial PO2. RVSP was linearly related to right
ventricular mass (r 5 0.78). Oxidative capacity (O2 consumption) of homogenates of right and left ventricles from both
groups of rats was measured with one of the following
substrates: pyruvate, glutamate, acetate, and palmitoyl-Lcarnitine. Oxidation of all substrates was significantly greater
in the left than in the right ventricle in normal rats but not in
hypoxia-adapted animals, where it was the same, within the
experimental error. O2 consumption by the left ventricle was
greater in control than in experimental rats, but right
ventricular O2 consumption was similar in the two groups.
Maximal reaction velocity of cytochrome-c oxidase was about
the same in the two ventricles, and there were no significant
differences between control and hypoxia-adapted animals.
HPLC analyses showed significantly higher aspartate levels
and aspartate-to glutamate concentration ratios in both
ventricles of hypoxic rats than in corresponding tissues from
controls, indicative of a decreased flux through the malateaspartate shuttle under conditions of O2 limitation. Myocardial glutamine levels were lower in hypoxic rats, and glutamine-to-glutamate concentration ratios decreased, although
primarily in the pressure-overloaded right ventricle. These
findings indicate that normal energy metabolism in the left
ventricle differs from that in the right and that the differences, particularly those of amino acid metabolism, are
markedly influenced by chronic exposure to hypoxia.
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CARDIAC ENERGY METABOLISM AND HYPOXIA
The respective RNAs were then pooled, and their concentration was determined by absorbance at 260 nm. For Northern
blotting, a 20-µg aliquot of total RNA was added to two
volumes of medium containing 50% formamide, 2.2 M formaldehyde, 13 running buffer (40 mM MOPS, 10 mM sodium
acetate, 1 mM EDTA, pH 7.0), 0.04% xylene cyanol, 0.04%
bromphenol blue, 5% glycerol, and 10 mg/ml ethidium bromide, heated to 65°C, and electrophoresed through a 1.0%
agarose gel. The latter contained 2.2 M formaldehyde and 13
running buffer. RNA quality and loading were checked by
ultraviolet illumination. The gels were blotted and fixed to
Hybond N1 positively charged nylon membranes (Amersham,
South Clearbrook, IL). Northern hybridizations were performed as described previously (11, 18). Blots were air dried,
exposed to autoradiography film (XAR, Kodak, Rochester,
NY) at 280°C with intensifying screen for 1–7 days, and
scanned (model SI densitometer, Molecular Dynamics, Sunnyvale, CA). Values (derived from densitometric pixel volume) were normalized to the signal generated from ribosomal
protein L28. Data were expressed as relative mRNA levels
calculated as percentages of the relevant control value (assigned a value of 100%).
Oligodeoxyribonucleotides. Five pairs of gene-specific oligonucleotides were synthesized using an ABI 392 Medium
Throughput DNA/RNA Synthesizer (Perkin Elmer/Applied
Biosystems, Foster City, CA; numbers correspond to description of probe preparation): 1) 58-CAAAATGCCAAGGAAATCTTAACCC, 2) 58-GACAGTAGCTTTGCTGTTGGTCT,
3) 58-GAGAAGGCCTACCAAATCCTGATG, 4) 58-AGGGGCGACCGCATGCGTCTC, 5 ) 58-TCAGCTGATTTATAATCTTCTAAAGG, 6) 58-AGAAGTCAGAGTCACCTTCACAA,
7) 58-AAAACTCATTGCACCAGTTGCGG, 8) 58-ATCATCCTTTAGCTTCTGGTTGATA, 9) 58-ATGTCTGCGCATCTGCAATGGATG, and 10) 58-TCAGGAGCTCTTGGTGGGGGAGG.
Probe generation. cDNA for ribosomal protein L28, rat
hexokinase I, rat hexokinase II, rat lactate dehydrogenase A,
and rat lactate dehydrogenase B were generated via RT-PCR
using standard PCR conditions (39) and the following genespecific primers: hexokinase I (485-bp PCR product, oligonucleotides 1 and 2), hexokinase II (477-bp PCR product,
oligonucleotides 3 and 4), lactate dehydrogenase A (913-bp
PCR product, oligonucleotides 5 and 6), lactate dehydrogenase B (919-bp PCR product, oligonucleotides 7 and 8), and
ribosomal protein L28 (416-bp PCR product, oligonucleotides
9 and 10). All gene fragments were cloned into pT7Blue, and
the identity of each insert was confirmed by sequence analysis. Probe DNA was prepared by PCR with use of purified
plasmid containing the cloned cDNA fragment of interest as
template and the appropriate gene-specific primer pair. PCR
products corresponding to the expected sizes were purified
from agarose gels and labeled to high specific activity with
deoxy-[32P]CTP (New England Nuclear, Boston, MA), as
described previously (63).
Statistical analyses. Values are means 6 SE unless stated
otherwise. Statistical significance between groups was determined with Student’s t-test or ANOVA followed by the Newman-Keuls test. Significant differences were established at
the 0.05 level of confidence.
RESULTS
Physical characteristics. Table 1 compares some general characteristics and hemodynamic measurements
of rats maintained in the hypoxic colony (experimental)
with those of rats kept in room air (control). The
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artery were catheterized for measurement of systemic pressure and for sampling of arterial blood gases, respectively. A
pressure transducer (2-Fr, Millar, Houston, TX) was inserted
into the right ventricle via the jugular vein for measurement
of intraventricular pressure. The animals were tracheotomized but breathed room air spontaneously. At the end of
each experiment, a bilateral thoracotomy was performed to
remove the heart, which was rinsed in saline. The ventricles
were separated, weighed, and dried overnight at 90°C for
determination of dry weight.
Oxidative capacity. In a parallel study the heart was
excised immediately after decapitation and placed in a vial
containing ice-cold saline. The ventricles were separated on a
plastic petri dish cooled in ice. The ventricular walls, excluding the septum, were minced and homogenized in ice-cold
medium (1:10 wt/vol) containing 250 mM sucrose, 50 mM
Tris, and 1 mM EDTA, pH 7.4. The latter process, which
causes cellular disruption, involved two steps: an initial
treatment in a Polytron (model PT 3000, Brinkman Instrument; 30-s pulse) followed by homogenization (6 passes) in a
Teflon-glass Potter-Elvehjem homogenizer. To prevent proteolysis, the homogenates were kept on ice and used within 3–4
h of preparation.
Oxidative capacity was determined with a Clark-type O2
electrode in a stirred reaction vessel thermostated at 37°C.
An aliquot of the homogenate was added to medium consisting of 130 mM KCl, 20 mM K2HPO4, pH 7.2, and one of the
following substrates in combination with malate (1 mM):
glutamate, pyruvate, acetate (all at 10 mM), or palmitoyl-Lcarnitine (5 mM plus BSA, 5:1). Maximal respiratory rates
were elicited by addition of 250 nmol of ADP.
Enzyme activities. Cytochrome-c oxidase activity was measured polarographically (17) as described above in a medium
containing 50 mM KH2PO4 and 0.1 mM EDTA, pH 7.4. The
substrate was 0.04 mM cytochrome c, 0.63 mM N,N,N8, N8tetramethyl-p-phenylenediamene, and 12.5 mM sodium ascorbate.
Maximal velocities of hexokinase and lactate dehydrogenase were measured in homogenates of right and left ventricles prepared from separate hearts as described above in
nine volumes of cold medium consisting of 50 mM KH2PO4, 2
mM MgCl2, 1 mM EDTA, and 0.5 mM dithiothreitol, pH 7.4.
After preparation, homogenates were kept on ice; they were
used within 4 h. Reaction rates were monitored spectrophotometrically (model Lambda 18, Perkin-Elmer, Norwalk, CT) at
340 nm at room temperature. The activities of hexokinase
and lactate dehydrogenase were assayed according to Hansford (16) and Wahlefeld (65), respectively.
Amino acid analysis. Amino acids were determined in
neutralized extracts of blood (plasma) and heart homogenates
with HPLC (24). Blood was collected in heparinized tubes and
centrifuged (Sorvall Instrument/DuPont, Newtown, CT) at
low speed (1,500 g at 4°C) to separate serum. An aliquot of the
latter was quenched with an appropriate volume of perchloric
acid (final concentration 0.6 M) and, after removal of the
precipitated protein, neutralized with 2.5 M KHCO3. Samples
were obtained from animals used for measurements of oxidative capacity. Protein was measured by the biuret reaction
with BSA as a standard.
Measurements of mRNA abundance, RNA preparation, and
analysis. Steady-state mRNA levels were measured in three
independent determinations by Northern blot analysis. For
each experiment, tissues were obtained from two to five rats
that were adapted to hypoxia or had been kept in room air for
equivalent periods of time (aged matched). Samples were
stored at 280°C before use. Total RNA was extracted from the
tissues by using Ultraspec Reagent (Biotecx, Houston, TX).
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CARDIAC ENERGY METABOLISM AND HYPOXIA
Table 1. Physiological parameters in normoxic and hypoxic rats
Normoxia
Hypoxia
7 days
14 days
21 days
$28 days
PaO2 , Torr
PaCO2 , Torr
pHa
MAP, mmHg
83.8 6 1.9 (27)
43.5 6 1.4 (27)
7.40 6 0.01 (26)
79 6 2 (37)
83.0 6 4.0 (3)
87.5 6 4.9 (7)
79.5 6 3.1 (4)
82.6 (2)
36.9 6 2.7 (3)
37.5 6 1.9 (7)*
28.7 6 3.4 (4)†
37.4 (2)
7.32 6 0.02 (3)*
7.30 6 0.02 (7)‡
7.36 6 0.01 (4)
7.3 (2)
100 6 10 (3)*
96 6 6 (10)†
102 6 5 (8)‡
105 6 9 (3)*
HR, beats/min
RVSP, mmHg
Hct, %
366 6 11 (37)
29 6 2 (36)
50 6 1 (11)
453 6 9 (3)
433 6 13 (10)†
431 6 15 (8)*
403 6 23 (3)
48 6 3 (3)†
61 6 4 (10)‡
76 6 5 (8)‡
78 6 6 (3)‡
73 (1)
79 6 1 (9)‡
83 6 2 (8)‡
89 6 1 (3)*†
Values are means 6 SE for number of animals in parentheses. Rats were housed in standard cages in room air or in Plexiglas chambers in a
10% O2 atmosphere for 7–36 days. PaO2 and PaCO2 , arterial PO2 and PCO2 ; pHa , arterial pH; MAP, mean arterial pressure; HR, heart rate;
RVSP, right intraventricular peak systolic pressure; Hct, hematocrit. Significantly different from normoxia: * P , 0.05; † P , 0.01; ‡ P , 0.001.
Fig. 1. Effect of chronic hypoxia on right ventricular mass and its
relation to right intraventricular systolic pressure. Measurements
were obtained from urethan-anesthetized rats exposed to hypoxia for
7–36 days. Right ventricle was isolated, rinsed in saline, and dried
overnight at 90°C. Each point represents an individual animal.
mals over the time course of the experiments; hence, all
values were averaged to give a single control. By
contrast, marked changes resulted from hypoxia. Body
weight was less in hypoxia-adapted than in normoxic
animals at all time points, and a decrease was seen
after 1 day. Moreover, hypoxic animals did not show
evidence of weight gain (cf. 1 day vs. 41 days). There
was a rise in left ventricular mass when normalized to
body weight; this effect was small and most apparent at
41 days [cf. maximal value, i.e., 2.28 6 0.33 (n 5 3),
obtained at 41 days with value for normoxic rats, i.e.,
1.63 (n 5 1), or value from Table 1, i.e., 1.82 6 0.03].
However, the weight of the right ventricle rose steadily
during the first 2 wk of hypoxic exposure and resulted
in a near doubling in the ratio of ventricular mass to
whole body mass at that time; this relation changed
minimally with continued hypoxia.
Oxidative capacity. Homogenates, and not isolated
mitochondria, were used to determine rates of substrate utilization (per gram of tissue). This was done for
the following reasons: 1) isolation of mitochondria
yields a fraction of their total content, whereas the aim
of the study was evaluation of the ‘‘total’’ oxidative
capacity of the intact tissue in situ, 2) mitochondria
isolated from ‘‘stressed’’ muscles may be more ‘‘fragile’’
and, consequently, might sustain more damage during
isolation than the organelles from ‘‘healthy’’ tissues,
and 3) the wet weight of the normoxic right ventricle,
typically ,190 mg, precluded isolation of enough mitochondria to measure in duplicate oxidation of all substrates and activity of the cytochrome oxidase.
In normoxic rats, respiration was significantly greater
(P , 0.05), regardless of the type of substrate, in
homogenates from the left than from the right ventricle
(Table 3, Fig. 2). On average, the difference represented
an ,30% greater oxidative capacity.
Homogenates of left ventricle from hypoxic animals
oxidized all substrates at much slower rates than those
obtained from the corresponding muscle of rats kept in
room air (Fig. 2). The decrease occurred after only 24 h
of hypoxia and continued, progressively, until 14 days.
From this time onward, oxidation rates remained low
and relatively constant. For example, when glutamate
served as the metabolic fuel, its rate of oxidation fell
from 29.3 6 1.4 to 26.6 6 1.0 µatm O2 · min21 · g wet wt21
in 24 h and to 19.6 6 1.0 µatm O2 · min21 · g wet wt21
(i.e., by .30%) after 14 days of hypoxia (Table 3); it was
22.6 6 2.3 µatm O2 · min21 · g wet wt21 after 41 days in a
low-O2 atmosphere.
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animals were exposed to 10% O2 for 7, 14, 21, and $28
days.
When the animals were brought out of the chamber,
anesthetized, and permitted to respire on room air,
arterial PO2 was similar in control and hypoxia-adapted
groups. However, there were notable differences in
several other parameters. Arterial PCO2 was decreased
and arterial pH was modestly acidic in the experimental group. Hematocrit was increased by 50% at 14 days,
a typical hypoxic adaptation. Heart rate and mean
arterial pressure were higher by ,15 and 25%, respectively, when averaged across the duration of hypoxia
(after 7 days values did not rise further) than in
controls. Right intraventricular peak systolic pressure
(RVSP) was about twofold greater by 14 days of hypoxia
(P , 0.01) and continued to increase until ,21 days.
When individual measurements of RVSP from all experimental animals were plotted as a function of right
ventricular mass (Fig. 1), a straight-line relationship
was obtained. This shows that the rise in right ventricular pressure is associated with a marked increase in
right ventricular dry weight (r 5 0.78).
Table 2 summarizes the effect of hypoxia on body
weight and cardiac mass in relation to the total body
weight. These results are from animals exposed to low
PO2 for various periods of time and then used for
measurement of oxidation rates. There was little change
in body weight (range 454–600 g), and the heart-tobody weight ratio remained constant in normoxic ani-
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CARDIAC ENERGY METABOLISM AND HYPOXIA
Table 2. Effect of chronic hypoxia on body weight and cardiac mass
Hypoxia
Normoxia
Body wt, g
Ventricular wet wt/body wt, mg/g
Right
Left
1 day
7 days
14 days
20–36 days
.41 days
505 6 15
403 6 21‡
346 6 47§
325 6 24§
370 6 15§
402 6 19§
0.46 6 0.03
1.82 6 0.04
0.56 6 0.09
2.11 6 0.07
0.75 6 0.06†
2.01 6 0.38*
1.09 6 0.06§
2.00 6 0.09
1.11 6 0.07§
1.97 6 0.01
1.13 6 0.09§
2.28 6 0.33
Values are means 6 SE for 3–10 animals, except where noted otherwise (*n 5 2). Data were obtained from animals used for measurement of
substrate oxidation, except those from left ventricle of normoxic rats. Significantly different from normoxia: † P , 0.05; ‡ P , 0.01; ‡ P , 0.001.
days, each n 5 1) were within the range of those shown
in Fig. 3A.
The maximal activity of lactate dehydrogenase in
right and left ventricles of normoxic rats was 98.8 6 4.5
and 120.7 6 5.3 µmol/g wet wt, respectively (Fig. 3B).
Neither the absolute values nor the difference between
the two chambers (,27%) was altered by hypoxia.
The level of expression for hexokinase I and II and
lactate dehydrogenase A and B was evaluated to determine whether hypoxia adversely affected the distribution of the isozymes. Consistent with the enhancement
of hexokinase activity, there was an increase in the
relative abundance of mRNA for hexokinase I and II in
the ventricles of hypoxia-adapted rats (Fig. 4). The
latter changes were apparent after 21 days, but not
after 14 days (data not shown), in low O2. For hexokinase I the levels were increased by 55 6 0.1 and 25 6
0.1% in right and left ventricles, respectively, relative
to normoxic values. The corresponding elevations in
expression of hexokinase II mRNA were 72 6 0.2 and
47 6 0.3%. In the same hearts, levels of lactate
dehydrogenase mRNA were modestly affected by hypoxia; lactate dehydrogenase A was increased by 37 6
0.1 and 30 6 0.1% in the right and left ventricle,
respectively, whereas the levels of transcript for the B
isoform were essentially unchanged.
Amino acid metabolism. A comparison of the total
concentrations of the amino acids (Table 4) in plasma
Table 3. Effect of chronic hypoxia on substrate oxidations in right and left ventricle
Hypoxia
Pyruvate
Right
Left
Glutamate
Right
Left
Acetate
Right
Left
Palmitoyl-L-carnitine
Right
Left
Cytochrome c oxidase
Right
Left
Normoxia
1 day
7 days
14 days
20–36 days
.41 days
24.0 6 1.0
30.0 6 1.2
22.8 6 0.8
27.0 6 0.9
22.4 6 2.7
23.8 6 1.3†
19.2 6 1.1
20.1 6 1.1§
25.2 6 1.1
22.3 6 1.3§
21.6 6 1.5
22.1 6 1.5§
22.7 6 0.8
29.3 6 1.4
22.2 6 0.7
26.6 6 1.0
23.3 6 2.9
22.9 6 1.8
20.5 6 1.3
19.6 6 1.0‡
25.4 6 1.5
22.7 6 1.5†
22.6 6 1.9
22.6 6 2.3†
5.9 6 0.5
8.0 6 0.4
5.2 6 0.6
6.3 6 0.9†
5.3 6 0.5
5.8 6 0.2†
5.2 6 0.4
4.9 6 0.3§
6.4 6 0.2
5.5 6 0.3‡
5.7 6 0.8
5.8 6 0.5‡
18.5 6 0.9
23.7 6 1.5
17.2 6 0.5*
22.3 6 2.2*
18.3 6 1.7
18.4 6 0.7†
14.8 6 0.9
16.1 6 1.8‡
14.4 6 1.2
15.3 6 1.0§
10.7 6 1.4§
15.5 6 0.2§
415 6 39
488 6 26
488 6 26
509 6 9
433 6 18
477 6 33
393 6 12
409 6 25
437 6 18
404 6 16
399 6 28
414 6 32
Values are means 6 SE for 3–10 experiments/group, except where noted otherwise (n 5 2). Units are µatm O · min21 · g wet wt21. Normoxic
values were obtained from animals maintained in room air for a period of days coincident with that for hypoxic values. All substrates were
combined with 1 mM malate. Significantly different from normoxia: * P , 0.05; † P , 0.01; ‡ P , 0.001.
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By contrast, long-term exposure to hypoxia was not
associated with any decline in respiratory activity of
the right ventricle with pyruvate, glutamate, or acetate
(Fig. 2, Table 3). However, oxidation of the long-chain
fatty acid palmitoyl-L-carnitine was markedly decreased. With the latter substrate, O2 consumption fell
from the normoxic value of 18.5 6 0.9 to 10.7 6 1.4
µatm O2 · min21 · g wet wt21 in animals kept in hypoxia
for .41 days. This represented a change of .40%.
Maximal activity of cytochrome-c oxidase was nearly
the same in homogenates from left and right ventricles
and was essentially unaffected by long-term hypoxia
(Table 3).
Glycolytic activity. Because oxidative capacity reached
an apparent nadir in the left ventricle after 14 days of
hypoxia, markers of glycolytic capacity were evaluated
beginning at this time point. Maximal velocity of
hexokinase (one of the rate-limiting steps in the glycolytic pathway) was similar in homogenates of right and
left ventricles (5.5 6 0.6 and 5.6 6 0.6 µmol/g wet wt,
respectively) from normoxic rats. Animals adapted to
hypoxia for 14 days showed a marked increase in
myocardial enzyme activity (Fig. 3A); the rise was to
9.9 6 0.9 µmol/g wet wt (P , 0.01), i.e., by 80%, in
homogenates of the right ventricle and to 8.2 6 0.7
µmol/g wet wt (P , 0.05), i.e., by 46%, in preparations
from the left ventricle. Measurements in hearts of
animals exposed to longer periods of hypoxia (28 and 40
CARDIAC ENERGY METABOLISM AND HYPOXIA
H75
from normoxic and hypoxia-adapted rats showed no
difference between the two groups. A small rise, 29%, in
aspartate concentration and a slight decrease, 14%, in
glutamine concentration in samples from hypoxic rats
were statistically not significant. Nevertheless, these
changes were consistent with those obtained from
cardiac muscles. The sum of the collective amount of
the amino acids in right and left ventricles was for the
most part unchanged by long-term hypoxia (Table 4).
For aspartate, however, levels in right and left ventricles were 57% (P , 0.01) and 91% (P , 0.01) greater
than in the corresponding controls. Glutamate concentrations in neither plasma nor heart were significantly
affected by hypoxic adaptation. The aspartate-toglutamine ratio, however, increased in right and left
ventricles by 70 and 94%, respectively, in response to
long-term hypoxia. Right and left ventricular glutamine concentration was 36% (P , 0.01) and 10% lower
than in the respective muscles of normoxic rats. Consequently, the glutamine-to-glutamate ratio declined from
1.6 to 1.1 in the right ventricle of the hypoxic rat, but
essentially no change in this parameter occurred on the
left side (1.3 to 1.2).
DISCUSSION
The heart, like other metabolically highly active
tissues, is considered to be very sensitive to acute
episodes of hypoxia. Nonetheless, mammals including
humans can endure prolonged periods of O2 deprivation, for example, during ascent to high altitude. Many
species have adapted quite well during evolution to
these seemingly harsh conditions. The purpose of the
present investigation was to characterize alterations in
fuel metabolism in the right and left ventricle caused
by relatively long-term (days) exposure of animals to a
low-O2 environment. Our major findings were threefold: 1) The normally enhanced oxidative capacity of
the left ventricle, relative to its right counterpart, was
diminished by hypoxic adaptation. Oxidation of differ-
Fig. 3. Effect of chronic hypoxia on maximal
activities of glycolytic enzymes in rat heart.
Maximal activities of hexokinase (A) and lactate dehydrogenase (B) were measured spectrophotometrically at 21°C. Right ventricle
was dissected free from left ventricle and
septum (discarded) in ice-cold media. Isozyme
profiles were not determined. Values are
means 6 SE (n 5 3–5).
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Fig. 2. Effect of chronic hypoxia on
substrate oxidation in rat heart. Values
at time 0 represent combined measurements from all normoxic animals maintained in room air as controls for various periods of hypoxia. Malate (1 mM)
was added in conjunction with each
substrate to ensure that tricarboxylic
acid cycle was not substrate limited.
Substrate and ventricle orientation (i.e.,
right or left) are indicated. Data obtained from animals kept in hypoxia for
41–53 days were combined and designated 41 days. Values are means 6 SE
[n 5 3–10 animals/group, except for
palmitoyl-L-carnitine (Palm-L-carn) at
1 day of hypoxia, where n 5 2].
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CARDIAC ENERGY METABOLISM AND HYPOXIA
Fig. 4. Abundance of mRNAs for hexokinase and
lactate dehydrogenase in right and left ventricles of
normoxic and hypoxia-adapted rats. Northern blots
are representative of triplicate samples obtained
from RNAs extracted from hearts of 2–5 animals. RV
and LV, samples from right and left ventricle, respectively; HK and LDH, hexokinase (I or II) and lactate
dehydrogenase (A or B). L28 served as a control.
gether, the alterations described above suggest that
limitation of O2 availability results in a reduced capacity to synthesize ATP via oxidative phosphorylation in
the left, but not the right, ventricle. Perhaps more
importantly, the changes in amino acid metabolism are
indicative of significant disturbances within the metabolic pathways for energy production, which, to our
knowledge, have not been previously described for
these conditions. Hence, a simple enhancement of
glycolysis is not the only change in substrate utilization
in the heart resulting from long-term hypoxia.
Chronic hypoxic exposure leads to pulmonary hypertension, which, in turn, results in marked hypertrophy
(for recent clinical examples see Refs. 35 and 38) and,
as is often the case, eventual failure of the right
ventricle. In the present study a linear correlation was
obtained between RVSP and right ventricular dry
weight. This compensatory increase in muscle mass is a
typical response to a sustained elevation of pulmonary
pressure that imposes a chronic physical and metabolic
overload on the ventricle.
A first step toward understanding the potentially
deleterious effects of chronic overload is an examination of mitochondrial morphology and/or function. Early
investigations of the overloaded ventricle indicated a
loss of mitochondrial volume per cell (4, 71) and disappearance of mitochondrial cristae (72). Mitochondrial
oxidative capacity was decreased (34, 57, 70, 71) or
Table 4. Effect of chronic hypoxia on myocardial and plasma levels of amino acids
Normoxia
Asp
Glu
Gln
[Asp]/[Glu]
[Gln]/[Glu]
S (Asp 1 Glu 1 Gln)
Total amino acids
Right
ventricle
Left
ventricle
13.7 6 1.2
29.6 6 3.4
46.4 6 4.1
0.5 6 0.06
1.6 6 0.09
89.6 6 7.3
266 6 20
9.6 6 0.6
32.7 6 2.9
43.3 6 3.5
0.3 6 0.03
1.3 6 0.03
85.6 6 6.4
255 6 17
Hypoxia
Plasma
21 6 2
129 6 7
710 6 41
859 6 42
4,381 6 63
Right
ventricle
Left
ventricle
21.5 6 1.8†
28.9 6 3.2
29.9 6 2.6†
0.8 6 0.12*
1.1 6 0.05‡
80.3 6 4.9
266 6 21
18.3 6 1.9†
33.6 6 3.3
38.8 6 2.6
0.6 6 0.1*
1.2 6 0.08
90.7 6 4.8
285 6 19
Plasma
27 6 3
121 6 9
613 6 41
761 6 37*
4,271 6 229
Values are means 6 SE for 6 normoxic and 7 hypoxic animals (samples correspond to those in Table 3). Units are expressed in tissue as
nmol/mg protein and in plasma as µM. Total includes (in addition to those listed) the following amino acids: asparagine, serine, histidine,
glycine, threonine, taurine, alanine, methionine, valine, phenylalanine, ileucine, leucine, and lysine, concentrations of which were unaltered
by hypoxia. [Asp], [Glu], and [Gln], aspartate, glutamate, and glutamine concentrations. Significantly different from normoxia: * P , 0.05;
† P , 0.01; ‡ P , 0.001.
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ent carbon substrates, including pyruvate, palmitoyl-Lcarnitine, and glutamate, was compromised in the left
ventricle within 24 h of hypoxic exposure and remained
reduced when animals were maintained in a low-O2
atmosphere. By contrast, substrate oxidation in the
right ventricle was, on average, unaffected by chronic
hypoxia. The sole exception was catabolism of longchain fatty acid, normally the preferred substrate in
the heart, which was decreased. This change, unlike
that found in the left ventricle, did not become apparent until 14 days of hypoxia, even though a marked
elevation of right ventricular work expressed as a rise
in intraventricular pressure and an associated increase
in right ventricular mass was noted by 7 days. 2) The
maximal velocity of one of the rate-limiting enzymes of
glycolysis, hexokinase, was markedly enhanced in both
ventricles, although the increase was modestly greater
on the right side. A substantial rise in the relative
abundance of mRNA for hexokinase I and II was also
found, but the changes in enzyme activity and its
transcripts were not temporally aligned. 3) The intracellular levels of aspartate and the aspartate-to-glutamate concentration ratio were increased in both hypoxic ventricles. Although the levels of glutamine
decreased in these tissues, the glutamine-to-glutamate
concentration ratio was changed only in the right
ventricle, thereby distinguishing the effects of hypoxia
from those of chronic pressure overload. Taken to-
CARDIAC ENERGY METABOLISM AND HYPOXIA
The left ventricle, unlike the right, underwent a
moderate loss of oxidative capacity in response to
chronic hypoxia. The reduction was such that absolute
rates of oxidation became similar in both ventricles,
thereby eliminating the differences between the chambers seen in the normoxic rats (see also Ref. 25). The
general decline of oxidative capacity in the left ventricle of the hypoxia-adapted rat suggests that the
number of mitochondria per myocyte is reduced or the
activity (or content) of key enzymes within the metabolic pathways of the mitochondria is decreased (or
downregulated) in an apparently coordinated manner.
Supporting the latter notion are data which show that
hypoxia simultaneously decreases the maximal velocity of several enzymes of the tricarboxylic acid cycle and
cytochrome aa3 content of rat skeletal muscle cells and
mouse lung macrophages in culture (48). Exposure of
mitochondria isolated from embryos of Artemia franciscana (brine shrimp) to anoxic media has been shown to
result in rapid decline of protein synthesis (31); a
similar effect, a reduction by $90%, was reported as an
early response to hypoxia in turtle hepatocytes (32, 33;
for review see Ref. 19). Protein biosynthesis is an
ATP-demanding process that is very sensitive to a lack
of the nucleotide and a decrease in the ratio of triphosphate to diphosphate nucleotides (23). It is possible,
therefore, that a fall in the synthesis of the relevant
polypeptides is responsible for the fall in oxidative
capacity of the left ventricle described here. Interestingly, incubation of brine shrimp mitochondria with the
respiratory chain inhibitors cyanide and antimycin A
had little effect on protein synthesis, which suggests
that the process may be regulated directly by O2
concentration.
An alternative explanation for the decline in oxidative capacity of the left ventricle is that it occurs in
response to a decrease in energy demand and/or substrate availability, which accompany, or result from,
exposure to low PO2. The content of mitochondrial
enzymes within tissue is not necessarily an inherent
property of a particular cell type but, rather, may be
coupled, over the time average, to the cellular need for
ATP (9, 52). For example, increasing energy demand by
chronic endurance-type exercise (21) or by thyroid
hormone treatment (41) enhances mitochondrial cytochrome content in skeletal muscle and in the heart,
respectively. By contrast, limb immobilization lowers
ATP requirements by the affected musculature, and, as
a consequence, oxidative capacity falls (21), whereas
severe restriction of substrate supply, i.e., starvation,
results in a marked loss of mitochondrial proteins (60).
It has been shown that ascent to high altitude results in
weight loss, diminished food intake, altered absorption
of nutrients, lethargy, and modifications of protein
synthesis (for review see Ref. 27). Moreover, normobaric or hypobaric hypoxia was found to depress whole
body O2 consumption and temperature (14; for brief
review see Ref. 37). The hypoxia-adapted animals also
exhibited signs of growth retardation (Table 1). Although neither whole body O2 consumption, motor
activity, nor food intake was monitored in this study, it
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unaffected in some animal models (46, 59, 61) and in
biopsies of the human heart (6). The present study,
unlike many earlier reports, evaluated the progression
of metabolic changes in the stressed, overloaded right
ventricle and the ‘‘nonoverloaded’’ left ventricle. Our
findings show that the oxidative capacity of the former,
measured with a range of substrates, is not altered by
chronic pressure overload. These data are consistent
with the lack of change in the maximal velocity of
cytochrome-c oxidase, the terminal, rate-limiting reaction of the electron transport chain. Oxidation of palmitoyl-L-carnitine, however, was decreased in the right
ventricle from 14 days of hypoxia onward, declining
42% compared with control after 41 days. This decline
was also found in the hypoxic left ventricle, but a
statistically significant change was noted as early as 7
days of hypoxia. Although the direction of these changes
is in agreement with the results of Kinnula and Hassinen (29) on mitochondria from 7-day hypoxic rats, the
latter authors had to use more severe limitation in O2
delivery (40.8 kPa) to detect a significant fall in fatty
acid oxidation.
The mechanism of the reduction of fatty acid catabolism is not known. Previous studies in models of
pressure (47) and volume (8) overload showed a fall in
capacity to oxidize long-chain fatty acids that was
associated with reduced myocardial levels of carnitine.
Oxidation of medium-chain fatty acids, which diffuse
freely into the inner mitochondrial space, however, was
unaffected by chronic overload (8). The latter finding
suggested that the decrease in utilization of long-chain
fatty acids was caused by the inability of these molecules to cross the inner mitochondrial membrane for
lack within the cardiac myocyte of adequate carrier,
carnitine (47, 68). Our data obtained in the presence of
an adequate (5 mM) level of carnitine indicate that a
component, or components, of fatty acid catabolism
must also be adversely affected by long-term hypoxia.
Mitochondrial metabolism of fatty acids is a complex
process. It utilizes several proteins before the electrons
enter the respiratory chain; they include two transporters, carnitine acyltransferases I and II, and four
enzymes, acyl-CoA dehydrogenase (plus electrontransferring flavoprotein), enoyl-CoA hydratase, b-hydroxyacyl-CoA dehydrogenase, and acyl-CoA acetyltransferase. In principle, each of these molecules could
be influenced by prolonged hypoxia. However, because
the enzymes involved in b-oxidation resemble the proteins of the ‘‘proper’’ respiratory chain, whereas catabolism of palmitoyl-carnitine is the one that is specifically
decreased in the right ventricle, one could postulate
that chronic lack of O2 and/or consequences thereof
target one of the inner membrane transfer proteins.
Indeed, changes in the activity of carnitine palmitoyltransferase I have been noted previously (66) after a
5-h incubation of neonatal cardiac myocytes in substrate- and (essentially) O2-free media. Irrespective of
the mechanism, the present data suggest that limitation of O2 availability, rather than chronic pressure
overload, is a more important factor in determining the
final level of tissue utilization of long-chain fatty acids.
H77
H78
CARDIAC ENERGY METABOLISM AND HYPOXIA
dehydrogenase. Lactate dehydrogenase, which is ‘‘better’’ suited for anaerobic metabolism, has been shown
to be induced by hypoxia in cultured cells (12, 48) and
the human heart (15).
Our results show a marked, about twofold, increase
of myocardial aspartate concentration and a lowering
of glutamine concentration in both ventricles, which
suggest that metabolism of these two amino acids is
interrelated. Amino acids are not major substrates for
generation of ATP in the heart, although it has been
postulated that in hypoxia-ischemia myocardial glutamate helps provide high-energy phosphates through
substrate-level phosphorylation (45, 54, 69). However,
glutamate could also elicit beneficial effects in hypoxia
by other mechanisms. This amino acid is produced in
the cardiac muscle from glutamine [the high level of
which in plasma ensures an abundant supply to the
myocardium (30)] by a phosphate-activated glutaminase (40, 43) and can provide 2-oxoglutarate via the
action of glutamate dehydrogenase or aminotransferases. Increased provision of 2-oxoglutarate protects
against depletion of the tricarboxylic acid cycle intermediates and, hence, preserves oxidative capacity (7).
Administration of glutamine has been shown to be
cardioprotective, i.e., to preserve mechanical function
and levels of high-energy phosphates, during acute
periods of ischemia-reperfusion (28). The decrease in
the glutamine-to-glutamate concentration ratio in the
right ventricle of the hypoxic animals may be indicative
of a greater contribution by glutamine to support the
increased ATP requirements of the overloaded tissue.
On the other hand, enhanced operation of alanine
aminotransferase allows glycolysis to proceed without
accumulation of lactic acid (with alanine as the end
product that is readily lost via circulation), whereas
generation of aspartate by aspartate transaminase
supports the malate-aspartate shuttle. The latter is the
main mechanism in the heart for transport of reducing
equivalents from the cytosol to the mitochondrion (53).
Our finding that the aspartate-to-glutamate concentration ratios in hearts from hypoxic animals were increased indicates (56) that flux through the malateaspartate shuttle was decreased under O2-limited
conditions.
The molecular mechanisms and cellular processes
responsible for transforming cells such as cardiac myocytes, which are normally sensitive to hypoxia, to cells
that are more tolerant of O2 deprivation are not known.
Unicellular organisms are capable of adjusting the
levels of energy-producing proteins, in particular the
terminal oxidases, toward aerobic or anaerobic respiration (cytochrome bo to cytochrome bd), depending on
the availability of O2 (for review see Ref. 5). Recently, it
has been reported that the DNA-binding activity of
transcription factors involved in coordination of mitochondrial protein expression in eukaryotes, i.e., the
nuclear respiratory factors NRF-1 and NRF-2 (10; for
review see Ref. 55), is modulated by the redox state of
the cell (36). In addition, low levels of O2 activate a
transcription factor, termed hypoxia-inducible factor,
HIF-1a. The latter, which affects the upregulation of
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cannot be ruled out that locomotion and other specific
ATP-requiring processes were suppressed during hypoxic adaptation. In this case, a fall in mitochondrial
enzyme activity of the left ventricle might be expected
to result from a lowering of cardiac work. This conclusion is consistent with the suggestion of Hochachka and
co-workers (19) that suppression of metabolic work and
sparing of O2 for ATP generation is a more efficient
survival strategy than enhancing ventilatory rate for
O2 convection.
During O2 deprivation, substrate-level phosphorylations within the glycolytic pathway and the tricarboxylic acid cycle may supplement the need for ATP
generated by oxidative mechanisms. In the present
study, hexokinase activity was elevated by nearly twofold in the right ventricle, which should have augmented glycolytic energy production and compensated,
to some extent, for the loss of oxidative capacity. The
relative abundance of mRNA for hexokinase I and II
was also increased in the same chamber, suggesting a
rise in the content of the relevant protein. However,
this followed, rather than preceded, the change in
activity. Similar apparent lack of synchronization for
hexokinase II has been reported previously for skeletal
muscle subjected to chronic electrical stimulation (20).
Such behavior indicates that a simple precursorproduct relationship may not apply to all conditions. In
response to changing energy demands, stimulation of
hexokinase activity may arise from association of the
enzyme with the mitochondrial membrane, thereby
taking advantage of locally produced ATP (44). It is also
possible that each enzyme operates best at a certain
range of velocities, and only when that is exceeded
must the amount of the protein itself increase (9). This
ensures maintenance of almost constant, optimal activity per unit of enzyme and explains the temporal
relationship between hexokinase activity and the
amount of its message seen in the present work.
Enhancement of glycolytic capacity seen here in
hypoxia-adapted animals likely represents a response
to chronic ventricular overload and to hypoxia per se.
Earlier studies have indicated that, depending on the
prevailing hormonal state, phosphorylation of glucose
is rate limiting for its utilization during sustained
contractile activity (20, 26). Expression of GLUT-1, the
minor transporter isoform, was shown previously to be
greater in the hypoxic (14 days of adaptation) than in
the normoxic rat heart and significantly higher in the
right than in the left ventricle, suggesting an ‘‘additive’’
effect of pressure overload and hypoxia (58). A similar
trend for hexokinase mRNA expression and enzyme
activity was found in the present work. Increased
glycolytic capacity resulting from chronic overload, but
in the absence of hypoxia, has been reported previously
by Taegtmeyer and Overturf (62). In the latter study,
this was accompanied by a change in the proportion of
lactate dehydrogenase isozymes, i.e., from the cardiac
to the skeletal muscle type (see also Refs. 3 and 13). In
our hands, the total activity of lactate dehydrogenase
was unaffected. There was, however, an apparent upregulation in both ventricles of the A isoform of lactate
CARDIAC ENERGY METABOLISM AND HYPOXIA
erythropoietin, vascular endothelial growth factor, and
a number of glycolytic enzymes (5, 42, 67), may also be
under redox control (22). Therefore, it is conceivable
that O2 directly, or via its influence on the cellular
metabolic state, regulates the expression of proteins
within the energy-producing pathways, thus enabling a
defensive response to hypoxic environments. A greater
understanding of the molecular mechanism(s) underlying these adaptive changes may offer improved strategies for therapeutic interventions needed for O2-related
pathology.
Received 2 April 1998; accepted in final form 11 September 1998.
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