(Circulation. 2000;102:3124.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Hibernation in Noncontracting Mammalian Cardiomyocytes
From the Department of Biochemistry, The University of Western Australia, Nedlands, Australia.
Correspondence to Peter Arthur, PhD, Department of Biochemistry, The University of Western Australia, Nedlands 6907, Australia. E-mail: parthur{at}cyllene.uwa.edu.au
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Abstract |
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Background—The aim of the present study was to establish whether isolated neonatal mammalian cardiomyocytes were capable of downregulating energy-using processes other than contraction while maintaining metabolic stability when oxygen availability was reduced.
Methods and Results—Metabolic response of cardiomyocytes was investigated under moderate (5 to 6 µmol/L) and severe (2 to 3 µmol/L) forms of hypoxia. Cells exposed to oxygen concentrations of 5 to 6 µmol/L exhibited rates of oxygen consumption, which were decreased to 64% of normoxic rates. Rates of cellular energy usage were decreased because this reduced rate of oxygen consumption was not associated with either decreased intracellular ATP and phosphocreatine concentrations or a compensatory switch to glycolysis. When cells were exposed to oxygen concentrations of 2 to 3 µmol/L, rates of oxygen consumption decreased to 9% of normoxic rates. This decreased rate of oxygen consumption was associated with energetic stress, because a significant switch to glycolysis occurred and intracellular phosphocreatine concentrations were decreased by 40%. Rates of cellular energy usage were further decreased as indicated by stable intracellular ATP concentrations.
Conclusions—Our results suggest that isolated cardiomyocytes are capable of downregulating energy-consuming processes other than contraction when oxygen supply is decreased. Regions of myocardial tissue are also capable of downregulating metabolic activity during ischemia by shutting down contractile activity (myocardial hibernation). We suggest that metabolic downregulation associated with myocardial hibernation may not be exclusively due to reduced rates of contractile activity. Other energy-using processes (eg, protein synthesis, mRNA synthesis, ion channel activity, and proton leak) may also be shut down.
Key Words: hibernation • hypoxia • metabolism • myocytes • respiration
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Introduction |
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The term "hibernating myocardium" is used to describe clinical observations in which a region of intact myocardium remains viable through a decrease in contractile function when myocardial blood flow is reduced and that at reperfusion completely recovers contractile function.1 Myocardial hibernation with functional recovery has been demonstrated clinically in patients undergoing coronary artery bypass surgery2 3 4 5 and in various animal models.6 7 8 However, such metabolic downregulation has been measured directly only in animal models, by determination of rates of oxygen consumption and lactate output and of intracellular ATP and PCr concentrations.8 9 10
A similar response, "oxygen
conformance,"11 has been
observed in various isolated cell preparations, including adult rat
hepatocytes11 and
cardiomyocytes12
and embryonic chick
cardiomyocytes.13 14
Budinger et al13 demonstrated
that suspensions of isolated embryonic chick cardiomyocytes
were capable of deceasing their rates of oxygen consumption and
contractile activity at oxygen concentrations as high as 
63
µmol/L. The authors concluded that isolated embryonic chick
cardiomyocytes responded to reduced oxygen availability by
downregulating energy-using pathways associated with
contraction.
The abilities of ischemic regions of the myocardium to undergo hibernation and of isolated cardiomyocytes to conform to hypoxic oxygen concentrations suggest that cardiomyocytes contain oxygen sensors involved in regulation of contractile activity. Studies of hibernating myocardium suggest that only contractile activity is downregulated during ischemia. Because contractile activity does not always immediately recover after revascularization,5 15 it seems likely that other energy-using processes may also be shut down. A study by Piper et al16 supports this suggestion, given that rates of energy consumption in quiescent adult rat cardiomyocytes were shown to be reduced under anoxic conditions. However, rates of ATP usage were measured only under anoxic conditions, and as a consequence intracellular ATP concentrations were not stable. The purpose of the present study was to determine whether isolated mammalian cardiomyocytes were capable of downregulating energy-using processes other than contraction while maintaining metabolic stability when exposed to moderate (5 to 6 µmol/L) and severe (2 to 3 µmol/L) forms of hypoxia. Our results show that rates of cellular energy usage were decreased under both forms of hypoxia because no compensatory switch to anaerobic glycolysis occurred and intracellular ATP concentrations remained stable. We suggest that contractile activity is not the only energy-using process downregulated during hypoxia in cardiomyocytes.
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Methods |
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Isolation of Cardiomyocytes
Neonatal cardiomyocytes were isolated from the hearts of 1-day-old Sprague-Dawley rats by use of a method described previously.17
Oxygen Consumption Measurements
Cells were resuspended in a Tris-buffered
Krebs-Henseleit solution (137 mmol/L NaCl, 5.4 mmol/L KCl,
1.0 mmol/L NaH2PO4,
0.8 mmol/L MgSO4, 2.0 mmol/L
CaCl2, 5.0 mmol/L Tris, 5.5 mmol/L
glucose, 10 µmol/L verapamil, and 50 µmol/L
NiCl2; pH 7.4 at 37°C), and rates of oxygen
consumption were measured in either the perfusion (flow-through)
system18 or the closed-cell
chamber.19
Analytical Methods
Intracellular concentrations of ATP and
phosphocreatine (PCr) were measured by capillary electrophoresis as
described previously.17
Lactate content in samples of perfusate from the flow-through
system were measured bioluminescently as described
previously.20 Rates of
lactate accumulation in samples from the closed-cell chamber were
assessed spectrophotometrically by use of a method described
previously.18
Calculation of ATP Turnover
Rates of ATP turnover were calculated by determining
yield of ATP from rates of oxidative phosphorylation
(assuming a P:O ratio of
2.58),21
anaerobic glycolysis (assuming 1 mol of ATP is produced per
mole lactate) and ATP and PCr depletion.
Measurement of Glucose Uptake
Rates of glucose uptake were determined in the
closed-cell chamber by calculating rates of glucose oxidation, lactate
production, and glycogen accumulation under normoxic and anoxic
conditions. Rates of glucose oxidation were measured by the method of
Guppy et al.22 Rates of
lactate production were measured by use of a method described
previously.23 Intracellular
glycogen concentrations were measured as described
previously.24 25
Measurement of Glucose Uptake in the Presence
of Fatty Acids
Rates of glucose uptake were additionally measured in
the presence of palmitate 130 µmol/L and oleate 81 µmol/L under
normoxic conditions. The fatty acid mixture was made up in medium 199
according to the method of Guppy et
al.26
Measurement of Lactate Oxidation
Rates of lactate oxidation were measured in the
presence of 1 mmol/L lactate in the closed-cell chamber under
normoxic conditions. Cell suspension (2 mL) was preincubated with
L-[U-C14 ]lactate (2 µCi)
for 30 minutes. Rates of
14CO2
production were determined according to the method of Guppy et
al.22
Respiratory Inhibition With Myxothiazol
Rates of oxygen consumption were measured in the
presence of myxothiazol in the closed-cell chamber. Myxothiazol was
incrementally added to the cell suspension until the rate of oxygen
consumption was inhibited by 95% to 98%. A final myxothiazol
concentration of 6 µmol/L was typically required to achieve this
level of respiratory inhibition.
Statistical Analysis
Results are expressed as mean±SEM. Data were
analyzed by Student’s
t test or repeated ANOVA, with
statistical differences determined by least significant difference post
hoc tests. Data were considered significantly different when
P<0.05.
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Results |
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Neonatal Cardiomyocytes as an Experimental Model for Studying Differences in Normoxic and Hypoxic Cardiac Energy Metabolism
We measured rates of glucose oxidation in the presence and absence of fatty acids and measured rates of lactate oxidation to determine whether isolated suspensions of neonatal cardiomyocytes respond similarly to the whole heart with respect to fuel usage. Rates of glucose uptake in the presence of fatty acids (0.41±0.04 nmol · min-1 · 10-6 cells) were 66±13% lower than rates measured in the absence of fatty acids (2.1±1.3 nmol · min-1 · 10-6 cells; P<0.05; n=3). When provided with lactate as an alternative fuel to glucose, isolated cardiomyocytes oxidized lactate at a rate of 0.10±0.02 nmol · min-1 · 10-6 cells. These results suggest that isolated suspensions of neonatal cardiomyocytes represent a valid model for the study of cardiac energy metabolism, because cells were capable of oxidizing lactate27 and rates of glucose uptake were significantly inhibited when measured in the presence of fatty acids.28
Effect of Extracellular Oxygen Concentration on
Oxygen Consumption
Isolated neonatal cardiomyocytes decreased
their rates of oxygen consumption over a range of oxygen concentrations
in the flow-through system
(Figure 1
).
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Effect of Moderate Hypoxia (5 to 6
µmol/L) on Rates of Oxygen Consumption, Glycolytic Activity, and
Cellular Energy State
Reduced rates of oxygen consumption could be maintained
for 1 hour when neonatal cardiomyocytes were maintained at
5 to 6 µmol/L in the flow-through system
(Figure 2). Rates of oxygen uptake at 5 µmol/L were 64±2%
of rates at 100 µmol/L and remained decreased after 1 hour, at
66±2% of reoxygenated values
(P<0.05). Calculated rates of
lactate output at 5 µmol/L were significantly higher than rates at
100 µmol/L (1.2±0.3 versus 0.9±0.2
nmol · min-1 · 10-6
cells at 5 and 100 µmol/L, respectively;
P<0.05), whereas rates of
lactate output after 1-hour incubation were not significantly higher
than reoxygenated rates (1.1±0.2 versus 1.0±0.2
nmol · min-1 · 10-6
cells at 6 and 100 µmol/L, respectively). Rates of ATP turnover at 5
µmol/L were 68±2% of rates at 100 µmol/L and after 1 hour of
hypoxia were 69±2% of reoxygenated rates
(P<0.05;
Figure 2).
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), lactate output (
), and ATP turnover (
). Measurements were
taken as described. Oxygen concentration surrounding cells was adjusted
to 100 µmol/L before being decreased to 5 µmol/L; cells then were
maintained at 5 to 6 µmol/L for 1 hour before being
reoxygenated to 100 µmol/L.
*P<0.05 vs rates of oxygen
consumption, lactate output, and ATP turnover at initial oxygen
concentration of 100 µmol/L;
n=7.Results from experiments in the closed-cell chamber revealed that magnitude of metabolic downregulation was dependent on duration of hypoxic exposure. Oxygen concentration in the closed-cell chamber decreased at a rate proportional to the rate of oxygen consumption by the cells contained within it. This process allowed measurements to be taken more rapidly: oxygen concentration usually decreased from 100 to 5 µmol/L in 15 minutes in the closed-cell chamber versus 60 minutes in the flow-through system. Rates of oxygen consumption at 5 µmol/L were 82±3% of rates measured at 100 µmol/L (P<0.05; n=3). Degree of shutdown in oxygen consumption between the systems was significantly different (P<0.05) for closed-cell chamber (n=3) versus flow-through (n=7) experiments. Rates of oxygen consumption under normoxic conditions in the closed-cell chamber (1.5±0.1 nmol · min-1 · 10-6 cells) compared well to rates measured in the flow-through system (2.2±0.2 nmol · min-1 · 10-6 cells).
Intracellular ATP and PCr concentrations were measured to
establish whether decreased rates of ATP turnover were associated with
reduced rates of ATP usage. No significant differences existed in
intracellular ATP and PCr concentrations between normoxic and hypoxic
conditions with the flow-through system
(Figure 3). When experiments were performed in the
closed-cell chamber, ATP and PCr concentrations did not change
significantly between 100 and 5 µmol/L. Rates of oxygen consumption
and degree of metabolic downregulation were also measured
in the absence of contractile inhibitors and were not
significantly different than results presented above (data not
shown).
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) and PCr (
Effect of Severe Hypoxia (2 to 3
µmol/L) on Rates of Oxygen Consumption, Glycolytic Activity, and
Cellular Energy State
In experiments with the flow-through system, cellular
rates of oxygen consumption at 3 µmol/L were 13±2% of rates at 100
µmol/L (P<0.05), and after
hypoxic incubation for 1 hour, rates remained decreased, at 9±2% of
reoxygenated rates
(P<0.05;
Figure 4). Rates of lactate output were significantly higher
under hypoxic conditions
(Figure 4). Rates of ATP turnover at 3 µmol/L were 36±2%
of rates at 100 µmol/L, and after incubation for 1 hour at 2 to 3
µmol/L, rates remained decreased, at 40±2% of the
reoxygenated value
(P<0.05;
Figure 4).
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Rates of oxygen consumption at 2 µmol/L were 64±3% of rates at 100 µmol/L when measured in the closed-cell chamber (P<0.05; n=3). This degree of shutdown in oxygen consumption was significantly less than when measured at 2 to 3 µmol/L in the flow-through system (P<0.05; n=3 for closed-cell experiments and n=4 for flow-through experiments).
No significant difference in ATP concentrations existed
between normoxic and hypoxic conditions
(Figure 5). Intracellular PCr concentrations decreased
significantly when oxygen concentrations were decreased to 2 to 3
µmol/L and with reoxygenation recovered to 96±13%
of initial measurement
(Figure 5).
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Rates of Glucose Uptake Under Normoxia and
Anoxia
Rates of glucose uptake were measured in the
closed-cell chamber so that we could determine whether increased rates
of lactate output under hypoxia were metabolically
significant. Normoxic rates of glucose uptake were 2.1±1.3
nmol · min-1 · 10-6
cells compared with 0.9±0.1
nmol · min-1 · 10-6
cells under anoxia. Lactate production accounted for a
significantly increased proportion of glucose uptake when oxygen
concentrations were reduced from normoxia to anoxia: 21±7% and
99±1%, respectively (P<0.05;
n=3). Intracellular glycogen accumulated at a rate of 0.9±0.8
nmol · min-1 · 10-6
cells under normoxic conditions and was degraded at a rate of 0.5±0.1
nmol · min-1 · 10-6
cells under anoxic conditions.
Effects of Myxothiazol on Rates of ATP
Depletion
The closed system was used to determine whether the
mechanism by which cardiomyocytes downregulated energy
usage at 2 to 3 µmol/L was dependent on energetic stress. Respiratory
rates of cells maintained under normoxic conditions (100 µmol/L) were
inhibited by 95% to 98% with myxothiazol. Rates of ATP turnover were
calculated and compared with rates at oxygen concentrations of 100, 5,
and 3 µmol/L
(Figure 6). Rates of ATP turnover in the presence of
myxothiazol were 84% lower than rates at 100
µmol/L.
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Discussion |
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The present study is the first to suggest that energy-using processes other than contraction can be downregulated by isolated mammalian cardiomyocytes over a range of oxygen concentrations (2 to 70 µmol/L). Cardiomyocytes held in suspension generally are assumed not to exhibit contractile activity. However, evidence suggests that stirring stimulates contractile activity in suspensions of cardiomyocytes.29 For this reason, all experiments were performed in the presence of the contractile inhibitors verapamil (10 µmol/L) and NiCl2 (50 µmol/L).30 31
Effects of Different Degrees of Hypoxia
(5 to 6 µmol/L Versus 2 to 3 µmol/L) on Oxidative
Phosphorylation, Glycolytic Activity, and Cellular
Energy State
We found that isolated noncontracting neonatal
cardiomyocytes decreased rates of oxygen consumption in
response to reduced oxygen availability. Rates of oxygen consumption
were decreased at oxygen concentrations 
70 µmol/L. Our results
compare well with others that used contracting embryonic chick
cardiomyocytes in which rates of oxygen consumption were
decreased at oxygen concentrations as high as 63
µmol/L.13 In contrast to
our findings, several studies have shown that rates of mitochondrial
oxygen consumption by isolated adult rat cardiomyocytes do
not begin to decrease until extracellular oxygen concentrations are
much lower: 0.6,32
6,33
12,12 and 20
µmol/L.34 In these studies,
adult cardiomyocytes were exposed to only brief periods of
hypoxia. Research has suggested that metabolic
downregulation is a time-dependent
effect,13 and a previous
study has demonstrated that isolated hepatocytes display
oxygen conformance only when exposed to hypoxia for >2
hours.11 Exposure of adult
cardiomyocytes to such a brief period of hypoxia
may therefore explain why these cells did not exhibit oxygen
conformance at oxygen concentrations as high as 70 µmol/L. For this
reason, we performed studies with 2 different experimental systems. In
the closed-cell chamber, cardiomyocytes were exposed to
hypoxia for only brief periods (<1 minute), whereas in the
flow-through system, cells were subjected to hypoxia for
substantially longer periods (60 minutes). Our results show that the
magnitude of metabolic shutdown at 2 to 5 µmol/L was
significantly greater when experiments were performed in the
flow-through system as opposed to the closed-cell chamber. These
findings support the suggestion that metabolic
downregulation is a time-dependent effect and may help to explain the
differences in oxygen conformance between adult and neonatal
cardiomyocytes.
A potential source of error in our measurements could have been introduced by presence of either intracellular oxygen gradients or oxygen gradients within our experimental setup. However, we do not believe that either of these factors have affected our results, because intracellular oxygen gradients have been measured in resting adult cardiomyocytes and were found to be <0.2 µmol/L32 . Rates of oxygen consumption by platelets have been measured in the flow-through system and did not decrease until oxygen concentrations fell to <5 µmol/L.35 These results suggest that significant oxygen gradients do not exist within neonatal cardiomyocytes or within our experimental system.
Cardiomyocytes responded to oxygen concentrations of 5 to 6
µmol/L by decreasing their rates of oxygen consumption by 35%. This
decreased rate of oxygen consumption could be maintained for 
1 hour
and was fully reversible on reoxygenation. Cellular
viability can be maintained when aerobic ATP production is
decreased if anaerobic ATP production is increased
or if cellular energy usage is reduced. Our results indicate no
compensatory switch to anaerobic glycolysis despite rates
of ATP turnover that were reduced by 32%. Because intracellular ATP
and PCr concentrations remained stable, we conclude that rates of ATP
usage must have been reduced to match rates of ATP
production.
The second part of the present study examined how cardiomyocytes responded to a more-severe degree of hypoxia (2 to 3 µmol/L). Cardiomyocytes exposed to oxygen concentrations of 2 to 3 µmol/L exhibited rates of oxygen consumption that were decreased to 9% of rates at 100 µmol/L. These decreased rates of oxygen consumption were associated with stable ATP concentrations, decreased PCr concentrations, and a significant switch to anaerobic ATP production. Our results show that isolated cardiomyocytes respond to near-anoxic oxygen concentrations by downregulating energy-using processes other than contraction and that this downregulation is associated with energetic stress as indicated by decreased PCr concentrations and increased lactate concentrations.
Energetic Stress Provides the Signal for
Metabolic Downregulation at Oxygen Concentrations of 2
µmol/L
The present results suggest that energetic stress
may act as a signal to downregulate energy usage when oxygen
concentrations fall to less than 3 µmol/L. We created energetic
stress under normoxic oxygen concentrations by inhibiting rates of
oxygen consumption by 95% to 98% with myxothiazol, an
inhibitor of electron flow through complex III in the
mitochondria. If cells were not capable of downregulating
metabolic activity under these conditions, then ATP stores
would be rapidly degraded and death would ensue. In the presence of
myxothiazol, cells remained viable and further decreased rates of ATP
turnover.
Summary
The present results show that
cardiomyocytes are capable of responding to reduced oxygen
availability and that this response is dependent on severity of
hypoxia. Cardiomyocytes responded to oxygen concentrations of 5
to 70 µmol/L by downregulating energy-using processes. This
metabolic downregulation was not associated with energetic
stress. At oxygen concentrations less than ~3 µmol/L,
cardiomyocytes were capable of further reducing rates of
energy usage. However, this additional downregulation was dependent on
the presence of energetic stress.
Because isolated suspensions of cardiomyocytes seem capable of shutting down energy-using processes other than contraction and in view of clinical studies that show that contractile activity does not always immediately recover after revascularization,5 15 metabolic downregulation associated with myocardial hibernation seems unlikely to be due to decreased rates of contractile activity exclusively. Other energy-consuming processes that may be downregulated include protein synthesis, mRNA synthesis, ion-channel activity, and proton leak.36 Shutting down these energy-using processes when oxygen supply is reduced may provide a means of conserving ATP and thus making it available for processes necessary for survival. Mechanisms by which changes in oxygen concentration are sensed in regions of hibernating myocardium and in isolated suspensions of cardiomyocytes are unclear but may involve cytochrome c oxidase14 37 38 or various intracellular signaling molecules, including nitric oxide39 40 and reactive oxygen species.41
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Acknowledgments |
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The present study was supported by the National Health and Medical Research Council of Australia, the Raine Medical Research Foundation, and an Australian Research Council grant. We thank Associate Prof Michael Guppy for his useful comments and for help with the measurement of glucose oxidation.
Received April 28, 2000; revision received July 12, 2000; accepted July 14, 2000.
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T. M. Casey, J. L. Pakay, M. Guppy, and P. G. Arthur Hypoxia Causes Downregulation of Protein and RNA Synthesis in Noncontracting Mammalian Cardiomyocytes Circ. Res., April 19, 2002; 90(7): 777 - 783. [Abstract] [Full Text] [PDF]
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