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(Circulation Research. 1997;81:69-75.)
© 1997 American Heart Association, Inc.

Articles

Mitochondrial Response to Heart Rate Steps in Isolated Rabbit Heart Is Slowed After Myocardial Stunning

C.J. Zuurbier, , J.H.G.M. van Beek

From the Laboratory for Physiology (C.J.Z., J.H.G.M. van B.), Institute for Cardiovascular Research, Vrije Universiteit, Amsterdam, the Netherlands, and the Center for Bioengineering (C.J.Z.), University of Washington, Seattle.

Correspondence to Dr J.H.G.M. van Beck, Laboratory for Physiology, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, Netherlands.

	Abstract

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Abstract The oxidative capacity of mitochondria isolated from myocardium is undiminished after myocardial stunning, which is remarkable because stunning affects many other cellular functions. The aim of the present study was to assess the mitochondrial oxidative response in intact rather than isolated myocardium. The mean response time of mitochondrial O₂ consumption to heart rate steps (t_mito) was measured before and after 15-minute ischemia or high-flow hypoxia in isolated rabbit hearts. The t_mito was calculated from the time course of venous O₂ tension to steps in heart rate, with corrections made for diffusion and vascular transport delay. Isovolumic hearts were perfused with Tyrode's solution at 37°C. Developed left ventricular pressure at 35 minutes of reperfusion was decreased significantly to 67±3% after ischemia (mean±SEM, n=8) and to 79±6% after hypoxia (n=8) relative to the control condition (n=8), without increased cellular creatine kinase release. Before ischemia or hypoxia, t_mito was 4.3±0.3 seconds. During reperfusion after ischemia or hypoxia, the increase in t_mito (by 62±10% and 64±18%, respectively) was significantly larger than that in time controls (24±12% increase). The major determinant of decreased contractility and slower mitochondrial response appeared to be O₂ deprivation and/or reintroduction rather than other consequences of stopped flow. O₂ consumption at a given rate-pressure product was not increased after ischemia or hypoxia, indicating undiminished cardiac contractile economy. Brief ischemia or hypoxia, resulting in stunning, was associated with a slowing of the in vivo mitochondrial oxidative response, indicating that energy transfer and/or signaling between energy-consuming sites and mitochondria is affected in stunned myocardium.

Key Words: stunning • mitochondria • ischemia • hypoxia • creatine kinase

	Introduction

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Reversible contractile dysfunction of cardiac muscle after brief ischemia has been termed myocardial stunning.^{1 2} Disturbances in excitation-contraction coupling, contractile apparatus, membrane structures, cytoskeleton, collagen, and cellular bioenergetics have all been proposed as potential causes of stunning.^{3 4} Although many cellular functions are affected during stunning, mitochondrial function seems to be remarkably resistant. Consequently, it is believed that the decreased contractile performance is not caused by depressed mitochondrial function.³

The present study sought to determine whether stunning affects mitochondrial oxidative function in vivo. The reasons for reexamining this question are as follows: (1) The Ca²⁺ overload, which may be an important cause of stunning, may also affect mitochondrial inner membrane permeability and induce the so-called permeability transition pore,⁵ which in turn may affect mitochondrial function. (2) During reperfusion after ischemia, the intramitochondrial Ca²⁺ concentration is elevated, and Ca²⁺ is important for the regulation of mitochondrial metabolism.⁶ (3) Mitochondrial CK activity, assayed in vitro, is significantly diminished after very brief ischemia, and this enzyme is thought to play a key role in the regulation of mitochondrial ATP synthesis.^{7 8}

Mitochondria isolated from stunned myocardium show an undiminished maximal rate of respiration.⁹ However, extrapolation of results from isolated mitochondria to the intact heart might be misleading because of possible selectivity of the isolation procedure (most of the mitochondria are lost), the unavoidable change in the interface between mitochondria and cytosol,⁷ and the impossibility of studying the regulation of mitochondrial O₂ consumption through signals in the cytoplasm, including Ca²⁺. For these reasons, we decided to challenge the idea that mitochondrial function is not affected during stunning by assessing the function of the mitochondria in their natural intracellular environment rather than by studying the organelles after isolation.

To study mitochondrial function in intact myocardium, we determined the t_mito.¹⁰ This recent technique quantifies mitochondrial function at submaximal levels of workload and O₂ consumption, which is important, because testing the mitochondria at maximal capacity might be confounded by O₂ supply limitation in intact myocardium. The t_mito represents the delay between ATP hydrolysis and mitochondrial ATP synthesis. A large t_mito means a potentially large depletion of high-energy phosphate stores (such as PCr) during quick increases in ATP hydrolysis. This in turn indicates low mitochondrial function, resulting from restricted capacity to phosphorylate ADP or to impaired energy and/or signal transfer between sites of ATP hydrolysis and the mitochondria.

New features of the present study are the direct analysis of mitochondrial function in intact myocardium (rather than in isolated mitochondria) and the assessment of the dynamic adaptation of oxidative phosphorylation to quick changes in cardiac workload in stunned myocardium.

	Materials and Methods

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Heart Perfusion
New Zealand White rabbits of both sexes, weighing 2.7±0.3 kg (mean±SD), were anesthetized with intramuscularly administered 10 mg/kg fluanisone and 0.3 mg/kg fentanyl citrate (Hypnorm, Janssen Pharmaceutica) supplemented with pentobarbital sodium (10 mg/kg IV). After opening the thorax and injection of intravenous heparin (2500 IU IV), the aorta was cannulated in situ, and perfusion was started before excision of the heart. The hearts were perfused according to the method of Langendorff at 37°C with Tyrode's solution containing (mmol/L) NaCl 128.3, KCl 4.7, CaCl₂ 1.36, MgCl₂ 1.05, NaHCO₃ 20.2, NaH₂PO₄ 0.42, and glucose 11.0, gassed with 95% O₂/5% CO₂. Adenosine (10^-5 mol/L) was added to the perfusate to obtain maximal vasodilation, which is advantageous for our method of determining the t_mito.¹⁰ The right atrium was closed by ligation of the caval veins, and the venous effluent left the heart via the right ventricle and the cannulated pulmonary artery. Thebesian venous effluent from the ventricular wall was drained from the left ventricular lumen by a cannula pierced through the apex. For assessment of contractile performance, a water-filled latex balloon was inserted into the left ventricle and connected to a Statham P23 Db pressure transducer (Statham Instruments). The atrioventricular node was crushed with forceps, resulting in a spontaneous heart rate below 120 bpm. Hearts were then electrically stimulated via two electrodes on the right ventricle, with the pacing rate set between 100 and 120 bpm, just above the spontaneous heart rate. Although we aimed for a basal pacing rate of 100 bpm, 29% of all hearts (3 control, 2 ischemic, and 2 hypoxic hearts) were paced with a basal rate between 110 and 120 bpm because the atrioventricular block did not result in a lowering of the spontaneous heart rate to 100 bpm. Despite these exceptions, the basal heart rate will be indicated as 100 bpm for brevity. Details of preparations and procedures have been described previously.^{10 11}

Measurement of Response Time of O₂ Consumption
An extensive description of the technique is given in Reference 10¹⁰ . In short, the venous mean response time (t_v) is determined from the time course of the venous O₂ tension to a step in heart rate (see Fig 1C). Subsequently, the mean time necessary for diffusion and vascular transport of O₂ between the mitochondria and the O₂ electrode (t_transport) is subtracted from t_v. This allows us to obtain the true t_mito (t_mito=t_v-t_transport). The t_transport can be determined experimentally from the venous O₂ response to small steps in perfusion flow or arterial O₂ tension.^{10 11}

View larger version (14K): [in this window] [in a new window]   Figure 1. Heart rate step in isolated rabbit heart. Typical example of time courses of left ventricular pressure (LVP, A), of the product of heart rate and peak systolic pressure (RPP, B), and of coronary venous O2 tension (C). Pacing rate (given in bpm) was changed at the arrows.

RPP, which is defined as heart rate (1/period between beats) times systolic pressure, is used as an index of metabolic demand on a beat-by-beat basis.^{11 12} Steps in metabolic demand are brought about by switching from one heart rate to another. After such steps in heart rate, the mean t_RPP is negative, because RPP reaches a steady state after an initial overshoot^{11 12} (Fig 1A and 1B). Previous studies have also shown that there is a small initial increase ("initial deflection") in venous O₂ tension after an upward step in heart rate because of a transient increase in venous outflow (Fig 1C; the opposite is found after a downward step in heart rate). The t_mito is corrected for t_RPP and the initial deflection.^{10 11 12}

Experimental Protocol
End-diastolic pressure was set at 5 mm Hg by adjusting left ventricular balloon volume. The hearts were perfused at constant flow throughout the experiment after setting flow to obtain an initial perfusion pressure of 80 mm Hg, measured just above the aortic cannula with a Statham P23 Db pressure transducer. Coronary O₂ tensions in arterial inflow and venous outflow were continuously monitored by two Clark-type electrodes (time constant, 1 second), calibrated before and after the experiment.¹⁰ O₂ concentrations were calculated by multiplying the O₂ tension by the O₂ solubility of 1.34 µmol O₂·liter Tyrode's solution^-1·mm Hg^-1 at 37°C. Data were recorded on paper and simultaneously digitized and stored on a personal computer (Olivetti PC). O₂ consumption (in µmol·g_dw^-1·min^-1) is calculated as the product of flow and the arterial-to-venous O₂ concentration difference.

After a 30-minute period of preparation, the hearts were paced at the basal heart rate of 100 bpm, and a series of different tests in fixed order were executed over the next 30 minutes: (1) four different steps in heart rate, in a random order, stepping up from the basal heart rate to test heart rates of 140, 170, 200, and 230 bpm and back, to measure the venous response time; (2) downward steps in perfusion flow and, separately, in arterial O₂ tension at 100 bpm, for two purposes: to calculate the O₂ transport time from the venous O₂ transient response and to assess the sensitivity of O₂ consumption to small reductions in O₂ supply; (3) similar steps in perfusion flow and in arterial O₂ tension at 230 bpm to assess effect of heart rate on O₂ transport; and (4) an indicator-dilution step with Evans blue bound to albumin at 100 bpm and at 230 bpm to assess intravascular transit time.

Subsequently, during the next 15 minutes, three different groups received different treatments: a control group (n=8) received normal flow and 95% O₂; an ischemic group (n=8) underwent total global no-flow ischemia; and a hypoxic group (n=8) received normal flow, but with perfusate gassed with 95% N₂/5% CO₂, which resulted in an arterial PO₂ of 19.1±6.7 mm Hg at the level of the aortic cannula. During this 15-minute intervention period, all hearts were continuously paced at 100 bpm, and the volume of the balloon in the left ventricle was constant. The hearts of the ischemic and hypoxic groups were submerged in Tyrode's solution kept at 37°C and gassed with 95% N₂/5% CO₂ to prevent inward O₂ diffusion across the heart's outer surface.¹³ After this period, the ischemic and hypoxic hearts were reperfused or reoxygenated, respectively, for 20 minutes at preischemic flow rates with Tyrode's solution gassed with 95% O₂/5% CO₂, and the control hearts were perfused as before for 20 minutes. For the ischemic group, the flow was gradually increased to the preischemic flow rate within the first minute of reperfusion. In the next 30 minutes (minutes 20 through 50 of reperfusion), tests 1 through 4, described above, were repeated. The venous effluent was collected every 10 minutes during the first 40 minutes of reperfusion, starting within the first minute of reperfusion, for determination of venous CK efflux to assess cell membrane damage.¹⁴

Statistical Analysis
Data are presented as mean±SEM, except when indicated otherwise. Two-way (by treatment group and by time) ANOVA for repeated measurements was performed for diastolic and developed pressure during the reperfusion period and for CK release during the time course of experiment. One-way ANOVA was performed to test whether changes of variables (value at the beginning of the experiment minus the value at the end of the experiment) after ischemic or hypoxic treatments or after normoxic perfusion for the same time (control) differed significantly from zero and whether these changes differed significantly among treatment groups. ANOVA was followed by planned comparisons among class means¹⁵ when a significant main effect (P<.05) was found. The P values for the planned comparisons were adjusted according to Bonferroni (P/n, for n comparisons).

	Results

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No differences among groups were observed for wet weight of the hearts at the end of the experiment (8.46±0.18 g), for dry weight, determined after 2 days of storage at 70°C (1.46±0.03 g), or for average coronary flow (10.8±0.4 mL·min^-1·g^-1). Perfusion pressure at the beginning of the experiment was 81.5±8.3 mm Hg. The increase in perfusion pressure with constant flow during the 1.5-hour experiment amounted to 8.0±2.7, 25±3.9 (significant versus control), and 9.6±4.8 mm Hg for the control, ischemic, and hypoxic groups, respectively.

During 15-minute high-flow hypoxia, diastolic pressure increased continuously, whereas no increase was found during ischemia (Fig 2). Although diastolic pressure was increased in the ischemic group for the first 35 minutes of reperfusion, no significantly different diastolic pressures were observed among the three groups at the end of reperfusion (65 minutes). After 3 minutes of ischemia, developed left ventricular pressure had declined to 5% of the preischemic level, whereas the hypoxic hearts continuously developed pressure, although developed pressure was only 10% of the prehypoxic value after 15 minutes of hypoxia (Fig 2). During the reperfusion period, both hypoxic and ischemic hearts showed a similar incomplete recovery in developed pressure. No significant differences among the three groups were observed in CK release during reperfusion (average, 0.22±0.05 U·min^-1·g_dw^-1), indicating that 15-minute ischemia or hypoxia does not result in cell necrosis in this model.

View larger version (22K): [in this window] [in a new window]   Figure 2. The end-diastolic pressure (EDP) and the developed left ventricular pressure (DLVP [systolic pressure minus diastolic pressure]) as a function of time for isolated hearts. DLVP is normalized to 100% for the mean value at t=0, which corresponds to 96, 99, and 105 mm Hg for control, ischemia, and hypoxia, respectively. Points represent mean±SEM; error bars often fall within the symbol. *Statistically significant difference compared with control (P<.05). #Statistically significant difference between hypoxia and ischemia (P<.05).

When the perfusion flow was decreased by 9.2±0.3% to test the effect of O₂ supply on aerobic metabolism, measured O₂ consumption was decreased significantly by 2.8% at 230 bpm, but not at 100 bpm. After arterial O₂ content was lowered by 6.9±0.3%, O₂ consumption was found to be decreased significantly by 2.5%, both at 100 bpm and at 230 bpm. The measured decrease in O₂ consumption after the small reductions of O₂ supply was the same in all three groups and did not change during the experiment. We used the transport time derived from the flow step at 100 bpm, where O₂ consumption is stable during supply reduction, to correct the venous O₂ response to heart rate steps for transport delay between mitochondria and the venous O₂ electrode. The Table shows this transport time of O₂ for the three groups at the beginning and end of the experiment. The relative changes in transport time during the course of the experiment were small and not significantly different among the groups.

View this table: [in this window] [in a new window]   Table 1. Mean Transport Time in Control, Ischemic, and Hypoxic Hearts

The t_RPP ranged from -0.4 to -0.9 seconds, and the correction time for the initial deflection of the venous O₂ tension ranged from -0.06 to -0.08 seconds for the heart rate steps to 140 and 230 bpm at the beginning of the experiment, respectively. No change in the initial deflection was observed over the duration of the experiment for all three groups. The t_RPP increased in the course of the experiment (+0.7 seconds), and this was similar for all groups. The t_mito, corrected for t_RPP and the initial deflection, is presented as a function of the test heart rate (Fig 3). Because t_mito was not significantly different between the upward and downward steps in heart rate, the average of both is given. At the beginning of the experiment, t_mito ranged from 3.2 to 4.8 seconds and was not significantly different among the four heart rates or the three treatment groups studied. Paired analysis within groups demonstrates that independent of heart rate, t_mito becomes significantly larger during the experiment for the ischemic (P=.002) and hypoxic (P=.01) groups but not for the control group (P=.35). The increases in t_mito observed after ischemia (2.3 to 3.0 seconds for different heart rate steps; overall, 62±10% increase) and after hypoxia (1.7 to 2.9 seconds; 64±18% increase) were both significantly larger than the small increase found for the control group over the same duration of the experiment (0.3 to 1.0 seconds; 24±12% increase). The increase in t_mito was not significantly different between ischemic and hypoxic groups. We conclude that 15-minute ischemia or hypoxia resulted in a slowing down of the adaptation speed of mitochondrial O₂ consumption to steps in heart rate.

View larger version (14K): [in this window] [in a new window]   Figure 3. The tmito as function of test heart rate. The heart was paced up to the test heart rate from 100 bpm (see "Materials and Methods"). Separate panels show results for the control, ischemic, and hypoxic groups. The tmito was corrected for the contribution of the initial deflection, tRPP, O2 diffusion, and vascular transport. Values were determined at the beginning of the perfusion period before any ischemia or hypoxia (open symbols), and the measurements were repeated toward the end of the experiment (solid symbols), which was after ischemia or hypoxia, where applicable. For control, the measurement was repeated at exactly the same time point at the end of the experiment after uninterrupted normoxic perfusion. #Statistically significant difference between begin and end value within each group (P<.05). *Statistically significant difference in the change in tmito during the experiment compared with the change observed for control (P<.05).

O₂ consumption as a function of RPP before and after ischemic or hypoxic interventions, and at the same time points for the control group, is illustrated in Fig 4. Linear fits were generated between O₂ consumption and RPP for each group. At the beginning of the experiment, before interventions, the y intercept was 8.44±1.74, 11.26±1.50, and 11.92±2.00 µmol·g_dw^-1·min^-1 and the slope was 0.73±0.06, 0.67±0.03, 0.66±0.06 nmol·g_dw^-1·mm Hg^-1 for the control, ischemic, and hypoxic groups, respectively. Paired analysis within groups showed that no changes in slope occurred in the control group (P=.85) and in the ischemic group (P=.65), whereas in the hypoxic group a significant increase to 0.87±0.08 nmol·g_dw^-1·mm Hg^-1 was observed (P=.03). The intercept decreased after ischemia to 6.39±1.26 µmol·g_dw^-1·min^-1 (P=.04) and after hypoxia to 4.20±2.31 µmol·g_dw^-1·min^-1 (P=.01), but the decrease seen in the control condition (to 6.37±2.21 µmol·g_dw^-1·min^-1) was not significant (P=.11). The best test for effects of hypoxia or ischemia on the RPP-O₂ consumption relation, comparing the change in slope and intercept after ischemia or hypoxia to the change over the same duration of the control experiment, yielded no significant effects for ischemia or hypoxia (P=.18 and .08 for group effects on changes in intercept and slope, respectively).

View larger version (12K): [in this window] [in a new window]   Figure 4. Myocardial O2 consumption vs RPP for control, ischemic, and hypoxic groups. Values of RPP and O2 consumption are mean±SEM for each heart rate. Measurements were performed before ischemia or hypoxia (open symbols) and repeated after ischemia or hypoxia (solid symbols). For the control group, measurements were repeated after continuous normoxic perfusion, at same time point as for hypoxic and ischemic groups (solid diamond).

	Discussion

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The major finding in the present study is that after 15 minutes of ischemia or hypoxia in the isolated rabbit heart, sufficient to cause myocardial stunning, cardiac mitochondrial O₂ consumption adapts slower to changes in metabolic demand induced by heart rate steps. Because the maximal oxidative capacity of isolated mitochondria stimulated with ADP is not yet depressed after such brief ischemia or hypoxia,^{9 16 17 18 19} we propose that energy transfer and/or signaling across the cytosol between sites of ATP consumption and the mitochondria is affected by the brief ischemia or hypoxia. The slower adaptation of mitochondrial energy production in these stunned hearts means that when ATP hydrolysis increases quickly, there is a potentially larger depletion of high-energy phosphates, mainly of PCr,^{12 20} and an increased accumulation of breakdown products of ATP, at least in the first phase of the transient response to elevation of cardiac workload. Consequently, the slower mitochondrial response means a lower free energy of ATP hydrolysis and less free energy available for contraction and ion pumping. This finding for the transient response corresponds with a ³¹P-NMR study involving isolated rat hearts in which steady state levels of ADP and P_i concentration increased much more with elevated workload after myocardial stunning by ischemia, whereas no changes in ADP and P_i concentration were found with these elevated workloads in control hearts given glucose or palmitate as substrate, and only small changes were found when pyruvate was given.²¹

Mitochondrial Capacity and Response Time
The t_mito has been hypothesized to be inversely related to the MAC,^{10 22 23} and this relation has been found experimentally in various frog skeletal muscle fiber types and isolated rabbit hearts at low temperatures.^{23 24} If this inverse relation holds for the normothermic heart, a depression of MAC caused by brief ischemia or hypoxia could explain the increase in t_mito.

After 15 minutes of ischemia and 15 to 30 minutes of reperfusion, the MAC of isolated cardiac mitochondria, measured as mitochondrial state 3 respiration during maximal stimulation by ADP, was unchanged in many studies.^{16 17 18 19} Mitochondria isolated after 15 minutes of hypoxia without reperfusion²⁵ or after 15 minutes of ischemia followed by 5 minutes of reperfusion even showed increased MAC.¹⁶ Flameng et al⁹ observed an undiminished MAC in mitochondria isolated from reperfused ischemic rabbit hearts after 10 to 20 minutes of ischemia, under conditions directly comparable to those of the present study. We conclude that the oxidative capacity of mitochondria assessed in vitro by maximal stimulation with ADP is not depressed after brief ischemia or hypoxia. Moreover, in our most recent work in the isolated rabbit heart under similar experimental conditions, infusion of oligomycin (a mitochondrial ATP synthase inhibitor) reduced MAC by 50%,²⁶ but failed to cause a significant increase in t_mito (3±13%; n=4; heart rate steps, from 120 to 220 bpm). Taken together, these results indicate that a decrease in MAC does not explain the observed increase in t_mito and suggest that the increase in response time is caused by retardation of the signal coming from the ATP-consuming sites through the cytosol to the mitochondria.

CK and the Response Time
In all studies showing no depression of MAC after brief ischemia or hypoxia (see above), isolated mitochondria were tested by adding ADP, which bypasses the mitochondrial CK and enters the mitochondria directly. When both creatine and ADP were added, a decreased production of high-energy phosphates (ATP+PCr) was observed in mitochondria isolated after 15-minute ischemia followed by 30-minute reperfusion,¹⁸ whereas MAC tested with ADP alone was not affected in the same study. Further, the activity of mitochondrial CK has been shown to be directly depressed after 10- to 20-minute ischemia followed by 30-minute reperfusion.⁸

The signal from the myofibrils that stimulates oxidative phosphorylation in the mitochondria may be mediated via CK isoforms localized in myofibrils and mitochondria.⁷ The ADP generated during contraction in the myofibrils may not be able to stimulate the mitochondria directly,²⁷ and creatine rather than ADP may be the predominant form of phosphate acceptor that reaches the mitochondria.²⁸ Phosphorylation of creatine by mitochondrial CK may generate most of the ADP that enters the mitochondria to stimulate oxidative phosphorylation in the intact cell. Ischemia followed by reperfusion causes production of reactive oxygen species, which is an important cause of stunning.³ Reactive oxygen species cause inhibition of CK isozymes in the myofibrils and cytosol²⁹ and in the mitochondria.⁸ Therefore, decreased activity of CK may explain the increased t_mito observed in the present study.

Other Possible Determinants of the Response Time
Although the evidence pointing to decreased CK activity as cause of the slower mitochondrial response seems strong, alternative explanations should be considered. Ca²⁺ entry into the mitochondrial matrix^{6 30} and modification of mitochondrial F₁-ATPase activity via inhibitory proteins^{31 32} are considered to be potential regulators of oxidative phosphorylation. Very recently, it was shown directly that Ca²⁺ entry into the mitochondrial matrix causes reduction of NAD⁺, which may stimulate myocardial oxidative phosphorylation.³⁰ However, this NAD⁺ reduction does not take place in the first 30 seconds after cardiac workload is increased and is preceded by strong NADH oxidation. Although hypoxia decreases F₁-ATPase activity, this is quickly reversible upon reoxygenation.^{31 32} The half-time of activation of F₁-ATPase following elevation of cardiac workload is also >30 seconds.³¹ The time courses of NAD⁺ reduction and of the increase in F₁-ATPase activity are compatible with the measured slow entry of Ca²⁺ into the cardiac mitochondria³³ and are too slow to affect our t_mito, which describes the first phase of the transient response. The increase of t_mito after stunning is therefore very likely not caused by slower entry of Ca²⁺ into the mitochondrial matrix or slower regulation of F₁-ATPase activity.

The time constant for the change of P_i and PCr is significantly shorter (by 70%) than t_mito.¹² It has been hypothesized that transient buffering of changes in phosphate metabolites by glycolytic ATP synthesis in or near the myofibrils may retard the signal to the mitochondria.^{10 12} The unidirectional flux of phosphate groups across the glycolytic enzymes GAPDH and phosphoglycerate kinase is increased after 18-minute ischemia in the isolated rat heart, as found with ³¹P-NMR saturation transfer measurements.³⁴ Glycolytic buffering of ADP and P_i may cause greater retardation of the signal between myofibrils and the mitochondria after ischemia. Whether t_mito is retarded by enhanced glycolytic buffering or depressed CK activity, or both, remains unclear and requires further investigation using enzyme inhibitors.

Myocardial Economy
Although increased O₂ consumption relative to mechanical developed force has often been found in stunned cardiac tissue (see reviews in References 35 and 36^{35 36} ), no increase was encountered in other studies.^{21 37} In the present study, we found no significant increase, and there even seemed to be a nonsignificant opposite tendency. Elevated O₂ consumption at fixed RPP has been observed in stunned hearts at the time of development of contracture during ischemia, but not earlier.²¹ Contracture was not evident during the 15 minutes of ischemia in the present study, and stunning was perhaps not severe enough to cause uncoupling between O₂ consumption and force development.³⁶ The absence of mitochondrial uncoupling is in line with ³¹P-NMR saturation transfer measurements showing that the mitochondrial ATP synthesis–to–O₂ consumption ratio was unchanged after 18 minutes of ischemia in isolated rat hearts.³⁴

Mitochondrial Function and Depressed Contractility
Depressed cardiac contractility after myocardial stunning is usually not considered to be caused by diminished mitochondrial function.³ On the other hand, partial inhibition of MAC in intact rabbit heart by infusion of oligomycin resulted in considerably depressed cardiac contractility and energy turnover, under conditions comparable to those in the present study.^{26 38} In the intact cell, where a major energetic stimulus pathway to the mitochondria may be mediated via CK isozymes,^{7 39} depressed function of CK may lead to decreased myocardial energy turnover and contractility.⁴⁰ It is not yet clear whether failure of transcytosolic transport of high-energy phosphates and depression of MAC may lead to depressed contractility via common mechanisms.

We found that at lower than physiological temperatures, after 40 minutes of anoxia at 20°C²³ and after 25 minutes of ischemia at 28°C,⁴¹ t_mito was not increased in stunned myocardium, despite the reduction of contractile function. This suggests that a slower mitochondrial response is not a necessary condition for stunning and supports the multifactorial origin of stunning.

Methodological Considerations
The t_mito cannot be determined in in situ blood-perfused hearts, and the present study was therefore performed in isolated hearts. O₂ limitation has been observed in some,^{42 43 44} but not all,⁴⁵ isolated crystalloid-perfused heart studies. We tested whether O₂ consumption was limited by supply in our preparation by imposing small reductions of O₂ concentration or perfusate flow⁴² and found that O₂ supply was not limiting at low heart rates (see also Reference 12¹² ). However, t_mito was not different at high and low heart rates (Fig 3), and possible moderate limitation by O₂ supply at high heart rates is apparently of no consequence. Because a change in O₂ consumption may affect the determination of transport time,¹⁰ we derived the transport time from the small flow steps at 100 bpm where there was no O₂ limitation.

The recovery of pressure development and changes in t_mito were similar after hypoxia and ischemia, suggesting that O₂ deprivation and/or reintroduction is the major determinant of these responses rather than other aspects of ischemia, such as interruption of removal of waste products and carbon substrate supply. The increase in diastolic pressure observed during hypoxia but not during ischemia reproduces previous findings in the isolated rabbit heart.⁴⁶ This difference has been attributed to the collapse of the coronary blood vessels during ischemia, leading to loss of the "erectile effect" on chamber distensibility, and to acidosis, which develops during ischemia but not during hypoxia and reduces myofibrillar calcium sensitivity.⁴⁶

Conclusions
A brief period of ischemia or hypoxia, causing contractile stunning without cell necrosis, resulted in a slowing of the mitochondrial oxidative response to steps in cardiac workload. Our results corroborate that cardiac mitochondria are not uncoupled after brief ischemia or hypoxia. We propose that intracellular signaling and/or energy transfer across the cytosol between the energy-consuming ATPases and the mitochondria, possibly mediated by CK isozymes, are transiently affected by the O₂ deprivation and/or reintroduction that accompanies brief ischemia or hypoxia.

	Selected Abbreviations and Acronyms

 

CK = creatine kinase dw (as subscript) = dry weight MAC = mitochondrial aerobic capacity NMR = nuclear magnetic resonance PCr = phosphocreatine RPP = rate-pressure product tmito = response time of mitochondrial O2 consumption to steps in cardiac workload tRPP = response time for RPP

	Acknowledgments

 
This study was supported by Netherlands Heart Foundation grants 91.120 and D94.016. Dr van Beek is an Established Investigator of the Netherlands Heart Foundation. We are grateful to M.H. van Wijhe for technical assistance in performing the CK assay, to W. Gerrissen for animal preparation, and to G.J. Harrison and B. de Groot for discussion of the manuscript.

Received August 29, 1996; accepted April 7, 1997.

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