INTRODUCTION

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BOTH THERMODYNAMIC AND KINETIC models of oxidative phosphorylation regulation in the heart incorporate regulatory roles for myocardial ADP, P_i, NADH, and O₂ (3, 8). O₂ extraction by the heart is greater than in most other organs, with resting coronary venous PO₂ commonly as low as 20-25 mmHg in the normal in vivo heart (32). Because the very high level of myocardial O₂ extraction during basal conditions limits the ability to increase O₂ uptake by augmenting extraction, increases of O₂ demands during exercise or other stress must be met by parallel increases of coronary blood flow (32). Furthermore, reductions of coronary blood flow by as little as 10-20% in the intact in vivo heart cause decreases of contractile performance, suggesting that even modest reductions of myocardial perfusion can result in O₂ insufficiency (31). Kreutzer and Jue (18) examined the critical O₂ level at which O₂ availability failed to meet cellular energy demands in isolated perfused rat hearts working at 25°C. Using ¹H-NMR spectroscopy to assess the degree of myocardial myoglobin desaturation and to calculate intracellular PO₂ during progressive decreases of O₂ in Krebs-Henseleit perfusion buffer, they observed that the phosphocreatine (PCr) level began to decrease at an intracellular PO₂ of ~2 mmHg and fell to 80% of the basal value at an intracellular PO₂ of 1.8 mmHg (18). Further reductions of perfusate oxygenation resulted in sharp further decreases of PCr. Since these data were obtained in isolated hearts perfused with non-Hb-containing buffer at 25°C, it is unclear whether a similar relationship would exist in the intact heart in vivo. Consequently, the present study was carried out to examine the effect of decreasing myocyte oxygenation on myocardial high-energy phosphate (HEP) content in the intact heart in vivo. Graded reductions of coronary blood flow were produced to document the threshold level of myocyte oxygenation at which perceptible reductions of the PCr-to-ATP ratio (PCr/ATP; corresponding to an increase of myocardial free ADP) occurred and to examine the effect of progressive reductions of myocyte oxygenation on myocardial HEP content.

	METHODS

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Studies were performed in 10 adult mongrel dogs of either gender weighing 20-27 kg. All experimental procedures were approved by the University of Minnesota Animal Resources Committee. The investigation conformed to the Guide for the Care and Use of Laboratory Animals [DHHS Publ. No. (NIH) 85-23, revised 1985, Office of Science and Health Reports, Bethesda, MD 20892].

Experimental preparation. The dogs were anesthetized with pentobarbital sodium (30-35 mg/kg bolus followed by 4 mg · kg¹ · h¹ iv), intubated, and ventilated with a respirator with supplemental O₂ to maintain arterial blood gases within the physiological range. A heparin-filled 3.0-mm-OD polyvinyl chloride catheter was introduced into the right femoral artery and advanced into the ascending aorta. A left thoracotomy was performed through the fourth intercostal space, and the heart was suspended in a pericardial cradle. A heparin-filled 3.0-mm-OD catheter was introduced into the left ventricle (LV) through the apical dimple and secured with a purse-string suture. A similar catheter was inserted into the left atrium through the atrial appendage. A 1.5- to 2.0-cm segment of the proximal left anterior descending (LAD) coronary artery was dissected free, and a hydraulic occluder constructed of 2.7-mm-OD polyvinyl chloride tubing was placed around the artery. A silicone-elastomer catheter (0.75 mm ID) was placed into the LAD distal to the occluder by the method of Gwirtz (11). The region of the LV that became cyanotic on inflation of the occluder was determined by visual inspection, and a 28-mm-diameter NMR surface coil was sutured onto the pericardium overlying the ischemic area. The pericardial cradle was then released, the heart was allowed to assume its normal position, and the surface coil leads were connected to a balanced tuned circuit. Animals were placed on a water-filled thermal jacket connected to a circulating water bath to maintain rectal temperature at 37-38°C and placed into the magnet.

NMR general methods. Measurements were performed in a 400-cm-bore 4.7-Tesla magnet interfaced with a SISCO (Spectroscopy Imaging Systems, Fremont, CA) console. The LV pressure signal was used to gate NMR data acquisition to the cardiac cycle, while respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions (22, 24). ³¹P- and ¹H-NMR frequencies were 81 and 200.1 MHz, respectively.

NMR deoxymyoglobin methods. The method for ¹H-NMR detection of the proximal histidyl N- proton resonance of deoxymyoglobin (Mb-) has been described in detail (5). Briefly, a single-pulse collection sequence with a Gaussian pulse (1 ms) was used to selectively excite the N- proton signal of the proximal histidyl of Mb-. This frequency-selective pulse provided sufficient water suppression because of the large chemical shift difference between the water resonance and Mb- (>14 kHz). A short repetition time (TR = 35 ms) was used because of the short T1 value of Mb-. Each spectrum is acquired within 6 min (10,000 free induction decays). Although the short T1 of Mb- and the fast acquisition prevent gating of data acquisition to the cardiac cycle, signal loss as a result of heart motion was negligible because of the inherently broad line width of the Mb- peak. The Mb- resonance was detected at 71-72 ppm (relative to water) and remained virtually constant during the study protocol. No other resonances were detected within a 10-ppm region. We previously demonstrated that because of the short T2 relaxation times of the - and -subunits of deoxyhemoglobin, these resonances are not NMR visible in canine myocardium in vivo at 4.7 Tesla when a 1-ms Gaussian excitation pulse is used (5). Thus Mb- is detected with no interference from deoxyhemoglobin.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Sensitivity profile for the 1H-NMR signal across the left ventricular (LV) wall. Sensitivity for detection of 1H nuclei is proportionate to the height of the curve. x-Axis, distance, with the epicenter of the magnet at 0.0 cm; arrow, location of the surface coil; dashed vertical lines, epicardial (Epi) and endocardial (Endo) boundaries of the LV wall. Data demonstrate that the deoxymyoglobin resonance (Mb-) signal originates from all layers of the LV wall, with somewhat higher contribution from the mid- to the inner myocardium.

Spatially localized ³¹P-NMR technique. ³¹P-NMR spectra were acquired in late diastole with a pulse repetition time of 6-7 s. This repetition time allowed full relaxation for ATP and P_i resonances and ~90% relaxation for the PCr resonance. PCr resonance intensities were corrected for this minor saturation. RF transmission and signal detection were performed with a 28-mm-diameter surface coil. A capillary containing 15 µl of 3 M phosphonoacetic acid was placed at the coil center to serve as a reference. The proton signal of the water resonance was used to homogenize the magnetic field and to adjust the position of the animal in the magnet so that the coil was at or near the magnet and gradient isocenter. This was accomplished using a spin-echo experiment with a readout profile. The information gathered in this step was also utilized to determine the spatial coordinates for spectroscopic localization. Chemical shifts were measured relative to PCr, which was assigned a chemical shift of 2.55 ppm relative to 85% phosphoric acid at 0 ppm. Spatial localization across the LV wall was performed with the RAPP-ISIS/FSW method. The technical details of this method, including voxel profiles, voxel volume, and the accuracy of spatial localization obtained in phantom studies and in vivo, have been published elsewhere (12, 24, 26). Briefly, signal origin was restricted by using B₀ gradients and adiabatic inversion pulses to an 18 mm × 18 mm column coaxial with the surface coil and perpendicular to the LV wall. Within this volume, the signal was further localized by using the B₁ gradient to five voxels spanning the LV wall from epicardium to endocardium. Each set of spatially localized transmural spectra consisted of a total of 96 scans accumulated in a 10-min block. Resonance intensities were quantified by using integration routines provided by SISCO software. The values for PCr and ATP in each voxel were normalized to those present in the basal state, and the PCr-to-ATP ratio (PCr/ATP) was determined for each voxel. P_i resonances were also measured, and the ratio of P_i to PCr (P_i/PCr) was calculated.

Myocardial blood flow measurements. Myocardial blood flow was measured using 15-µm-diameter microspheres labeled with ⁵¹Cr, ⁸⁵Sr, ⁹⁵Nb, and ⁴⁶Sc. Microsphere suspension containing 2 × 10⁶ microspheres was injected through the left atrial catheter, while a reference sample of arterial blood was withdrawn from the aortic catheter at a rate of 15 ml/min beginning 5 s before the microsphere injection and continuing for 120 s. Radioactivity in the myocardial and blood reference specimens was determined using a gamma spectrometer (Packard Instruments, Downers Grove, IL) at window settings chosen for the combination of radioisotopes used during the study. Activity corrected for overlap between isotopes and for background was used to compute blood flow as milliliters per gram of myocardium per minute.

Myocyte oxygenation. Myocyte oxygenation was estimated from the myoglobin O₂ saturation-PO₂ relationship as follows

(1)

where Mb-O₂ and Mb- are fractional contents of oxymyoglobin and deoxymyoglobin, respectively, PO₂ is the intramyocyte PO₂, and (PO₂)₅₀ is the PO₂ at which myoglobin is half-saturated with O₂ (2.38 mmHg at 37°C) (27). Therefore, if the integral of the Mb- resonance intensity measured during complete coronary occlusion represents total myoglobin content (in the Mb- form), then the resonance integrals determined during other experimental conditions can be normalized relative to this value to result in Mb- expressed as a fractional value of total myoglobin content. The fractional content of myoglobin O₂ during each experimental period can be calculated from the relationship Mb-O₂ = 1  Mb-. The fractional contents of oxymyoglobin and Mb- and the temperature-corrected (PO₂)₅₀ can then be employed to calculate intracellular PO₂ using Eq. 1. Because of continuing collateral blood flow, it is unlikely that myoglobin would be completely deoxygenated during coronary occlusion. Consequently, calculations were performed assuming that the Mb- resonance measured during coronary occlusion represented 90 or 95% of the total myoglobin, and the Mb- resonances obtained during the study were normalized in relation to that value. Similarly, the fractional myoglobin O₂ content in the basal state (i.e., at a time when no Mb- resonance was detected) could not be directly determined (5). On the basis of a recent report using reflectance spectroscopy in open-chest swine which demonstrated that myoglobin O₂ saturation is >92% under basal conditions, we calculated intramyocardial PO₂ values assuming that the fractional Mb- content in the basal state was 90 or 95%. We also carried out calculations assuming a basal Mb- O₂ saturation of 80%, although a value this low is unlikely, since we have found that the present technique can detect ~10% fractional content of Mb- (unpublished observations).

Study protocol. Aortic and LV pressures were measured with fluid-filled pressure transducers positioned at midchest level and recorded on an eight-channel direct-writing recorder (Coulbourne Instrument, Lehigh Valley, PA). LV pressure was recorded at normal and high gain for measurement of end-diastolic pressure. Hemodynamic measurements and ³¹P- and ¹H-NMR spectra were first obtained under basal conditions. Midway through the 20-min data acquisition period, a microsphere injection was performed for determination of myocardial blood flow. After completion of baseline measurements, the hydraulic coronary occluder was slowly inflated with a micrometer-driven syringe to reduce distal LAD pressure to ~60 mmHg (stenosis I). When a stable coronary pressure had been maintained for 5 min, ³¹P and ¹H spectra were obtained, and a microsphere injection was performed. The occluder was then further inflated to decrease distal coronary pressure to ~50 mmHg (stenosis II), and all measurements were repeated. Measurements were then obtained at a coronary pressure of ~40 mmHg (stenosis III). Finally, the LAD was completely occluded (total occlusion), and all measurements were repeated. In three animals, hearts were stained with triphenyltetrazolium chloride to ensure that the experimental protocol did not result in myocardial necrosis. In these animals, coronary reperfusion was allowed for 2 h after completion of the protocol before the hearts were harvested. The hearts were then sectioned into 1-cm-thick layers parallel to the mitral valve annulus and incubated for 15 min in 4 g of triphenyltetrazolium chloride dissolved in 400 ml of Sörenson's buffer at 37°C. No evidence of infarct was observed in any of these hearts.

Data analysis. Hemodynamic data were measured from the chart recordings. ³¹P spectra were analyzed as described above. Transmural blood flow distribution was determined from the microsphere measurements. Data were analyzed with one-way ANOVA for repeated measures. P < 0.05 was considered significant. When a significant result was found, individual comparisons were made using the method of Scheffé.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Hemodynamic and blood flow data. Hemodynamic and myocardial blood flow measurements during each experimental condition are shown in Table 1. Heart rate tended to increase and mean aortic pressure tended to decrease with increasing stenosis severity, although these changes did not achieve statistical significance. LV systolic pressure decreased significantly during stenosis II and III, as well as during total occlusion (each P < 0.05). LV end-diastolic pressure increased significantly with application of stenosis I and underwent a further significant increase during total occlusion (P < 0.05). Coronary perfusion pressure progressively decreased as the degree of stenosis was increased.

Myocyte oxygenation and HEP levels. Typical ¹H and ³¹P spectra are shown in Figs. 2 and 3. To take into account differences in absolute blood flow between animals (reflecting variations in the rate of basal state metabolism), blood flow for each individual animal was normalized to the basal state value. As shown in Fig. 4, reductions of myocardial blood flow produced by progressive increases in stenosis severity resulted in parallel stepwise decreases of myoglobin O₂ saturation. The relationship between myoglobin saturation and blood flow was well described by the linear regression equation shown in Fig. 4A (R² = 0.89, P < 0.01). When intracellular PO₂ was calculated from the Mb- measurements, an exponential relationship was observed between myocardial blood flow and intracellular PO₂ (Fig. 4B).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Typical 31P-NMR spectra showing myocardial high-energy phosphate (HEP) and Pi resonances from 1 heart under baseline conditions (A), during coronary stenosis that reduced distal coronary pressure to 40 mmHg (B), and during total left descending coronary artery occlusion (C). 2,3-DPG, 2,3-diphosphoglycerate; PCr, phosphocreatine.

View larger version (6K): [in this window] [in a new window]   Fig. 3.   Interleaved 1H-NMR spectra obtained from the same heart as in Fig. 1 showing myocardial Mb- resonances under baseline conditions (A), during coronary stenosis that reduced distal coronary pressure to 40 mmHg (B), and during total left descending coronary artery occlusion (C).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Myoglobin percent saturation (A) and calculated myocyte PO2 (B) plotted against myocardial blood flow (MBF) normalized to the prestenosis control value. It was assumed that the Mb- resonance measuring total coronary occlusion represented 95% of the total myoglobin and that the fractional myoglobin O2 content in the basal state was 90%.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   PCr (A) and ATP (B) normalized to the prestenosis control value plotted against myocyte PO2. PCr decreased to 80% of the control value when intracellular PO2 had decreased to 4.4 mmHg, while ATP was reduced to 80% of the control value at PO2 of 1.1 mmHg. It was assumed that the Mb- resonance measuring total coronary occlusion represented 95% of the total myoglobin and that the fractional myoglobin O2 content in the basal state was 90%.

	DISCUSSION

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In a previous study using ¹H-NMR, we found that no Mb- was visible in in vivo canine or swine myocardium operating at basal or elevated work states, despite the observation that HEP levels fell during high work states (34). This suggested that, even at high work states, O₂ availability was nonlimiting and indicated that HEP changes that occur at high workloads cannot be ascribed to O₂ limitation. The present study was carried out to define the relationships between myocardial blood flow, myocyte PO₂, and HEP levels in in vivo myocardium under conditions where limited O₂ availability does limit the rate of oxidative phosphorylation.

The effect of decreased O₂ availability on myocardial HEP has been reported previously in isolated working rat hearts perfused with Krebs-Henseleit buffer at 25°C. Using progressive reductions of the perfusate flow rate while intracellular PO₂ was determined from the degree of myoglobin oxygenation using ¹H-NMR spectroscopy, Kreutzer and Jue (19) observed that when the intracellular PO₂ fell to ~4 mmHg, contractile performance decreased and myocardial lactate production occurred. At this time, myocardial O₂ consumption and HEP remained normal. Not until intracellular PO₂ had fallen to 2 mmHg was there a perceptible decrease in myocardial PCr and O₂ consumption. The findings suggested that an increase in myocardial NADH was able to compensate for the decreased PO₂ at PO₂ values between 4 and 2 mmHg, so that the PCr concentration and rate of O₂ consumption were maintained. When using ¹H-NMR measurements of the Mb- resonance for calculation of intracellular PO₂, assumptions must be made concerning the degree of myocardial O₂ saturation during basal conditions as well as the fraction of myoglobin that is deoxygenated during total coronary occlusion. In the dog, a small amount of collateral inflow continues during total arterial occlusion, so that complete myoglobin desaturation is unlikely. Within reasonable limits, the degree of myoglobin O₂ saturation assumed during total coronary occlusion had a very small effect on the calculated PO₂ values at which PCr fell to 80 or 50% of the basal level. However, assumptions concerning the degree of O₂ saturation during basal conditions did influence the calculated critical PO₂ values for PCr and ATP. Thus, if myoglobin was assumed to be 90 or 95% saturated during basal conditions, (PO₂)₈₀ values for PCr were 4.3-4.5 mmHg. However, if myoglobin was assumed to be only 80% saturated during basal conditions, then calculated (PO₂)₈₀ values were decreased by one-half. We believe that the values obtained when myoglobin saturation during basal conditions is assumed to be 90-95% saturated are more likely to be correct for three reasons. First, the ¹H-NMR technique utilized in this study is sufficiently sensitive to detect a proton resonance when myoglobin is >10% desaturated. Second, using data obtained with in vivo reflectance spectroscopy, Arai et al. (1) determined that myoglobin O₂ saturation during basal conditions is on the order of 92%. Finally, Arai et al. observed that infusion of adenosine to increase coronary blood flow and augment O₂ delivery resulted in no change in the myoglobin signal, consistent with the interpretation that myoglobin O₂ saturation is >90% during basal conditions. Using the assumption that myoglobin is 90-95% saturated with O₂ during basal conditions yields values at which PCr was reduced to 80% [(PO₂)₈₀] and 50% [(PO₂)₅₀] of 4.3-4.5 and 1.0-1.2 mmHg, respectively. These values are substantially higher than in the isolated rat hearts, where a perceptible decrease of PCr occurred only when coronary flow rates were decreased sufficiently to reduce intracellular PO₂ below 1.5 mmHg, with (PO₂)₈₀ of 1.1 mmHg and (PO₂)₅₀ of 0.5 mmHg (19). In a subsequent study from the same laboratory, the coronary flow rate was maintained constant while hypoxia was produced by reducing perfusate PO₂; in this situation, the persistently high coronary flow rate would result in washout of lactate and prevent acidosis (18). With this experimental model, slightly higher critical PO₂ values were obtained; PCr began to decrease at an intracellular PO₂ of 2 mmHg, with a (PO₂)₈₀ of 1.8 mmHg. Nevertheless, the intracellular PO₂ values calculated to produce reductions of PCr were higher in the in vivo canine heart in the present study than in the isolated perfused rat hearts. It is possible that reduced metabolic rates of rat hearts perfused at 25°C, compared with normothermic in vivo conditions, could contribute to a lower critical PO₂ for PCr in the perfused hearts.

The response of myocardial HEP levels to decreased coronary perfusion could be altered by changes of myocardial O₂ demands; an active decrease of energy demands in response to O₂ limitation would allow the heart to accommodate to the decreased O₂ availability and delay the development of HEP changes. Gregg (10) demonstrated that myocardial O₂ consumption can be influenced by coronary flow and perfusion pressure. Pressure in the coronary arterial system has been proposed to act to distend the ventricle, thereby augmenting contractility and O₂ consumption by increasing sarcomere length (2). In addition, Kitakaze and Marban (17) observed that, in perfused ferret hearts, changes of coronary pressure and flow resulted in parallel changes in the calcium transient, implying a preload-independent mechanism for perfusion-related alterations of contractile force and O₂ demands. Zündorf and colleagues (35), using isolated rat hearts in which graded hypoxia was produced by decreasing the PO₂ of the perfusate buffer, found that reduced cytochrome aa₃ was not detected until contractile performance had fallen to 25% of control. These findings imply that decreased O₂ availability (despite constant coronary flow) also caused a decrease of contractile performance that allowed the heart to accommodate to the decreased O₂ availability and maintain myocardial energy balance.

Differences in the rate and magnitude of downregulation of myocardial energy demands in response to reductions of coronary perfusion might explain different critical PO₂ values for PCr between in vitro perfused hearts and in vivo hearts. There is evidence that such flow-mediated alterations of contractile performance are more prominent in isolated perfused hearts. Thus, Marshall (20) observed that when coronary flow was decreased in isolated rabbit hearts, tissue lactate did not increase until O₂ consumption and developed pressure had decreased by nearly one-half, while PCr and ATP did not decrease until O₂ consumption and systolic function had decreased to 20-30% of the control level. Similarly, Keller and Cannon (15), using ³¹P-NMR to measure myocardial HEP content in perfused rat hearts, found that modest reductions of coronary flow resulted in proportionate decreases of O₂ consumption and contractile performance without significant reductions of PCr. Adaptations of energy demands in response to reduced coronary perfusion have also been observed in intact animals, but these appear to be of lesser magnitude and to require a longer period of adjustment. Thus, in open-chest swine or dogs, 30-40% reductions of coronary blood flow initially caused a decrease of myocardial PCr, but this was followed by recovery to near-control values within 60 min, despite persistent hypoperfusion (7, 22). This response has been termed the early phase of myocardial hibernation. It is possible that a difference in response to decreased O₂ delivery could explain the difference in curves relating intramyocyte PO₂ to coronary perfusion between perfused rodent hearts and the in vivo hearts in the present study. This is supported by the finding of Kreutzer and Jue (19) in the isolated rat heart that not until coronary flow was decreased from 11 to <1 ml/min was there appearance of Mb- or detectable loss of PCr. In contrast, in the present study a 30% reduction of coronary blood flow caused a perceptible loss of PCr. The rapid ability of the isolated perfused rodent heart to decrease energy demands in response to reduced perfusion could explain a difference in critical PO₂ for PCr from the in vivo heart. An additional concern is whether deoxyhemoglobin present in the in vivo heart might interfere with ¹H-NMR spectroscopic measurements of Mb-. In a previous study from this laboratory, we demonstrated that because of the short T2 relaxation times of the - and -subunits of deoxyhemoglobin, they are not NMR visible in canine myocardium with the 1-ms excitation pulse used in the present study (5).

An important question is why any O₂ limitation should occur at intramyocyte PO₂ values that far exceed the apparent Michaelis-Menten constant of cytochrome oxidase with respect to O₂. Chance (4) observed in isolated myocardial mitochondria that the critical PO₂ at which oxidative phosphorylation began to decrease was ~0.01 mmHg. The finding in the present study that PCr levels began to decrease when intramyocyte PO₂ fell below 5 mmHg indicates that additional factors must influence oxidative phosphorylation in the intact heart. One possibility is that the high critical PO₂ in the intact heart might result, in least in part, from inhomogeneities of O₂ availability; the observed NMR measurements could represent an average of some areas of myocardium that are adequately perfused with some areas that are profoundly ischemic. Decreases of coronary blood flow are known to result in increased spatial heterogeneity of perfusion (23). A flow-limiting arterial stenosis causes redistribution of perfusion away from the subendocardium so that blood flow, HEP, and presumably myoglobin saturation are lowest in the subendocardium (25). This transmural redistribution occurs during hypoperfusion in in vivo hearts and isolated perfused rat hearts (33), so that differences in perfusion pattern could not account for differences in the flow-PO₂ relationship between the two experimental models. Furthermore, the sensitivity profile for detection of Mb- using the present ¹H-NMR spectroscopy system is essentially uniform across the LV wall, so that differences in the myocardial regions sampled between ³¹P- and ¹H-NMR spectra could not account for differences in the critical PO₂ values for PCr and ATP between the present in vivo and the perfused rat hearts.

Inhomogeneities of O₂ availability can also occur at the cellular level. Large PO₂ gradients exist between the erythrocyte and cytosol as the result of diffusional resistance to O₂ transport in the extracellular carrier-free region (13). Furthermore, high-resolution spectrophotometric measurements performed on single cardiac myocytes have demonstrated a steep spatial PO₂ gradient near the sarcolemma, while PO₂ values toward the center of the cell are nearly flat (30). Takahashi et al. (29) observed that as the extracellular PO₂ was decreased, NAD(P)H autofluorescence and myoglobin desaturation became heterogeneous, with greatest fluorescence remote from the sarcolemma in a pattern that was a mirror image to the local intracellular PO₂. A significant PO₂ gradient between the cytosol and the inner mitochondrial membrane could explain the difference between the Michaelis-Menten constant of cytochrome oxidase for O₂ in isolated mitochondrial preparations and the mean cytosolic PO₂ values that are associated with HEP alterations in the intact heart. However, taking into account the low O₂ flux density at the mitochondrial membrane, mathematical models of O₂ diffusion suggest very low perimitochondrial O₂ gradients (6). This is difficult to reconcile with reports that graded reductions of perfusate PO₂ in isolated rat cardiomyocytes resulted in parallel reductions of cytochrome aa₃ oxidation and myoglobin oxidation (coherence) (4, 14). Chance (4) and Ito et al. (14) postulated that since the mitochondrial membrane is impermeable to myoglobin, a significant PO₂ gradient can occur because O₂ must diffuse passively through a myoglobin free volume before it can react with cytochrome oxidase. Using ³¹P-NMR measurements of HEPs, they found that the phosphorylation potential began to decrease when myoglobin oxygenation fell to 80% of control (14). These results are remarkably similar to the present findings in the in vivo heart showing a linear relationship between myocyte oxygenation and PCr.

Ischemia can cause several changes that have opposing effects on the degree of heterogeneity of PO₂. Although perfusion heterogeneity increases when coronary flow is decreased (23), capillary recruitment occurs which can decrease the O₂ diffusion distance and reduce O₂ flux density (21). Since the red cell capillary transit time is inversely proportional to the blood flow rate, it is likely that the extracellular O₂ gradient would decrease as flow rates are reduced by the coronary stenosis. Furthermore, simulated ischemia caused a decrease in the magnitude of the intracellular PO₂ gradient in isolated cardiomyocytes, both as the result of a decrease in the rate of O₂ flux into the cell and a reduction of the rate of O₂ consumption (28). Myoglobin is able to facilitate O₂ diffusion in the cytosol based on a gradient of oxymyoglobin from the sarcolemma to the core of the cell. In the present study and in the report of Arai et al. (1), Mb- was not detectable during control conditions, indicating that myoglobin facilitation is not required to maintain O₂ availability for mitochondrial respiration. However, at the low PO₂ values that exist when flow is limited by an arterial stenosis, myoglobin-facilitated O₂ transport is likely to become increasingly important.

Summary. During graded reductions of coronary blood flow, perceptible loss of PCr first occurred at a mean intracellular PO₂ of ~5 mmHg. Further reductions of coronary blood flow resulted in a precipitous fall of PCr and PCr/ATP as well as progressive increases of P_i. The data indicate that, in the normal in vivo heart, O₂ availability plays an increasing role in regulation of oxidative phosphorylation when mean intracellular PO₂ values fall below 5 mmHg.