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Laboratory for Physiology, Institute for Cardiovascular Research, Vrije Universiteit, 1081 BT Amsterdam, The Netherlands
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We investigated the role of myoglobin (Mb) in
supplying O2 to mitochondria
during transitions in cardiac workload. Isovolumic rabbit hearts
(n = 7) were perfused retrogradely
with hemoglobin-free Tyrode solution at 37°C. Coronary venous
O2 tension was measured polarographically, and tissue oxygenation was measured with
two-wavelength near-infrared spectroscopy (NIRS), both at a time
resolution of ~2 s. During transitions to anoxia, 68 ± 2% (SE)
of the NIRS signal was due to Mb and the rest to cytochrome oxidase.
For heart rate steps from 120 to 190 or 220 beats/min, the NIRS signal
decreased significantly by 6.9 ± 1.3 or 11.1 ± 2.1% of the
full scale, respectively, with response times of 11.0 ± 0.8 or 9.1 ± 0.5 s, respectively. The response time of end-capillary
O2 concentration
([O2]), estimated from
the venous [O2], was
8.6 ± 0.8 s for 190 beats/min (P < 0.05 vs. NIRS time) or 8.5 ± 0.9 s for 220 beats/min
(P > 0.05). The mean response times
of mitochondrial O2 consumption
(
O2) were 3.7 ± 0.7 and
3.6 ± 0.6 s, respectively. The deoxygenation of oxymyoglobin
(MbO2) accounted for only
12-13% of the total decrease in tissue
O2, with the rest being physically
dissolved O2. During 11%
reductions in perfusion flow at 220 beats/min, Mb was 1.5 ± 0.4%
deoxygenated (P < 0.05), despite the
high venous PO2 of 377 ± 17 mmHg,
indicating metabolism-perfusion mismatch. We conclude that the
contribution of MbO2 to the
increase of O2 during heart
rate steps in saline-perfused hearts was small and slow compared with
that of physically dissolved
O2.
myoglobin; mitochondrial oxygen consumption; Gregg phenomenon
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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MYOGLOBIN (Mb) is a monomeric protein that acts as a short-term O2 buffer and might facilitate O2 diffusion in the cell (41). Steady-state function of Mb has been studied extensively (18, 19, 22-25, 27, 35), but in this report we focus on the role of Mb in mitochondrial O2 supply during transients in cardiac energy metabolism. When O2 consumption increases quickly, coronary flow increases and extra O2 is extracted from the capillaries. However, both processes are relatively slow, and O2 supply to mitochondria might be transiently buffered by dissociation of O2 from Mb or, especially in saline-perfused hearts, by physically dissolved O2 in the tissue. We assessed the contribution of oxymyoglobin (MbO2) to the increased O2 consumption during a step in heart rate by comparing the time course of Mb deoxygenation with the time courses of venous O2 tension and mitochondrial O2 consumption. If the increased O2 demand is mainly buffered by MbO2, the response time of mitochondrial O2 consumption (tmito) can be assessed more precisely by monitoring Mb oxygenation (40). This is of interest because tmito indicates the dynamics of cardiac metabolism and transcytosolic energy transfer speed in cardiac myocytes during steps in workload (7-9, 11-15, 36, 40, 45).
Near-infrared spectroscopy (NIRS) has been used to measure hemoglobin oxygenation in skeletal muscle and brain tissue (5, 30) and can be used to measure Mb oxygenation in isolated hearts, provided that hemoglobin has been removed (39), because the NIR absorption of hemoglobin and Mb is virtually the same. In principle, NIR absorption can distinguish among Mb, MbO2, and cytochrome-c oxidase (Cyt Ox) when three or more wavelengths are used, but the equipment becomes very costly, and the reliability of the Cyt Ox component is questionable because different algorithms and spectrometers give very different results (6). Therefore, we applied two-wavelength NIRS in the isolated heart, whereas the relative contributions of Cyt Ox and Mb to this NIRS signal were determined experimentally. NIRS was subsequently used to assess the dynamics of Mb deoxygenation during steps in heart rate.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the first group (group A) the relative contribution of the Cyt Ox redox state to the NIRS signal was determined in KCl (25 mmol/l)-arrested isolated rat hearts. Rat hearts were chosen for direct comparison with the previous NIRS study in rats (39) and because the NIR-absorbing molecules are well characterized in this species (44), allowing a quantitative comparison between the NIRS method and these independent measurements. In the second group (group B; isolated rabbit hearts) the response times of mitochondrial O2 consumption (tmito), venous O2 tension (tv), and Mb oxygenation (tMbO2) during heart rate steps were determined simultaneously. The time courses of tmito and tv cannot yet be measured in rat hearts because of size limitations. The relative contributions of Cyt Ox and Mb were also assessed in the rabbits for comparison with the rat data. All animals were treated according to the guidelines of the DEC (Animal Experimental Committee) of the Vrije Universiteit (Amsterdam, The Netherlands).
Group A
Preparation. Male Wistar rats (n = 6), weighing 512 ± 48 g (SD), were anesthetized with 8 mg/kg xylazine and 60 mg/kg ketamine and received 2,500 IU heparin intraperitoneally. The hearts were excised and perfused according to Langendorff at 37°C with a constant flow of 95% O2-5% CO2-saturated Tyrode buffer containing (in mmol/l) 108 NaCl, 25 KCl, 1.36 CaCl2, 1.05 MgCl2, 20.2 NaHCO3, 0.42 NaH2PO4, and 11.1 glucose. Adenosine (10 µmol/l) was added for maximal vasodilation. The perfusion pressure was set at 80 mmHg at the start of each experiment by adjusting roller pump speed, and flow was subsequently kept constant. The right atrium was closed by ligation of the caval veins.
An oncotic agent was not added because the present study is meant to be comparable with a series of previous studies (8, 9, 11-15, 36-40) on isolated hearts that were also perfused with buffer without an oncotic agent. In addition, hemoglobin had to be removed to measure Mb, and oncotic agents added with the goal to decrease edema in the absence of blood decreased the formation of edema in our preparation only a little (Ref. 2; C. J. Zuurbier, unpublished observations in our experimental model).Measurements. Constant sample flows of Tyrode solution were drawn from just above the aortic cannula and out of the pulmonary artery cannula through cuvettes containing 2-mm diameter Clark-type O2 electrodes to measure the arterial and venous O2 tension. The response times were 0.71 and 1.75 s for the arterial and venous O2 measurement systems, respectively (for mathematical definition of response time, see Ref. 40).
Mb oxygenation was measured (5, 39) with an NIR tissue O2 monitor (RUNMAN CWS 2000 unit; NIM, Philadelphia, PA); a tungsten bulb, positioned ~1 cm from the left ventricular (LV) surface, flashed with a 3-V Square wave at 0.42 Hz (duty cycle 25%). A light guide, barely touching the LV surface, conducted the NIR radiation emanating from the tissue to two silicon photo diodes fitted with filters at 760 and 850 nm (half-intensity bandwidth 20 nm). The tungsten bulb and light guide were separated by approximately one-third of the circumference of the heart. The difference signal between 760 and 850 nm gives tissue oxygenation.Experimental protocol. Mb in the heart was completely deoxygenated by switching to Tyrode solution saturated with 95% N2-5% CO2, which was additionally passed through a membrane oxygenator (VPCML, Cobe Laboratories, Lakewood, CO) in the perfusion line gassed with 95% N2-5% CO2 (step to N2). Subsequently, Mb was reoxygenated by perfusion with oxygenated Tyrode solution additionally passed through the oxygenator gassed with 95% O2-5% CO2 (step to O2). Finally, KCN (end concentration 2 mmol/l) was infused for complete reduction of Cyt Ox during perfusion with oxygenated solution so that Mb was fully oxygenated. The decline of the NIR signal during KCN infusion (Cyt Ox reduction, high Mb oxygenation) divided by the average amplitude of the NIR signal during the steps between N2 and O2 (Cyt Ox reduction, complete Mb deoxygenation) gives the relative contribution of the Cyt Ox redox state to the NIR signal.
Possible nonspecific effects of KCN on NIR absorption were tested in vitro; the addition of 2 mmol/l KCN to rabbit blood diluted with 0.9% NaCl solution (100 and 250 µmol/l hemoglobin) did not lead to reduction of the NIR signal (n = 2 for each hemoglobin concentration). Full reduction of Cyt Ox by blocking with KCN gives the same NIR spectral change as transitions to anoxia (21). Thus changes of the NIR signal after KCN infusion in the isolated heart were due to reduction of Cyt Ox and not to nonspecific effects of KCN on heme or Cyt Ox.Group B
Preparation. Male rabbits (n = 7), weighing 2.9 ± 0.2 kg (SD), were anesthetized with 10 mg/kg fluanisone and 0.32 mg/kg fentanyl citrate injected intramuscularly, supplemented with 30 mg pentobarbital sodium injected intravenously. The aorta was subsequently cannulated in situ, after intravenous injection of 2,500 IU heparin, for perfusion according to Langendorff at 37°C with a constant flow of 95% O2-5% CO2-saturated Tyrode buffer, in this case with 128.3 and 4.7 mmol/l NaCl and KCl, respectively. The left ventricle (LV) was drained via a cannula (ID 1.5 mm) in the apex. LV pressure was measured with a water-filled balloon connected to a pressure transducer (Statham P23 Db). The heart was paced with electrodes on the right ventricle after the atrioventricular node was crushed so that spontaneous heart rate was <80 beats/min. The rest of the preparation and perfusion was the same as that for rats.
Measurements. Arterial and venous O2 tensions and Mb oxygenation were measured as described for group A. However, the tungsten bulb of the NIR oximeter was flashed with 4 V instead of 3 V because the rabbit hearts were larger than the rat hearts.
Heart rate was instantaneously switched between two preset pace frequencies with a stimulator that was built in our workshop. Details of heart isolation, experimental setup, and measurements are described elsewhere (37, 38).Experimental protocol. The protocol consisted of two parts: the first part to assess the response times, and the second part to quantify the relative contributions of Mb oxygenation and Cyt Ox redox state to the NIR signal during full-scale changes in tissue oxygenation (step to N2 followed by step to O2).
In part 1, heart rate steps were made in random order from 120 beats/min to either 140, 160, 190, or 220 beats/min and, in each case, back to 120 beats/min. Subsequently, downward and upward steps in perfusion flow (10.7 ± 0.3%) and arterial O2 concentration ([O2]; 5.7 ± 0.8%) were made to characterize O2 transport delays (see below) (40). Finally, the indicator Evans blue, bound to albumin, was infused at 120 and 220 beats/min to measure the intravascular volume (40). In part 2, steps were made between O2 and N2. First, when the NIR signal stabilized after the step from O2 to N2, KCl was briefly infused (end concentration 25 mmol/l) to induce transitory cardiac arrest. A possible change of the NIR signal after the cessation of cardiac contraction would indicate that the signal was sensitive to movement artifacts. After stabilization of the NIR signal following the step to O2, KCl was infused once again to test whether changes in the NIR signal might occur, in this case due to movement artifacts as well as to an increase in Mb oxygenation due to lower O2 consumption. Different effects of KCl infusion during O2 and N2 can therefore indicate that Mb was not completely oxygenated at 120 beats/min. Finally, KCN was infused to completely reduce Cyt Ox (see protocol for group A).Response Times of Mitochondrial O2 Consumption, Mb Deoxygenation, and Arterial and Venous [O2] to Steps in Heart Rate and to Steps Between N2 and O2
The response time of mitochondrial O2 consumption to steps in heart rate (tmito) characterizes the adaptation speed of oxidative phosphorylation at the level of the mitochondria and has been described in detail (40). Briefly, the time course of the venous O2 tension after a step in heart rate (tv, see Fig. 1), is corrected for diffusion and vascular transport delay (ttransport), estimated from the time course of the response of venous O2 tension to stepwise reductions of arterial O2 tension and perfusion flow so that tmito = tv
ttransport. The
buffering of O2 by Mb is taken
into account in
ttransport by
using and assessing the effective
O2 solubility in tissue that
includes MbO2. The product of
systolic LV pressure and heart rate (rate-pressure product) shows an
initial overshoot during steps between different heart rates. The
tmito is
additionally corrected for this phenomenon (see Ref. 14 for details).
The response times of Mb oxygenation
(tMbO2)
during steps in heart rate and
N2-to-O2
transitions and response times of arterial and venous
[O2]
(ta and
tv) during
N2-to-O2
transitions were defined analogous to
tv (see Ref. 40
for exact mathematical definition). The
ta characterizes
the speed of switching solutions in the arterial perfusion system.
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Estimation of Response Time of End-Capillary [O2]
From the measured tv we subtracted the estimated transit time of O2 from end of the capillaries to the venous O2 electrode as follows. From the total intravascular volume, calculated from the transit time of Evans blue (see above), the volume of the aortic cannula (0.15 ml) and the anatomic intracapillary and intra-arterial volumes (0.035 and 0.038 ml/g wet wt, respectively) (40) are subtracted. The resulting volume of venules, veins, right ventricle, and pulmonary cannula divided by the flow gives the transit time from the end of the capillaries to the measurement site of venous O2 tension, which is subtracted from tv to obtain tend capillary.Contribution of MbO2 to Change in O2 Consumption
Vd, m (in ml/g wet wt) is the ratio of the change in total tissue O2 stores [
Q
(MbO2 plus physically dissolved
O2 pool)], caused by a
change in O2 consumption, to the
change in venous [O2]
(CvO2)
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Statistical Analysis
Mitochondrial, venous, and Mb response times were tested for differences with repetitive-measurements one-way analysis of variance, followed by Newman-Keuls post hoc tests. Unpaired Student's t-tests were used to determine whether Mb deoxygenation differed significantly from zero. The average O2 consumption before and after an intervention was compared with the O2 consumption during an intervention using Student's paired t-test. Data were given as means ± SE, unless indicated otherwise. Test results were considered significant at P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Nonblotted wet weights of rat and rabbit hearts were 3.2 ± 0.3 and
11.2 ± 0.8 g, respectively. Dry weights, measured after 2 days of
dehydration at 60°C, were 0.29 ± 0.02 and 1.38 ± 0.09 g,
respectively. Perfusion flow was 8.6 ± 0.9 and 7.4 ± 1.2 ml · min
1 · g
wet weight1 in rat and
rabbit hearts, respectively.
In the KCl-arrested rat hearts the NIR signal decreased after KCN
infusion by 25.1 ± 4.4% of the full change of the NIR signal during the
N2/O2
step, indicating that Cyt Ox contributed one-fourth to the NIR signal.
In rabbit hearts paced at 120 beats/min, this decrease was 31.6 ± 2.3% (P < 0.05; Figs.
2 and 3). In
the rabbit hearts, Mb oxygenation decreased significantly by 6.9 ± 1.3 or 11.1 ± 2.1% of the amplitude of the
O2-to-N2
transition during heart rate steps from 120 to 190 or 220 beats/min,
respectively (P < 0.05; see Figs. 1
and 2). In four of seven experiments the NIR signal increased by 11.9 ± 4.1% of that during the
O2-to-N2 transition when KCl was infused during
O2 perfusion and by 15.4 ± 6.5% when KCl was infused during
N2 perfusion (Fig. 2). In the remaining three experiments no change was observed during KCl infusion.
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The tmito,
tv,
tMbO2,
and
tend capillary
to steps in heart rate are given in Table
1. For the step to 190 beats/min, tMbO2
was significantly slower than both
tmito
(P < 0.001) and
tend capillary
(P < 0.05). For the step from 120 to
220 beats/min, tMbO2
differed significantly from
tmito
(P < 0.001) but not from
tend capillary
(P > 0.05).
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For the
O2-to-N2
and
N2-to-O2
-transitions, the
ta,
tv,
tMbO2
are given in Table 2. During
the
O2-to-N2
transition, tMbO2
was significantly (P < 0.001) larger
than tv, whereas the opposite occurred during the
N2-to-O2
transition (P < 0.01).
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During the reductions in perfusion flow (by 11%) and arterial
[O2] (by 6%) at 220 beats/min, there was a significant Mb deoxygenation by 1.5 ± 0.4 and 1.5 ± 0.4% of the full
N2/O2
transition, respectively (P < 0.05). Only during 11% perfusion reduction at 220 beats/min did O2 uptake decrease
significantly (P < 0.05; Fig.
4A).
During the reduction in perfusion flow at 120 and 220 beats/min,
systolic LV pressure declined significantly by 2.6 ± 0.9%
(P < 0.05) and 5.5 ± 0.8%
(P = 0.018). These decreases
in O2 uptake, systolic LV
pressure, and NIR signal occurred despite high venous
O2 tensions, which were 428 ± 15 mmHg at 120 beats/min and 371 ± 17 mmHg at 220 beats/min and
decreased by 5.7 ± 0.9 and 6.3 ± 1.1%, respectively, during
the 11% reduction in perfusion flow. For the 6% reduction in arterial
[O2], these values
were 457 ± 13 and 377 ± 17 mmHg and decreased by 8.3 ± 1.1 and 9.7 ± 1.4% for 120 and 220 beats/min, respectively.
The extent of Mb deoxygenation was not correlated to the decrease of
systolic LV pressure during the reduction of both arterial
[O2] (slope 0, P > 0.05) and perfusion flow (slope 1.5 ± 0.7, P > 0.05) (Fig.
4B).
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The contribution of MbO2 to the changes in total tissue O2 stores was calculated (see METHODS) to be 12 ± 3 or 13 ± 3% (P < 0.05) for heart rate steps from 120 to 190 or 220 beats/min, respectively.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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NIR Signal
We assessed the relative contribution of Cyt Ox to the NIR signal experimentally. Any differences in penetration depth of NIR radiation between 760 and 850 nm were included in the quantification. The experimentally assessed contribution of 25.1% corresponded closely to the calculated 23.9% (see Tables 3 and 4) for which identical penetration depth independent of wavelength is assumed. Differences in effective optical path lengths through the tissue were small, 1.8% larger at 754 nm than at 816 nm (3). The assumption of a 3% larger path length at 760 nm than at 850 nm changes the calculated Cyt Ox contribution from 23.9 to 22.5%. Because Cyt Ox reduction during hypoxia causes changes in the NIR difference signal in the same direction and proportional to changes in Mb deoxygenation (34), we may conclude that the NIR signal indicates relative changes in Mb oxygenation accurately.
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The higher Mb content of 190 nmol/g wet weight in rat heart compared with 160 nmol/g wet weight in rabbit heart could explain the higher relative contribution of Cyt Ox (31.6%) in rabbit heart (41). The value of 160 nmol/g wet weight in blood-perfused rabbit hearts (41) compares favorably with that of 122 nmol/g wet weight found in our saline-perfused hearts, given that saline perfusion decreases the dry-to-wet weight ratio by ~25% compared with that of the heart in situ. Changes in the NIR signal as small as 0.2% from full scale could be detected by assessing NIR signals before, during, and after an intervention. In four of seven experiments the NIR signal increased on average by 12% during KCl arrest with Mb in the oxygenated state. These changes are likely caused by motion artifacts, because similar changes were detected in these four experiments when KCl was infused during N2 perfusion. The apparent lack of increase of Mb oxygenation when O2 consumption was decreased by arresting the heart indicates that Mb is almost fully oxygenated at 120 beats/min. The motion artifact during KCl arrest is due to the loss of contractility and flaccidity that was visually apparent and might change the contact between the light guide and the cardiac surface; in three of seven experiments this artifact was absent. After the heart rate step to 190 or 220 beats/min, the NIR signal decreased in three of seven experiments after a delay of ~4 s (not shown), indicating that during steps in heart rate the NIR signal reflects tissue oxygenation rather than movement artifacts, because the latter should have led to immediate changes.
Response Times and Changes in Tissue O2 Stores
During the heart rate steps MbO2 was deoxygenated by only 7-11%, corresponding with a 12-13% contribution of MbO2 to the change in total tissue O2 stores during the increase in O2 consumption. Because of this small change in MbO2 and because physically dissolved O2 cannot be measured, it is not yet possible to improve the determination of the full time course of oxidative phosphorylation during steps in heart rate. However, the reader should be aware that for the determination of the response time tmito, the effect of Mb deoxygenation is taken into account (40). It is remarkable that Mb deoxygenation contributes relatively little to the transients in oxidative phosphorylation, given that the saline-perfused heart is often suspected to be poorly oxygenated (20, 37).From the NIR data and response times, the tissue [O2] can be calculated, but it is necessary to assume that O2 is distributed homogeneously in the tissue. Although this assumption is probably unrealistic, the calculation is still instructive. The binding constant of O2 for Mb (KMb) is taken to be 3.2 µmol/l (31). The intracellular [Mb] is 154 µmol/l, corresponding to 122 µmol per liter of tissue (intra- plus extracellular spaces, see METHODS). Intracellular MbO2 concentration therefore decreases by 16.9 µmol/l (11%) during the heart rate step from 120 to 220 beats/min, corresponding to 13.42 µmol per liter of tissue. This is 13% of the change in total tissue O2 stores in intracellular plus interstitial space (see RESULTS). The decrease of physically dissolved [O2] from 120 to 220 beats/min is therefore (87/13) × 13.42 µmol/l = 89.8 µmol/l. Given that a 89.8 µmol/l decrease in [O2] leads to an 11% decrease in Mb saturation, the hyperbolic relation Mb saturation = [O2]/([O2] + KMb) leads to two equations (for the Mb saturation at the two heart rates) with two unknowns that resolve to a quadratic equation with the positive root [O2] = 109.7 µmol/l at 120 beats/min. Tissue [O2] decreases to 19.9 µmol/l at 220 beats/min. According to this calculation, the Mb saturation would be 97.2% at 120 beats/min, close to full saturation as already concluded from the NIRS change during cardiac arrest. Mb saturation decreases to 86.2% at 220 beats/min. Thus, when homogeneous tissue [O2] is assumed, physically dissolved O2 represents 48.1% [109.7/(118.6 + 109.7)] of the total tissue O2 at 120 beats/min and 15.9% [19.9/(105.2 + 19.9)] at 220 beats/min. In conclusion, in saline-perfused rabbit hearts, the contribution of MbO2 to the increase in O2 consumption during a step in heart rate is small because of relatively high intracellular [O2], and physically dissolved O2 is more important as an O2 buffer than MbO2 in saline-perfused hearts.
The tissue [O2] is low compared with the venous O2 tension, which is >370 mmHg even at 220 beats/min. The arterial-to-venous [O2] gradient provides no explanation for the relatively low tissue [O2] because the intravascular [O2] is high in both arteries and veins in this preparation. The difference between venous and tissue [O2] can be explained by heterogeneity of the ratio between local metabolism and perfusion or by large diffusion gradients and diffusion times. Mb oxygenation follows arterial [O2] without a significant time delay after the step back from N2 to O2 (Fig. 2), indicating that the Mb is quickly reoxygenated without appreciable diffusion delay. If diffusion times for O2 into and out of the cell are similar, the delay in deoxygenation during the step from O2 to N2 is not due to diffusion delay but suggests that at least in some regions the tissue [O2] is decreased without leading to Mb deoxygenation, suggesting that the tissue [O2] is far above the KMb for Mb in these regions. This is further supported by the observation that during heart rate steps (Fig. 1) tend capillary lags tmito by only ~5 s, indicating that diffusion times are <5 s. However, a large part of this 5-s delay is actually due to perfusion-limited transport delay. During the step from O2 to N2, tMbO2 lags both tv and ta by 21 s, which is much more than can be explained by diffusion delay. This large delay is explained by assuming that the tissue [O2] is greater than the KMb for Mb in most of the tissue. During gradual tissue deoxygenation, venous and tissue [O2] decrease without appreciable Mb deoxygenation, and only when tissue [O2] reaches the steeply declining part of the Mb dissociation curve does Mb oxygenation also start to decline during O2 washout. Figure 2 shows that Mb oxygenation only starts to fall when the venous O2 tension has dropped by 80% during an arterial switch to N2. In addition, during the step to 190 beats/min, tMbO2 is significantly larger than tend capillary. In response to a step in heart rate, physically dissolved O2 diffuses to the mitochondria and end capillary O2 tension decreases before Mb oxygenation decreases with a delay, contributing only a minor part of the O2 used for the increased O2 consumption. It can be concluded that the large difference between the tissue [O2] and venous O2 tension cannot be explained by large diffusion times. However, it is possible that heterogeneity of perfusion relative to local O2 consumption does result in locations with low venular [O2]. Because the mixed coronary venous O2 tension is more strongly determined by the high-flow regions, a high venous O2 tension exists in the presence of regions with relatively low local intravascular O2 tensions.
At 120 beats/min, tissue [O2] in most regions is much higher than the KMb of Mb. The hypoxic areas at high heart rates occupy only a limited fraction of the heart; otherwise, venous O2 tension and average Mb oxygenation would be much lower. The hypoxic areas would be responsible for immediate local deoxygenation of MbO2 (in 4 of 7 hearts) on a step in heart rate to 220 beats/min and for the 1.5% deoxygenation during a 6% decrease in arterial [O2] at 220 beats/min. The large well-oxygenated part of the heart would prevent the NIR signal from decreasing more than ~11% at increased heart rate. Steenbergen et al. (32, 33) showed that, during high-flow and low-flow hypoxia, growing areas of high NADH fluorescence appeared surrounded by nonfluorescent areas, suggesting coexistence of hypoxic areas and well-oxygenated areas. The same phenomenon may develop in this study by increased pacing frequency. Indeed, patchy NADH fluorescence appeared in our rabbit heart preparation at very high heart rates (J. F. Ashruf, J. B. Hak, J. H. G. M. van Beek, and C. Ince, unpublished observations).
The tmito is ~2-3 times faster than tend capillary and tMbO2, strongly suggesting that the immediate adaptation of oxidative phosphorylation to a step in heart rate is not limited by O2 availability, because MbO2 is not yet appreciably decreased when O2 consumption has already reached a new, higher level. Instead, transcytosolic energy transfer speed between sites of ATP consumption and mitochondria seems to be a more important determinant of tmito. This is suggested by our recent observations in isolated rabbit hearts in which inhibition of mitochondrial aerobic capacity did not affect tmito but inhibition of creatine kinase accelerated tmito (7, 15).
During reductions of perfusion flow at 220 beats/min, O2 uptake, systolic LV pressure, and Mb oxygenation decreased. The question is whether this decrease in O2 uptake is due to the significant decrease in Mb oxygenation. Systolic LV pressure is not sensitive to Mb deoxygenation during reduction of arterial [O2] (Fig. 4B), suggesting that the reduction in systolic pressure is not caused by decreased tissue oxygenation per se. The decrease was previously suggested to be due to the garden hose effect: a change in perfusion pressure results in a change in the diastolic stretch for the myocytes surrounding the coronary vessels and thus causes a change in O2 demand (1). However, it has been shown that, during mild reductions of perfusion flow, inorganic phosphate (Pi) levels increase (10, 26). The decrease of systolic LV pressure we observed might be caused by local increases in Pi secondary to deteriorated metabolism-perfusion matching and localized severe tissue deoxygenation (Fig. 4B) caused by the decrease of perfusion pressure (4).
In conclusion, our data strongly suggest that in saline-perfused rabbit heart, at 37°C, increased O2 consumption in response to a step in heart rate is mainly buffered by physically dissolved O2, not by MbO2. Diffusion of O2 to the mitochondria does not limit the adaptation speed of oxidative phosphorylation to a step in heart rate. At 120 beats/min, tissue appears well oxygenated and global O2 supply appears sufficient. However, an increase in heart rate led to Mb deoxygenation despite maximal vasodilation, and O2 supply limitation of aerobic metabolism and contractility began to appear at high heart rates.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. de Groot, Laboratory for Physiology, Institute for Cardiovascular Research, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands (E-mail: b.de_groot.physiol{at}med.vu.nl).
Received 28 July 1998; accepted in final form 12 January 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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) or
perfusion flow (
) at 220 beats/min.