From Pulmonary and Critical Care Medicine, The University of
Chicago, Chicago, Illinois 60637
During myocardial hibernation, decreases in
coronary perfusion elicit inhibition of contraction, suggesting that
energy demand is attenuated. We previously found an inhibition of
contraction and O2 consumption during hypoxia (3%
O2; PO2 = 20 torr for >2 h) in
cardiomyocytes, which was reversible after reoxygenation. This study
sought to determine whether mitochondria function as cellular
O2 sensors mediating this response. Embryonic
cardiomyocytes were studied under controlled O2 conditions.
Hypoxia produced no acute decrease in mitochondrial potential as
assessed using tetramethylrhodamine ethylester (TMRE). Cellular [ATP]
was preserved throughout hypoxia, as assessed using the probe Magnesium
Green. Thus, ATP synthesis and utilization remained closely coupled. Cells adapted to hypoxia for >2 h exhibited a 4% increase in
mitochondrial potential upon reoxygenation, suggesting that a
partial inhibition of cytochrome c oxidase had
existed. To test whether the oxidase serves as an O2
sensor, azide was administered (1 mM) to simulate the
effects of hypoxia by lowering the Vmax of the
oxidase. The effects of azide on contraction and mitochondrial
potential mimicked the response to hypoxia. We conclude that partial
inhibition of cytochrome oxidase during hypoxia allows mitochondria to
function as the O2 sensor mediating the decreases in ATP
utilization and O2 consumption during hypoxia.
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INTRODUCTION |
Regional decreases in myocardial oxygen delivery have been shown
to result in decreased contractile activity and O2
consumption in a phenomenon termed hibernating myocardium.
For example, Arai et al. (1) induced a 30% reduction in
coronary blood flow in swine and observed an early depletion of ATP and
phosphocreatine (PCr)1 and an
increase in tissue lactate followed by a later recovery of PCr and
lactate, despite continued hypoperfusion and impaired contraction.
During progressive decreases in coronary blood flow, the same group
found that regional decreases in contractile function occurred without
a sustained decrease in the PCr to ATP ratio (2). Other investigators
have also found evidence of myocardial hibernation in intact hearts
(3-5). Collectively these results indicate that the myocardium can
develop significant contractile inhibition during reductions in blood
flow without apparent evidence of ischemia. This would appear to
represent an adjustment in ATP demand in response to a decrease in
regional O2 supply, which could protect the myocardium from
ischemic injury in states where blood flow is reduced more severely
(6). However, the mechanisms underlying this response are not fully
known (5).
The inference that intact myocardium can down-regulate energy
requirements and ATP demand during hypoxia suggests that cardiac myocytes may behave similarly. In contracting embryonic cardiomyocytes, we previously observed decreases in contractile motion and in the rate
of O2 uptake during prolonged moderate hypoxia
(PO2 = 20-40 torr for 1-2 h) (7). Moreover,
this inhibition was reversible within 3 h after return to
normoxia, which suggests that cardiac myocytes can detect moderate
hypoxia and initiate a suppression of ATP utilization in response.
Recently, Silverman et al. (8) found decreases in extent of
shortening in rat cardiac myocytes after incubation under 1%
O2 for 48 h, which is consistent with our observations
and reveals that this response is not unique to embryonic cells.
Collectively, these findings suggest that cardiomyocytes can respond to
moderate hypoxia by reversibly decreasing contractile activity, at
PO2 levels that should have been sufficient to
sustain mitochondrial respiration. Although hibernating myocardium is a
phenomenon of intact hearts by definition, studies of cellular responses to hypoxia may provide insight into the mechanisms involved in the intact ventricle.
A fundamental question in understanding the mechanism underlying the
response to hypoxia relates to how cardiac myocytes detect changes in
PO2. An ability to adjust cellular respiration
in response to PO2 implies the existence of a
sensor capable of detecting changes within the physiological range. It
is conceivable that such an O2 sensor could then activate a
signaling pathway leading to a down-regulation of contractile motion,
energy utilization, and oxygen demand. Recent evidence points to the
mitochondrial electron transport chain as a possible site of
O2 transduction (9). In this regard, we previously found a
reversible inhibition in cytochrome c oxidase
Vmax during hypoxia, as evidenced by
PO2-dependent decreases in
TMPD-ascorbate respiration (7). Moreover, kinetic studies of isolated
bovine heart cytochrome oxidase confirmed the existence of
PO2-dependent alterations in
Vmax (10). A
PO2-dependent change in the kinetic
activity of cytochrome oxidase could elicit changes in mitochondrial
redox state, which could confer a sensitivity to
PO2 and allow the mitochondria to act as the
cellular O2 sensor. In the present study we assessed the
effects of moderate hypoxia and reoxygenation on mitochondrial
transmembrane potential to determine whether changes in the function of
the oxidase were apparent in the intact myocyte, and whether under
normoxic conditions the cytochrome c oxidase inhibitor
sodium azide could mimic the reversible decrease in contraction
observed during hypoxia.
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MATERIALS AND METHODS |
Cardiac Myocyte Isolation--
Myocytes were isolated using a
method modified from Barry et al. (11) and previously
described (7). Briefly, hearts of 10-11-day-old chick embryos were
removed and placed in Hanks' balanced salt solution without magnesium
and without calcium (Life Technologies, Inc.). The ventricles were
minced, and the cells were dissociated using four to six cycles of
trypsin (0.025%, Life Technologies, Inc.) degradation at 37 °C with
gentle agitation. Trypsin digestion was halted after 8 min by
transferring dissociated cells to a trypsin inhibitor solution. After
filtering (100 µm), the cells were centrifuged for 5 min at 1200 rpm
at 4 °C and then resuspended in nutritive media (54% Barry's
solution (in mM: NaCl (116), KCl (1.3), NaHCO3
(22), MgSO4 (0.8), NaH2PO4 (1.0),
CaCl2 (0.87), glucose (5.6)), 40% M199 with Earle's salts
(Life Technologies, Inc.), 6% heat-inactivated fetal bovine serum and
penicillin (100 units/ml), and streptomycin (100 mg/ml)). These cells
were then placed in a large Petri dish in a humidified incubator (5%
CO2, 95% air at 37 °C) for 45 min to allow early
adherence of fibroblasts. The nonadherent cells were then enumerated
(hemacytometer), their viability confirmed at >85% (trypan blue) and
between 0.6 and 1.5 × 106 cells were plated on glass
coverslips (25 mm) in nutritive medium. Cell yield averaged 5-6 × 105 cells per embryo. Cells were maintained in a
humidified incubator for 2-3 days, at which point synchronous
contractions of the monolayer were noted. All experiments were
performed on spontaneously contracting cells at day 3 or 4 after
isolation, at which point cell viability was typically >99%.
Perfusion System--
Synchronously contracting cardiac myocytes
on 25-mm glass coverslips were placed in a stainless steel flow-through
chamber (2-ml volume). This flow-through chamber was inserted into a
heated platform (37 °C) (Warner Instruments) on an inverted
microscope stage. A water-jacketed glass equilibration column
(37 °C) mounted above the microscope stage was used to equilibrate
the perfusate to known oxygen tensions. The perfusate used during
experiments consisted of a buffered salt solution (in mM:
NaCl (117), KCl (4.0), NaHCO3 (18), MgSO4
(0.8), NaH2PO4 (1.0), CaCl2 (1.21), glucose (5.6)) that was bubbled with 5% CO2 at different
O2 concentrations. The gas used to control the
PO2 and PCO2 of the
perfusate was supplied by a calibrated precision mass flow controller
(Cameron Instruments). A short length of stainless steel or
polyethylethylketone tubing was used to connect the equilibration
column to the flow-through chamber to minimize the leakage of ambient
O2 into the perfusate. In some studies, the
PO2 in the chamber was confirmed using an optical phosphorescence quenching method (Oxyspot, Medical Systems Inc.) In those studies, a porphin-based dye (20 µM) bound
to albumin (5% w/v) was added to the perfusate (12, 13) and the
fiberoptic light guide was positioned above the upper glass coverslip
of the flow-through chamber. In those studies, the
PO2 in the chamber was determined from the
O2-dependent phosphorescence decay following a
pulse of excitation light generated by a xenon flash lamp.
Data Acquisition and Analysis--
The inverted microscope was
equipped for epifluorescent illumination and included a xenon light
source (75 W), a cooled 12-bit digital CCD camera (Princeton
Instruments), a shutter and filter wheel (Sutter), and appropriate
excitation and emission filters. Data were acquired and analyzed using
Metamorph software (Universal Imaging).
Contractile motion of cells was recorded at video rates on magnetic
tape using a high resolution video camera (Hammamatsu), as described
previously (7). Briefly, the cells were illuminated with visible light
at low intensity using Hoffman modulation optics (Modulation Optics,
Inc.). This optical system tended to accentuate the changes in surface
topology that were apparent during contraction. Approximately 1 min of
contractile motion was recorded at each stage for later analysis.
Recorded sequential video frames were digitized and pixels were
assigned an intensity value ranging from 0 to 255. For each pixel, the
absolute change in intensity was summed over ~250 frames. These
summed changes in intensity were summed for all pixels, providing a
single measure of motion in the field that consistently described the
motion that already was evident by inspection. The contraction analysis
was carried out as a series of macroinstructions using the Metamorph
software (Universal Imaging).
Measurement of Mitochondrial Transmembrane Potential--
The
mitochondrial transmembrane potential was measured using the cationic
dye tetramethylrhodamine ethylester (TMRE, Molecular Probes). This
fluorophore enters the cells and is accumulated in mitochondria
according to the Nernst equation (14, 15) and has been used previously
to assess relative changes in mitochondrial potential. Unlike with
rhodamine 123, we did not observe quenching of cellular fluorescence
with TMRE unless excessive dye was loaded into the cells. Excitation
(535 nm), dichroic (565 nm), and emission (610 nm) filters were used
(Chroma Technology). To minimize photobleaching of the dye, the
excitation intensity was attenuated with a neutral density filter, and
exposure times were limited to 100 ms. Coverslips with spontaneously
contracting cells were loaded for 1 h with TMRE (100 nM) in a humidified incubator at 37 °C. The cells were then placed in the flow-through chamber and continuously perfused with
the buffered salt solution containing TMRE (10 nM). After allowing 60-90 min for equilibration, a digital image was obtained of
a field of ~40 cells using a 40× oil immersion objective lens. Using
the data acquisition software, individual cells or clusters of several
(<10) cells were identified as regions of interest, and background was
identified as an area without cells or with minimal cellular
fluorescence. Subsequently, sequential digital images were obtained
every 1-3 min, and the average fluorescence intensity for all of the
cell regions and background was recorded for later analysis. TMRE
fluorescence intensity is reported as the average of the fluorescence
of all identified cell regions, less background, for each
coverslip.
Assessment of Cellular ATP Hydrolysis--
ATP has a greater
affinity for Mg2+ than does ADP, and the cytosolic ionized
magnesium concentration increases during ATP hydrolysis. The increases
in cytosolic [Mg2+] can be assessed using the
intracellular fluorescent indicator Magnesium Green (MgG), and the
behavior of this dye in cardiomyocytes has been studied in detail by
Leyssens et al. (16). We measured MgG fluorescence
(excitation 480 nm, emission 535 nm) in contracting cardiomyocytes
during prolonged hypoxia and reoxygenation to assess ATP hydrolysis.
Cells on coverslips were loaded with MgG in the acetoxymethyl ester
form (5 µM, Molecular Probes) for 30 min at 37 °C in a
humidified incubator. Subsequently, the cells were transferred to the
flow-through chamber and perfused with buffered salt solution under
controlled O2 and CO2 conditions. To abolish the capacity for anaerobic glycolysis, 2-deoxyglucose (20 mM) was added to the perfusate, along with pyruvate (5 mM) to support mitochondrial respiration. Cells continued
to contract spontaneously under these conditions. Fluorescent images
were collected for multiple regions of interest every 60 s, using
100-ms exposure times. Average fluorescence was then calculated for
each coverslip and analyzed as a percentage of initial values after
subtraction of background fluorescence. At the end of the study, the
mitochondrial uncoupler FCCP was added (5 µM) to induce
ATP hydrolysis to confirm activity of the probe.
Reagents--
In different experiments the cells were treated
with the mitochondrial uncoupler FCCP, the electron transport inhibitor
myxothiazol, the mitochondrial ATP synthase inhibitor oligomycin and
2,3-butanedione monoxime (BDM) (Sigma). Using a spectrofluorometer, all
of these compounds were found to exhibit insignificant fluorescence at 565 nm or quenching when excited at 530 nm (data not shown).
Statistical Analysis--
Replicate experiments were carried out
independently using separate coverslips with cells. Data were analyzed
using analysis of variance on the pooled data from the intervention and
control experiments. Statistical significance was determined at the
0.05 level. Data are presented as mean values ± S.E.
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RESULTS |
Effects of Prolonged Moderate Hypoxia on Contractile Motion in
Cultured Cardiomyocytes--
The contractile response to moderate
hypoxia and reoxygenation was studied in cultured cardiomyocytes (Fig.
1). After 2 h under normoxic
conditions (PO2 = 100 torr), the perfusate
PO2 was reduced to ~20 torr (3%
O2) for 3 h. No immediate effect on contraction was
noted, but within 1-2 h a significant decrease in motion was
consistently observed. When the perfusate O2 tension was
returned to 100 torr, no immediate effect on contractile motion was
noted. However, a progressive return of contraction developed over 2-3
h, and no significant difference from control levels was apparent at
3 h. These reversible decreases in contraction were similar to
those noted previously (7).

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Fig. 1.
Effect of hypoxia on total motion of
spontaneously contracting cardiomyocytes during perfusion with buffered
salt solutions under controlled O2 conditions. Hypoxia
(PO2 = 20 torr; 3% O2) was
initiated after base-line measurements. Recovery to
PO2 = 100 torr; (14% O2) was
initiated at 180 min, but restoration of contractile motion required
~3 h to reach base-line values (n = 4, p < 0.01).
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Effect of Prolonged Moderate Hypoxia on [ATP] in Contracting
Cardiomyocytes--
Previous studies of noncontracting cardiomyocytes
in suspension indicated that prolonged moderate hypoxia was associated
with a decrease in oxygen consumption rate without a decrease in
cellular ATP or phosphocreatine concentrations (7). To determine
whether ATP depletion occurs during prolonged hypoxia in contracting
cells, MgG fluorescence was used to assess ATP hydrolysis (16). To determine the capability of this dye to detect ATP hydrolysis, cells
loaded with MgG were imaged every 60 s during continuous normoxia
(Fig. 2). After 4 min, the mitochondrial
uncoupler FCCP was administered to limit ATP synthesis, and an abrupt
increase in fluorescence was noted, consistent with a rise in
intracellular Mg2+. In other cells loaded identically, the
ATP synthase inhibitor oligomycin (10 µg/ml) produced a similar
abrupt increase in fluorescence, as predicted (Fig.
3). To determine the effects of hypoxia
on ATP hydrolysis, cells loaded with MgG were imaged every 5 min during
2.5 h of incubation at PO2 = 20 torr or 100 torr. As shown in Fig. 4, a gradual loss
of fluorescence was noted in both groups, which appeared to be a
consequence of the progressive photobleaching behavior of the MgG
probe. However, no difference was noted between control and hypoxic
cells. Moreover, the absence of an increase in fluorescence during
hypoxia suggested that cytosolic [Mg2+] did not increase
as a result of increased ATP hydrolysis. However, when ATP synthesis
was halted by FCCP (5 µM) at the end of the study, a
marked increase in fluorescence indicating an increase in
[Mg2+] was noted.

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Fig. 2.
Effects of mitochondrial uncoupling on MgG
fluorescence in spontaneously contracting cardiomyocytes. Cells
loaded with MgG were perfused under normoxic conditions
(PO2 = 100 torr; 14% O2) and
administered FCCP (5 µM) to inhibit oxidative
phosphorylation. The marked increase in fluorescence suggests an
increase in Mg2+ released as a result of ATP
hydrolysis.
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Fig. 3.
Effects of mitochondrial ATP synthase
inhibition on MgG fluorescence in spontaneously contracting
cardiomyocytes. Cells loaded with MgG were perfused under normoxic
conditions (PO2 = 100 torr; 14% O2)
and administered oligomycin (10 µg/ml) to inhibit oxidative
phosphorylation. The marked increase in fluorescence suggests an
increase in Mg2+ release as a result of ATP
hydrolysis.
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Fig. 4.
Effects of hypoxia on MgG fluorescence in
spontaneously contracting cardiomyocytes. Under base-line
conditions, cells loaded with MgG were perfused with normoxic
(PO2 = 100 torr; 14% O2) solution.
At t = 30 min, perfusate PO2 was
reduced to 20 torr (3% O2) in the hypoxia group
while controls remained normoxic. At t = 180 min, the
mitochondrial uncoupler FCCP (5 µM) was administered to
elicit ATP hydrolysis. No statistical difference between groups was
detected (n = 3).
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Effect of Mitochondrial Inhibitors on Mitochondrial Membrane
Potential as Assessed Using TMRE Fluorescence--
Studies were
carried out to confirm that the fluorescent probe TMRE behaved in a
manner consistent with that expected for a mitochondrial potentiometric
dye. Administration of the mitochondrial uncoupler FCCP would be
expected to rapidly dissipate the mitochondrial electrochemical
gradient. As predicted, addition of FCCP (5 µM) to the
perfusate of contracting cardiac myocytes at t = 0 produced a rapid and marked decrease in fluorescence indicating a loss of potential (Fig. 5a).
Mitochondrial potential is generated by flux through the electron
transport chain, which is coupled to the extrusion of protons from the
matrix. Inhibitors of electron transport should decrease proton
extrusion, resulting in a decrease in the potential across the inner
mitochondrial membrane. Accordingly, the mitochondrial bc1 complex
inhibitor myxothiazol (50 ng/ml) was added to the perfusate of
contracting cardiac myocytes.(Fig. 5b). This produced a
significant decrease in TMRE fluorescence, consistent with a marked
decrease in potential.

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Fig. 5.
a, TMRE fluorescence in cardiomyocytes
administered FCCP (5 µM) to inhibit oxidative
phosphorylation. FCCP was administered at t = 0 s,
and cells were imaged every 60 s. Cells not given FCCP served as
time controls. Mitochondrial inhibition produced a significant decrease
in TMRE fluorescence, as predicted for a potentiometric dye
(n = 3). b, TMRE fluorescence in
cardiomyocytes administered myxothiazol (50 ng/ml) to inhibit oxidative
phosphorylation. Myxothiazol was administered at t = 0 s and cells were imaged every 60 s. Cells not given
myxothiazol served as controls (n = 3).
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Mitochondrial membrane potential is consumed by the
F1F0 ATP synthase. The
synthase allows protons to move down their electrochemical gradient as
they enter the matrix, resulting in the phosphorylation of ADP to ATP
using the resulting free energy release. Inhibition of ATP synthase
should therefore cause hyperpolarization of the mitochondrial membrane.
As shown in Fig. 6, addition of
oligomycin (10 µg/ml) to the perfusate of contracting cardiac
myocytes resulted in a significant increase in TMRE fluorescence.
Similarly, an inhibition of ATP utilization should cause a decrease in
the rate of ATP synthesis by limiting the supply of ADP to the
mitochondria, which should produce hyperpolarization of the
mitochondrial potential. To confirm this, BDM (30 mM) was
added to the perfusate of contracting cardiac myocytes at
t = 0 (Fig. 7). As an
inhibitor of Ca2+-dependent actin-myosin
interaction, BDM produced an immediate and significant increase in TMRE
fluorescence, which was accompanied by an immediate cessation of
contraction. Collectively, these results show that TMRE serves as an
appropriate qualitative measurement of mitochondrial membrane
potential.

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Fig. 6.
TMRE fluorescence in cardiomyocytes
administered oligomycin to inhibit oxidative phosphorylation.
Oligomycin (10 µg/ml) was administered starting at t = 0 s, and cells were imaged every 60 s. Cells not given
oligomycin served as controls. Mitochondrial ATP synthase inhibition
produced a significant increase in TMRE fluorescence, as predicted for
a potentiometric dye (n = 3). The later decrease in
fluorescence likely represents an artifact due to cell
deterioration.
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Fig. 7.
Effects of decreased ATP utilization on TMRE
fluorescence in spontaneously contracting cardiomyocytes. BDM
inhibits Ca2+-dependent actin-myosin
interaction in cardiac muscle, resulting in a decrease in ADP supply to
mitochondria. The subsequent decrease in ATP utilization should result
in a hyperpolarization of the mitochondrial membrane. BDM (30 mM) given at t = 0 s caused an immediate cessation of contractile motion, and elicited a sustained increase in fluorescence in the treated cells while fluorescence in the
control cells remained unchanged (n = 3).
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Effect of Hypoxia and Reoxygenation on Mitochondrial Membrane
Potential--
Mitochondrial membrane potential was assessed during
hypoxia under two different experimental protocols. In the first, TMRE fluorescence in contracting cells was measured in images obtained as
the perfusate O2 tension was decreased from 100 to 25 torr within 10 min (acute hypoxia). As shown in Fig.
8, acute hypoxia produced no acute change
in TMRE fluorescence, which suggests that membrane potential was
maintained. In the second, mitochondrial potential was recorded in
contracting cells that had been perfused for 2 h at
PO2 = 25 torr, as the PO2
was rapidly increased from 25 to 100 torr (acute reoxygenation). Fig.
9a shows that TMRE fluorescence increased significantly during reoxygenation, which suggests that an increase in membrane potential had occurred. This
relative hyperpolarization is consistent with an increase the rate of
electron transport during acute reoxygenation, compared with the rate
of ATP utilization. Interestingly, the change in membrane potential did
not occur until ~4 min after the equilibration mixture was switched
to normoxia. In separate studies, the PO2 within
the cell chamber was assessed under identical conditions of
reoxygenation using a phosphorescence-quenching method (12). Those measurements revealed that the PO2 within
the chamber reached a value of ~60 torr at 4 min, which suggested
that the delay in response was caused by the slow response in
reoxygenating the system (Fig. 9b).

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Fig. 8.
TMRE fluorescence in spontaneously
contracting cardiomyocytes perfused with normoxic
(PO2 = 100 torr; 14% O2)
solution. Cells were imaged every 60 s for 5 min, at which
point the PO2 in the perfusate was rapidly
decreased to 20 torr (hypoxia, 3% O2) or maintained at
normoxia. No significant difference in fluorescence response was
detected between the two groups (n = 3).
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Fig. 9.
a, TMRE fluorescence in spontaneously
contracting cardiomyocytes perfused continuously with hypoxic
media (PO2 = 25 torr) for 90 min. At
t = 0 s, the gas in the equilibration column was increased to PO2 = 100 torr (14%
O2, reoxygenation) or maintained at 25 torr (continued
hypoxia) while fluorescence was measured every 60 s for 600 s. A significant increase in fluorescence was detected in the
reoxygenation group compared with hypoxic controls (p < 0.05). b, change in PO2 within the
flow-through chamber after switching the gas mixture to normoxia at
t = 0 s.
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Effect of Azide on Contraction and Mitochondrial Potential--
To
test whether a partial inhibition of cytochrome oxidase function could
elicit the same response as moderate hypoxia, sodium azide (1 mM) was added to the perfusate in cells maintained under normoxic conditions. In preliminary studies this concentration of azide
was found to be sufficient to reduce the Vmax of
cytochrome oxidase, but too low to limit respiration in cells. When
azide was added, no immediate effect was seen but a progressive
suppression in contractile motion developed over 2 h (Fig.
10). Washout of the azide required
~10 min and was associated with a progressive return of contractile
motion that mimicked the response to hypoxia. To assess the effects of
azide (1 mM) on mitochondrial potential, cells loaded with
TMRE were imaged every minute for 5 min, at which point sodium azide (1 mM) was added to the perfusate (Fig. 11). No acute decrease in potential was
observed, suggesting that membrane potential was preserved. In other
cells loaded with TMRE and incubated with azide for 2 h, washout
of the azide was associated with an acute increase in membrane
potential, suggesting that a relative hyperpolarization of the membrane
had developed. These responses were similar to those seen during onset
and recovery from moderate hypoxia.

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Fig. 10.
Contractile motion of spontaneously
contracting cardiomyocytes perfused with normoxic
(PO2 = 100 torr; 14% O2)
solution. Azide (1 mM) was added after base-line
measurements at t = 0 min. At t = 2 h, azide was removed from the perfusate and cells were allowed
to recover for 2 h (n = 3).
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Fig. 11.
TMRE fluorescence in of spontaneously
contracting cardiomyocytes perfused with normoxic
(PO2 = 100 torr; 14% O2)
solution. Fluorescence images were obtained every 60 s for
700 s. In one group (Azide on), sodium azide (1 mM) was added to the perfusate at t = 300 s. A second group of cells had been continuously superfused with azide (1 mM) for 2 h prior to data collection. At
t = 300 s, azide was removed from the perfusate as
the cells continued to be imaged. A significant increase in
fluorescence was detected after washout of the azide (n = 3).
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DISCUSSION |
Myocardial hibernation is a chronic abnormality of contractile
function associated with coronary hypoperfusion (17). This dysfunction
is reversed if coronary blood flow is restored, which suggests that the
abnormality is not a consequence of ischemic cellular damage. Studies
using 31P NMR spectroscopy suggest inorganic phosphate
levels are not increased during hibernation, which supports the view
that the attenuation of contraction is not a consequence of a reduction in energy stores (5). However, the mechanisms underlying myocardial hibernation are not fully understood.
We previously reported that spontaneously contracting cardiomyocytes
down-regulate contractile motion and O2 consumption during prolonged (1-2 h) moderate hypoxia (PO2 = 25 torr) (7). Cells kept under those conditions for 24 h showed no
decrement in viability, suggesting that the inhibition was not a
consequence of cell damage. Similar findings of decreased contractile
motion with sustained viability have recently been reported in rat
cardiomyocytes cultured under 1% O2 for 48 h (8).
That study indicates that our results are not unique to embryonic
cells. Moreover, it extends our previous work by demonstrating that
hibernating cardiac myocytes were more tolerant to acute severe hypoxic
challenge, in terms of the time required to elicit ATP-depletion. These
studies suggest that a phenomenon similar to myocardial hibernation
develops in cardiac myocytes during prolonged hypoxia, which affords
protection from anoxic stress and is reversible within hours after
recovery to normoxic culture conditions.
Hypoxia Reversibly Decreases Contraction--
In this study,
cardiomyocytes superfused at PO2 = 20 torr
showed progressive decreases in contractile motion that reached 40% of
control levels within ~2 h. Recovery to PO2 = 100 torr was associated with a recovery of contraction, although
several hours were required to reach base-line levels. We previously
observed that cardiomyocytes maintained under normoxic conditions for
~6 h showed no significant changes in contractile motion, so the observed response to hypoxia cannot be explained by the effects of time
alone. During brief exposures to hypoxia, Rumsey et al. (18)
found that adult rat cardiac myocytes could maintain normal rates of
O2 consumption until the extracellular
PO2 fell to less than ~7 torr (18). Moreover,
the observations that cells continued to function normally during the
first hour of hypoxia and that the decreases in contraction were
reversible suggest that the effects of hypoxia we found could not be
explained by an O2 supply limitation of mitochondrial ATP
synthesis.
Nevertheless, to test whether hypoxia elicited ATP supply limitation,
we used Magnesium Green to assess ATP hydrolysis in contracting cells
during moderate hypoxia. The fluorescence of this dye increases with
the intracellular Mg2+ concentration, which increases
during ATP hydrolysis because ATP has a higher affinity for
Mg2+ than does ADP (16). Indeed, marked increases in
fluorescence were seen in normoxic cells treated with the mitochondrial
uncoupler FCCP or with the ATP synthase inhibitor oligomycin, both of
which should inhibit oxidative phosphorylation and cause ATP depletion. However, no increase in MgG fluorescence was seen during prolonged exposure to PO2 = 20 torr, suggesting that ATP
hydrolysis was minimal. We previously measured [ATP] and [PCr] in
quiescent cardiomyocytes and found that cellular levels were preserved
during prolonged moderate hypoxia. The present study extends those
results by showing that ATP levels are similarly preserved in
contracting cells during prolonged hypoxia.
To further test whether hypoxia elicited ATP supply limitation, we
assessed mitochondrial potential using the dye TMRE. Mitochondrial membrane potential provides the driving force for ATP synthesis. The
potential is generated by the supply of NADH through the matrix dehydrogenases and electron flux through electron transport chain. The
ATP synthase uses the energy from mitochondrial membrane potential to
synthesize ATP from ADP plus inorganic phosphate. In steady state, the
mitochondrial membrane potential therefore reflects a balance between
the rate of electron transport and the rate of ATP utilization by the
cell. An inhibition within the electron transport chain during hypoxia
would therefore result in a decrease in electron flux and a
depolarization of the membrane, while a sudden inhibition of ATP
utilization or an inhibition of the ATP synthase should produce a
relative hyperpolarization. Indeed, these responses were observed when
electron transport was inhibited with myxothiazol or when the ATP
synthase was inhibited with oligomycin. Therefore, if hypoxia limited
ATP synthesis by restricting mitochondrial electron transport, then a
profound decrease in membrane potential should have been evident at the
start of hypoxia in the TMRE studies. But mitochondrial potential was
maintained during acute hypoxia, which suggests that ATP utilization
and ATP synthesis remained closely matched. This conclusion is
consistent with the absence of ATP hydrolysis indicated by the MgG
data. Collectively, these findings suggest that hypoxia decreases
respiration in cardiomyocytes by activating a signaling pathway that
causes a reduction in contraction and ATP demand, rather than by
limiting ATP supply.
Hypoxia Causes a Partial Mitochondrial Inhibition--
Although
mitochondrial potential was not depleted during hypoxia, small
increases in potential were detected at reoxygenation. This suggests
that there was a rapid increase in electron transport without a
corresponding increase in ATP utilization. Such an increase would be
predicted if a partial inhibition of cytochrome oxidase were rapidly
removed at reoxygenation. In previous studies of the oxidase, we have
observed an immediate increase in Vmax upon reoxygenation after prolonged hypoxia (9, 10, 19). We therefore conclude that the changes in mitochondrial potential at reoxygenation reflect functional changes in the kinetics of the oxidase that develop
during prolonged hypoxia. These observations indicate that mitochondria
can respond to changes in O2 tension within the
physiological range, which could allow them to function as an
O2 sensor.
Cytochrome c Oxidase Serves as an O2
Sensor--
Sodium azide is a noncompetitive inhibitor of cytochrome
c oxidase (20). At high concentrations (5-10
mM) it can throttle respiration by limiting O2
consumption, resulting in an immediate cessation of contraction and
depletion of mitochondrial potential. By contrast, low concentrations
(
1 mM) merely reduce the Vmax of
the enzyme without limiting basal respiration. Our previous studies
indicated that prolonged hypoxia produces a reversible decrease in
Vmax of the oxidase (7, 9, 10), which is not sufficient to limit basal respiration. Thus, low concentrations of
azide could mimic the effects of hypoxia on the enzyme. Our observation
that azide during normoxia produced the same changes in contractile
function and mitochondrial potential seen during moderate hypoxia
implicates cytochrome oxidase as the oxygen sensor underlying the
functional response to hypoxia.
The observation that cells respond to physiological levels of
O2 suggests the existence of a signal transduction pathway
linking the sensor to the functional response. Some investigators have argued that cytochrome oxidase is ill-suited to function as an O2 sensor because its apparent Km for
O2 is <1 µM (8, 21). Therefore, at
physiological O2 concentrations the velocity of such an
enzyme system would remain independent of O2 until near-anoxic conditions were reached, limiting its ability to signal changes in the physiological range. However, changes in the
Vmax of the oxidase produced with hypoxia or
azide (1 mM) should increase the reduction state of
mitochondrial electron carriers upstream from the oxidase (19). It is
conceivable that subsequent redox-linked activation of second messenger
systems (22, 23) could then lead to an inhibition of ATP utilization
within the cell. Although not yet confirmed, the above sequence would
be consistent with (a) the previously observed changes in
cytochrome oxidase Vmax during hypoxia,
(b) the observed changes in mitochondrial potential during
hypoxia and reoxygenation, (c) the possible delay between onset of hypoxia or reoxygenation and the changes in cell motion and
ATP demand, and (d) the sustained coupling between
cellular respiration and ATP demand as indicated by membrane
potential and MgG. However, further study will be required to
fully identify the signaling cascade(s) mediating the functional
response.
Significance for Intact Tissues--
A number of similarities
between the response to hypoxia in cardiomyocytes and that seen in
hibernating myocardium should be noted. First, both appear to involve a
matching between ATP utilization and O2 supply without
evidence of ischemia. Second, both conditions are fully reversible upon
restoration of base-line conditions, without loss of cell viability or
necrotic damage. One notable difference is that contractile function in
hibernating myocardium recovers immediately upon restoration of flow
(24), whereas recovery of cardiomyocytes required 2-3 h after
restoration of normoxic conditions. Although the present study provides
insight into the mechanisms contributing to the response to hypoxia in cardiomyocytes, further work will be required to clarify the
relationship of these findings to the events in the intact hibernating
myocardium.