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J Appl Physiol 92: 1267-1276, 2002; doi:10.1152/japplphysiol.00194.2001
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Vol. 92, Issue 3, 1267-1276, March 2002

Low-O2 affinity erythrocytes improve performance of ischemic myocardium

Gösta Berlin1, Keith E. Challoner2, and Robert D. Woodson1,2

2 Section of Hematology, Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53792; and 1 Department of Transfusion Medicine, University Hospital, S-571 85 Linköping, Sweden


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

O2 transport and O2 diffusion interact in providing O2 to tissue, but the extent to which diffusion may be critical in the heart is unclear. If O2 diffusion limits mitochondrial oxygenation, a change in blood O2 affinity at constant total O2 transport should alter cardiac O2 consumption (VO2) and function. To test this hypothesis, we perfused isolated isovolumically working rabbit hearts with erythrocytes at physiological blood-gas values and P50 (PO2 required to half-saturate hemoglobin) values at pH of 7.4 of 17 ± 1 Torr (2,3-bisphosphoglycerate depletion) and 33 ± 5 Torr (inositol hexaphosphate incorporation). When perfused at 40 and 20% of normal coronary flow, mean VO2 decreased from the control value by 37 and 46% (P < 0.001), and function, expressed as cardiac work, decreased by 38 and 52%, respectively (P < 0.001). Perfusion at higher P50 during low-flow ischemia improved VO2 by 20% (P < 0.001) and function by 36% (P < 0.02). There was also modest improvement at basal flow (P < 0.02 and P < 0.002, respectively). The improvement in VO2 and function due to the P50 increase demonstrates the importance of O2 diffusion in this cardiac ischemia model.

blood oxygen affinity; oxygen dissociation curve; inositol hexaphosphate; isolated heart; rabbit


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ROLE OF O2 diffusion in O2 delivery remains a controversial and difficult area. It is well known that O2 flux from erythrocytes to cells of an organ depends on diffusion. Because the O2 pressure in cells, including cardiac myocytes, is only a few Torr (15), the O2 diffusion gradient depends heavily on the O2 pressure in the microvasculature at the point of its release from hemoglobin, a variable determined in part by the position and shape of the blood O2 dissociation curve (ODC). That changes in ODC position might enhance or limit O2 flow to cells in certain settings seems intuitively evident, given the existence of the Bohr phenomenon, the relationship between blood O2 affinity and hemoglobin concentration in mutant hemoglobins, the presence of higher O2 affinity in fetuses, the relative left ODC shift of animals native to high altitude, and the rise in 2,3-bisphosphoglycerate (BPG) and P50 (PO2 required to half-saturate hemoglobin) in anemia and low cardiac output states (7, 49). These observations are also consistent with the notion that the O2 pressure head is regulated in a range that does not greatly exceed what is needed for O2 flux. Indeed, in the case of myocardium, the fact that blood flow varies inversely with P50 (47) provides further support for this idea, as does the tight relationship between cardiac work and coronary flow. Nevertheless, experiments that provide unambiguous evidence of modulation of in vivo O2 off-loading by ODC shifts are comparatively sparse, and many experiments have shown only modest or no effect. Apart from its physiological significance, this is a matter of some importance in clinical medicine, given the changes in ODC position that are known to occur with cardiac disease, storage of red blood cells (RBCs), disturbances of acid-base balance and the like (40), as well as the possibility of therapeutic manipulation of the ODC (46).

A specific setting in which the O2 diffusion gradient could be of considerable importance is myocardial O2 delivery (50). Myocardial blood flow is characterized by a major degree of microheterogeneity, with flow rates in millimeter-range tissue volumes varying 6- to 10-fold under basal conditions (3, 5, 11, 12, 24, 41). This heterogeneity rises as tissue volume falls (12) and is greatest as the tissue volume analyzed approaches the domain of a single capillary (27). Local myocardial substrate uptake and O2 consumption (VO2) are also heterogeneous (24) and only somewhat matched to flow. When a major coronary vessel is constricted, downstream local flow also decreases but is initially random with respect to original local flow (9, 24). Anaerobic metabolism appears in foci with the greatest relative reductions in flow (24), a phenomenon believed to account for the patchiness of myocardial infarction after insults that reduce cardiac perfusion (3). Given that basal myocardial O2 extraction is normally high and locally variable (43), this could be simply because limited O2 extraction reserve caps VO2 sooner in local areas with higher extraction. Alternatively, if O2 diffusion between capillary units is of importance (50), one might expect dysoxia in loci that are most dependent on diffusion from adjacent regions. Accordingly, induced shifts of the ODC with other O2 transport variables held constant furnish a useful method to test the importance of local O2 diffusion in myocardial ischemia.

Several recent studies of ODC shifts on oxygenation of the heart and other tissues have been performed with the compound 2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxyl]-2-methylproprionic acid (RSR13). This molecule crosses the RBC membrane and interacts reversibly with hemoglobin, producing appreciable reductions in blood-O2 affinity (1). Results indicate that this drug may improve oxygenation when flow is blood decreased, particularly in models of ischemic heart disease and stroke (21, 29, 44, 45), implying that an increase in the O2 diffusion gradient may increase O2 flux. However, there is at least some evidence that RSR13 has effects on vascular tone other than those expected from the rightward ODC shift (Ref. 32 and Woodson, unpublished observations), although other observations have shown no such effect (29, 44). This could be a confounding variable, especially because vascular tone appears to mediate the microheterogeneity of blood flow (3, 4). In any case, it would be desirable to establish whether comparable effects of ODC shifts can be demonstrated when the ODC is shifted in other ways, particularly given the paucity of positive results in the literature.

These considerations prompted us to examine the role of O2 diffusion in cardiac ischemia, in which we tested the hypothesis that a shift in the ODC due to the presence of intraerythrocytic inositol hexaphosphate (IHP) would improve O2 diffusion and VO2 when the latter is limited by reduced O2 transport. We employed an isolated, isometrically contracting rabbit heart in these studies, a preparation widely used in studies of cardiac physiology and metabolism. The rabbit heart is known to display the same microheterogeneity of blood flow and metabolism observed in larger animals and humans (27, 35). This model allowed us to evaluate myocardial function and VO2 at a normal coronary flow rate and during ischemia when the erythrocyte (RBC) P50 was increased from a subnormal value to a supranormal one. Although other investigators have studied effects of altered RBC O2 affinity in the isolated heart, they employed quite different models and/or did not examine effects of altering P50 during ischemia.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of RBCs

Krebs-Henseleit buffer. Krebs-Henseleit buffer (KHB) was prepared as follows. The basic solution (in mM: 118 NaCl, 4.7 KCl, 2.75 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 0.52 Na2EDTA, 25 NaHCO3, and 11 dextrose and 1,000 U sodium heparin per liter) was equilibrated by bubbling with 95% O2-5% CO2 at room temperature. Bovine serum albumin (1.5%) was then added, and the solution was filtered (0.22 µm).

High-affinity RBCs (control cells). Human packed RBCs stored for 6-14 days in standard CPDA-1 solution (citrate-phosphate-dextrose-adenine) were washed three times in an isotonic saline solution (1,350 g, 5 min); the supernatant and the buffy coat were carefully removed. RBCs were then diluted with KHB. At this stage, the RBC solution was stored in a refrigerator overnight at a hematocrit of 30-40%. The following day, the RBCs were further washed twice in saline containing 10 mM CaCl2, 10 mM MgCl2, and 2 mM glucose. Base excess was corrected to ~0 meq/l (pH of 7.4 at PCO2 of 40 Torr) with addition of NaHCO3. The cells were diluted with KHB to give a hematocrit of 25%. The diluted RBC suspension was passed through a leukocyte removal filter (PALL RC100).

Low-affinity RBCs (IHP-loaded cells). Packed RBC units were stored for 6-14 days at 4°C. The cells were washed once in isotonic saline and then passed through a leukocyte removal filter (PALL RC100). After two more washes in isotonic saline, IHP was incorporated into the cells by the continuous-flow hypotonic dialysis technique, similar to that described by Teisseire et al. (38). The method was modified by reducing the flow rate of the RBCs through the hemodialyzer (Lundia 1C plate dialyzer) to 10 ml/min and by diluting the IHP solution with 0.15 M NaCl (1:1 vol/vol for the first 5 experiments and 1:1.5 for the subsequent experiments) to reduce the degree of P50 shift. After they were resealed, the cells were washed once in isotonic saline, once in hypotonic saline (240 mosmol/kgH2O) to lyse the most fragile cells, and two times in isotonic saline containing 10 mM CaCl2, 10 mM MgCl2, and 2 mM glucose. The RBCs were then diluted with KHB containing albumin (1.5%) and stored in a refrigerator overnight. On the day of perfusion, the cells were washed once in isotonic saline, once in hypotonic saline, and finally three times in saline with CaCl2, MgCl2, and glucose. The cells were then diluted with KHB with 1.5% albumin and NaHCO3 to achieve a hematocrit of 25% and pH 7.4. The IHP incorporation resulted in a P50 of 25-42 Torr (mean shift of 16.0 ± 5.1 Torr, range of 9-26 Torr). Mean recovery of RBCs was 61%. Supernatant hemoglobin concentration during perfusion was consistently below 0.1 g/dl, and the concentrations of ionized calcium, sodium, and potassium were within the normal range.

Isolated Heart Preparation

Experimental procedures were approved by the Animal Care Committee of the University of Wisconsin and were conducted in accord with the Guiding Principles in the Care and Use of Animals of the American Physiological Society and the Guide for the Care and Use of Laboratory Animals [DHSS Publication No. (NIH) 85-23]. Our method paralleled those used in other laboratories (25, 42). Male New Zealand White rabbits weighing between 1.5 and 2 kg were anesthetized with an 8:1 mixture of ketamine-xylazine administered intramuscularly and then were given 1,000 U of sodium heparin intravenously. The heart was quickly removed after an intravenous bolus injection of pentobarbital sodium (25-30 mg/kg). The heart was placed in a heated cabinet, the ascending aorta was immediately cannulated, and retrograde perfusion was started at once with either KHB solution (series A) or human RBCs suspended in KHB solution (series B). The time from sternal incision to cardiac perfusion was well under 1 min. A drain was created in the apex of the left ventricle (LV) by puncture with an 18-gauge needle to allow egress of blood from the Thebesian vessels. A cannulated, fluid-filled balloon connected to a pressure transducer was placed in the LV via a left atriotomy for measurement of LV pressure during isovolumic contraction. A second catheter was placed in the pulmonary artery to collect myocardial venous effluent. Aortic pressure was monitored by a pressure transducer connected to a stopcock inserted into the line just above the aortic cannula.

Perfusion Setup

A schematic diagram of the perfusion setup is shown in Fig. 1. The suspended RBCs were brought to physiological blood-gas concentration and temperature in a primary circuit. From a continuously stirred, covered reservoir, suspended RBCs were pumped at a relatively high rate (about 25 ml/l) through a membrane oxygenator (SciMed Life Systems, Minneapolis, MN) and a transfusion filter (PALL Ultipor) to a second similar overflow reservoir, from which they returned by gravity to the main reservoir. Red blood cells were then propelled by a second pump at the desired flow rate from the overflow reservoir, which also served as a bubble trap, to the heart cannula. Perfusate temperature was recorded by a needle probe in the aortic line just above the heart. Blood passing through the heart was not recirculated, which avoided influence of metabolites. The reservoirs were water-jacketed to maintain a perfusate temperature close to 37°C, and the entire apparatus was enclosed in a thermostated cabinet. The system was designed so as to avoid settling of RBCs, with the possibility of altered perfusate hematocrit, at any point in the circuit.


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  		Fig. 1.   Schematic diagram of perfusion setup. Suspended red blood cells (RBC) are continuously recirculated through oxygenators at a rapid rate in the 2 primary circuits (see text), from which they are pumped at the desired flow rate to the isolated heart. Perfusate temperature is registered by a needle probe (not shown) in the aortic inflow line just above the heart. The lower catheter, syringe, and transducer are for balloon inflation and left ventricular (LV) pressure measurement. The upper catheter and syringe are for anaerobic sampling of myocardial venous return. Except during sampling, venous return flows freely from the severed pulmonary artery and is discarded. A small LV apical stab wound allows drainage from the Thebesian circulation. The entire system is enclosed in a heated cabinet, and all reservoirs are water jacketed at 37°C (not shown). P50, PO2 required to half-saturate hemoglobin.

Series A. In this series, we used normal stored human RBCs ("control cells") to evaluate the reproducibility and sensitivity of the isolated heart model and to study the effects of ischemia on LV physiological parameters. Hearts (n = 9) were paced at a rate of 160-180/min (4-8 V, 10-ms pulse duration). They were initially perfused with KHB by gravity at a constant aortic pressure of ~90 mmHg. The intraventricular balloon volume was set to produce an end-diastolic pressure of 10 mmHg (2). The balloon volume was held constant during the experiment so that developed LV pressure [peak LV systolic pressure minus peak LV diastolic pressure (LVS-LVD)] reflected the contractile state of the myocardium. Hearts were allowed to stabilize for ~15 min under these conditions. Hearts that did not generate an LVS pressure of at least 60 mmHg or whose function declined during the stabilization period were discarded (2). About 20% of hearts were rejected for these reasons.

Perfusion by pump was then started with oxygenated RBCs at a constant flow rate of 9 ml/min. This corresponds to a perfusion rate of 2.1 ± 0.2 ml · min-1 · g ventricular wet weight-1 (mean ± SD), which is similar to the rate used by others in RBC-perfused isolated hearts (2, 20, 25) and close to the means reported for awake rabbits (28) and anesthetized, open-chest rabbits (17, 43). This flow rate produced a mean aortic pressure of 95 ± 22 mmHg. Ischemia was then induced by reducing the flow rate to 3.5 ml/min and then to 2 ml/min for at least 5 min. Hearts were allowed to recover for at least 5 min at a flow rate of 9 ml/min after each level of ischemia. Finally, flow was interrupted completely for 2 min (total ischemia), after which the flow rate was returned to 9 ml/min.

Series B. In this series, hearts (n = 12) were perfused with suspended control RBCs immediately after isolation at a flow rate of 9 ml/min, paced (130-180/min), and allowed to stabilize for ~15 min. Each heart was then perfused with control (high-affinity) and with IHP-loaded (low-affinity) RBCs at flow rates of 9.0 ml/min, 3.5 ml/min, and back to 9.0 ml/min. Arterial and venous samples were obtained in duplicate after at least 5 min of perfusion, and the results were averaged. The order of perfusion with control and IHP-loaded RBCs was randomly varied such that the order for half of the hearts was C9-IHP9-IHP3.5-C3.5-C9-IHP9, whereas the order for the other half was IHP9-C9-C3.5-IHP3.5-IHP9-C9, where C indicates perfusion with control cells, numbers indicate rates of perfusion (in ml/min), and IHP indicates perfusion with IHP-loaded cells. In most experiments, hearts were then exposed to total ischemia (no perfusion) for 2 min once (n = 10) or twice (n = 3), after which the flow rate was returned to 9.0 ml/min. Total experimental time including the stabilization period was 60-90 min.

Measurements

Heart rate and LV and aortic pressures were recorded continuously (Gould 481 strip-chart recorder). Duplicate arterial (oxygenated blood in the reservoir) and venous (pulmonary artery catheter) blood samples were taken after ~5 min at each flow rate for measurement of pH, PO2, PCO2 (Radiometer ABL 30, Copenhagen, Denmark), O2 content and saturation, and hemoglobin concentration (CO-oximeter, model 282, Instrumentation Laboratory, Lexington, MA). O2 content was determined from O2 saturation and hemoglobin concentration with allowance for dissolved O2. O2 extraction was expressed as follows: (arterial O2 content - venous O2 content)/arterial O2 content. VO2 was calculated as the product of perfusion rate (calibrated) and arteriovenous O2 content difference. LV-developed pressure was expressed as LVS-LVD. Cardiac work was expressed as the double product (LVS-LVD) × heart rate. ODCs were determined with either a Hemox Analyzer (TCS Medical Products) or with a Hem-O-Scan (Aminco) at 37°C and expressed at pH 7.4.

Histology

Three hearts from series A were examined histologically after perfusion with control RBCs. Muscle fiber structure was intact with normal striations and no visible edema at 1.5 h, the maximal time of any experiment. Compared with normal hearts, perfused hearts showed minimal, spotty hemorrhage in the LV myocardium, with a tendency of more hemorrhage with increasing perfusion time. These hemorrhages involved <5% of the myocardium. Only occasional punctate hemorrhages could be seen grossly. These changes are not surprising in light of absence of platelets and coagulation proteins in the perfusate and compare favorably with what others have observed grossly (M. Vogel, personal communication, and Ref. 42). By contrast, there was considerably more hemorrhage in the right ventricular wall. Because our study dealt only with LV function, we believe this did not affect our conclusions. The behavior and gross appearance of experimental hearts were similar to those of the histologically examined hearts. We found no other studies in which histopathology in this preparation was described.

Statistics

Duplicate values obtained for each parameter during each perfusion condition were first averaged. Differences in parameters with changes in flow rate at constant P50, and with changes in O2 affinity at constant flow rate, were examined by paired t-test. Differences as a function of P50 in series B following total ischemia were examined by unpaired t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Series A

Table 1 shows that arterial blood gases were close to the physiological range. Temperature averaged 35.2 ± 1.1°C. Figure 2 displays the relationship of LV-developed pressure, positive change in pressure over time (+dP/dt), and LV work as a function of flow rate (n = 9). At a normal flow rate of 9 ml/min (2.1 ± 0.2 ml · min-1 · g ventricular wet wt-1), mean value ± SD for LVS-LVD averaged 58 ± 6 mmHg and LV work was 9,463 ± 1,027 mmHg · beats · min-1. These values are in agreement with those of Apstein et al. (2), whose methodology closely paralleled ours. When total O2 transport was decreased to simulate ischemia by the reduction of flow rate to ~40% of the initial value (3.5 ml/min = 0.8 ± 0.1 ml · min-1 · g-1), to 20% (2 ml/min ± 0.4 ml · min-1 · g-1), and to 0% (total ischemia), there were progressive decreases in LV function. Thus LVS-LVD decreased from the starting value by 39, 53, and 77% with the three flow decrements, respectively (Fig. 2A; P < 0.01 or better for each decrement by paired t-test). Peak +dP/dt decreased by similar amounts (Fig. 2B; P < 0.01) or better, with changes in LV relaxation rate, as judged by negative peak dP/dt, paralleling changes in +dP/dt. Cardiac work decreased by 39, 52, and 77%, respectively (Fig. 2C; P < 0.01 or better). Myocardial O2 extraction increased by 73 and 163% at 40 and 20% of the initial perfusion rate, respectively (Fig. 2D; P < 0.001), whereas VO2 decreased by 37 and 46% (P < 0.001).

                              
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  		Table 1.   Arterial blood gas parameters and temperature



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  		Fig. 2.   Effect of various perfusion rates with control red blood cells and total ischemia on peak LV systolic pressure minus peak LV diastolic pressure (LVS-LVD; A), peak positive change in pressure over time (+dP/dt; B), LV work (C), and O2 extraction (D). A: *P < 0.001 vs. baseline control, §P < 0.05 vs. baseline control, dagger P < 0.01 vs. 3.5 ml/min, Dagger P < 0.001 vs. 2.0 ml/min. B: *P < 0.02 or better vs. baseline control, dagger P < 0.001 vs. 3.5 ml/min, Dagger P < 0.01 vs. 2.0 ml/min. C: *P < 0.001 vs. baseline control, §P < 0.02 vs. baseline control, dagger P < 0.01 vs. 3.5 ml/min, Dagger P < 0.02 vs. 2.0 ml/min. D: *P < 0.001 vs. baseline control, dagger P < 0.001 vs. 3.5 ml/min.

Hearts recovered completely with restoration of perfusion to the basal level after the two levels of partial ischemia; there were no statistically significant changes postischemia in any parameter from basal values. With restoration of perfusion to the basal level after total ischemia, which occurred at the end of the protocol, there was a 15% decrease from starting value in mean LVS-LVD (P < 0.05) and a 24% decrease in work (P < 0.02); arteriovenous O2 extraction and VO2 were unchanged.

Series B

Because this model responded to stepwise decreases in total O2 transport with parallel decrements in function, we evaluated the effect on function of ODC shifts in combination with changes in total O2 transport. Table 1 shows that arterial blood gases and temperature series in the circuits with control and IHP-loaded cells were virtually identical and close to the physiological range. Although an arterial PO2 slightly above the physiological level was employed, saturation of IHP-loaded cells, as expected, averaged 90 ± 2% (mean ± SD). P50 averaged 17 ± 1 and 33 ± 5 Torr, respectively (P < 0.001).

When perfused at 9 ml/min with control cells, LVS-LVD averaged 84 ± 21 mmHg, work (double product) was 13,173 ± 3,047 mmHg · beats · min-1, mean aortic pressure was 82 ± 28 mmHg, coronary vascular resistance was 42 ± 16 mmHg · ml-1 · min · g, O2 extraction was 3.1 ± 0.9 ml O2/dl, and VO2 was 0.062 ± 0.022 ml · min-1 · g-1. These values are shown as 100% in Fig. 3. When perfused with IHP-loaded cells, there were small but significant increases in LVS-LVD (P < 0.05), peak +dP/dt (P < 0.05), work (P < 0.02), O2 extraction (P < 0.002), and VO2 (P < 0.002). There were no significant changes in mean aortic pressure or coronary vascular resistance.


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  		Fig. 3.   Interaction of hemoglobin-O2 affinity and flow rate on LV-developed pressure (A), peak positive change in pressure over time (+dP/dt) (B), LV work (C), arteriovenous (A-V) O2 extraction (D), O2 consumption (VO2; E), and coronary vascular resistance (CVR; F) during control red blood cell perfusion (open bars) and high P50 [inositol hexaphosphate (IHP)-loaded] RBC perfusion (solid bars) at various coronary perfusion rates. Differences between control and high P50 perfusion at the same flow rate are as follows: *P < 0.05 or better, **P < 0.02 or better, ***P < 0.002 or better.

When the flow rate was reduced to 3.5 ml/min, simulating ischemia, mean LVS-LVD, +dP/dt, work, aortic pressure, VO2, and coronary vascular resistance decreased significantly, as in series A, whereas O2 extraction increased; this was true for control and for IHP-loaded cells in relation to their respective controls. Importantly, parameters of function and O2 delivery improved significantly (P < 0.01 or better) during simulated ischemia with IHP-loaded vs. control cells (Fig. 3). The increase in LV work and VO2 was sufficient to restore 17 and 20%, respectively, of the decrements due to this degree of ischemia.

Complete ischemia caused further significant decreases in functional parameters. Function during complete ischemia was independent of the type of RBC perfusion (control vs. IHP loaded) preceding the period of ischemia. Upon reperfusion after ischemia, function and VO2 improved significantly. These parameters were somewhat better when reperfusion was carried out with IHP-loaded cells, but the differences from reperfusion with control cells did not attain statistical significance.

Figure 4 depicts in vivo ODCs obtained by plotting arterial and venous O2 saturation and pressure on perfusate samples obtained under the various conditions described above. These curves redemonstrate the right shift measured in vitro for IHP-containing cells and show that the ODC is less steep. At 9 ml/min, mean venous O2 saturation was appreciably lower and mean venous PO2 appreciably higher with the IHP-loaded cells (Table 2, Fig. 4). This same pattern was observed at 3.5 ml/min. Accordingly, arteriovenous O2 content difference (Fig. 3) was significantly greater during perfusion with IHP-loaded RBC under both conditions and accounted for the significantly greater VO2 observed.


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  		Fig. 4.   In vivo O2 dissociation curves during control red blood cell perfusion () and IHP-loaded RBC perfusion (gray squares). Each point is the mean of duplicate arterial or venous samples obtained from individual hearts (series B) during basal or ischemic perfusion. Mean venous O2 saturation/venous PO2 values (venous point) for control cells at the basal perfusion rate (top ) and at the ischemic perfusion rate (bottom ) are shown. black-triangle, Corresponding values for perfusion with IHP-containing cells.


                              
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  		Table 2.   Venous PO2 and O2 saturation


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We employed a basal coronary flow rate (and resulting coronary perfusion pressure) to duplicate conditions of the in vivo rabbit heart (17, 28, 43). Our results for basal O2 extraction and VO2, as well as for LV-developed pressure and double product in this model, are virtually identical to those determined by Apstein et al. (2), whose conditions most closely paralleled ours, and are in good agreement with those of other workers (20, 25). In addition, our results clearly establish that this ex vivo heart preparation is sensitive to decreased O2 delivery due to ischemia, as there were stepwise decrements in myocardial function, increases in O2 extraction, and decreases in VO2 with each decrement in coronary flow. Because our protocol could produce stunning, which is observed in isolated hearts with somewhat longer periods of ischemia (29), it was important to document recovery of contractile function after successive bouts of ischemia. Indeed, we observed complete recovery after all periods of low-flow ischemia in series A and B, a finding that allowed us to use each heart as its own control for comparing low and high P50 RBCs. We did observe a modest reduction in contractile function after circulatory arrest. However, this was the last step of our protocol, after the data comparing effects of P50 during low-flow ischemia had been collected. Importantly, we achieved excellent matching of hemoglobin concentration, O2 saturation, and blood flow with low and high P50 RBC perfusions, so that ODC-related changes in microvascular PO2 was the only change in paired perfusions. Our protocol also avoided treatment-order bias because the order of perfusion with low and high P50 cells was varied. The protocol by its very design does not, of course, address global changes in blood flow that may result from a P50 change. It does, however, implicitly address any local changes in blood flow distribution that might result from a P50 shift.

Of considerable interest was the clear effect of a P50 increase of 16 Torr on cardiac function, O2 extraction, and VO2 when ischemia was present. This increase in P50 was sufficient to reverse 20% of the decrease in work and 36% of the decrease in VO2 due to ischemia. We believe this constitutes strong evidence that O2 delivery in this model is diffusion limited during ischemia, the increase in microvascular PO2 increasing the O2 diffusion gradient and improving O2 diffusion.

A number of previous investigators have examined the effects of a shift of the ODC on the coronary circulation, cardiac function and O2 uptake. In a small study, Ramo and colleagues (31) showed an effect of P50 on cardiac function. We showed in awake rats that a drop in P50 produced by exchange transfusion with low P50 blood (change in P50 of 21 Torr) caused coronary flow to increase by ~100% (47), a finding suggesting that myocardial perfusion is normally autoregulated in relation to microvascular PO2. Stücker et al. (36) showed a similar P50-coronary flow relationship in isolated, nonworking (Langendorff-type) rat hearts during perfusion with stored RBCs and resealed IHP-containing RBCs. In that study, coronary flow decreased by 36% (from 5.32 to 3.40 ml · min-1 · g wet wt-1) when P50 was raised from ~19 to 47 Torr at constant perfusion pressure, arterial O2 saturation, and hematocrit. Apstein et al. (2) also studied rabbit hearts contracting isometrically against a fluid-filled balloon during perfusion at fixed, constant flow, and arterial O2 content. Their study differed from ours in that they utilized a different method of increasing P50 (induction of BPG synthesis to supraphysiological levels), which would have yielded an ODC of a different shape (7, 38, 39, 49). Importantly, they studied the effect on O2 uptake and function of raising P50 during an isoproterenol infusion at fixed basal flow, whereas we studied hearts when flow was reduced well below basal levels. We are aware of no studies that address microheterogeneity of blood flow and its matching to metabolism under Apstein's conditions. However, it is known that the distribution pattern of local flow is maintained during adrenergic stimulation (13) and becomes less heterogeneous with increased work (26), the two conditions employed by Apstein et al.; by contrast, local flow patterns are completely disrupted during acute ischemia (9, 24). Apstein et al. nevertheless showed that O2 uptake and function improved with increased P50 under their conditions. Because their and our results are similar, it seems likely that increased O2 diffusion improves O2 flux in both settings despite presumed differences in the degree of perfusion heterogeneity. This serves to emphasize further the importance of O2 diffusion in myocardial oxygenation.

Also of note is our finding that significant changes in O2 extraction, VO2, and function resulted from the same P50 shift when the heart was being perfused at the basal rate at a P50 of 17 Torr, which suggests that O2 flux from microvessels to myocytes under these conditions was somewhat diffusion limited at the start. This finding is perhaps not unexpected because 17 Torr is a relatively low value for the rabbit (19) and because coronary blood flow is known to rise in intact animals when P50 falls (33). This finding is also consistent with data, albeit indirect, suggesting that O2 diffusion between adjacent capillary units occurs under normal conditions (50). However, any increased perfusion heterogeneity or unknown abnormalities that are due to the ex vivo preparation (edema, sluggish microvascular flow, and the like) would also be expected to benefit from a larger PO2 gradient.

Recently, the relationship between P50 and function has been examined in other cardiac models with the compound RSR13. These studies differed from the above in that P50 in all instances was raised from a normal level to an appreciably supranormal value. Thus Woods et al. (45) showed no effect of RSR13 (rightward P50 shift of 14 Torr) on function or on phosphocreatine and ATP concentrations in isolated, potassium-arrested rat hearts at basal perfusion. When perfused, however, at very low flow rates, there was significantly improved function and better preservation of the high-energy intracellular metabolites. Pagel and colleagues (29) examined recovery of function after myocardial stunning in dogs caused by repeated bouts of left anterior descending (LAD) occlusion without and with RSR13. They found better preservation of function when RSR13 was given in a dose sufficient to raise P50 from 33 to 46 Torr. Killgore et al. (21) showed improved function and morphology of dog hearts after cold cardioplegia when RSR13 is present. Weiss et al. (44) recently conducted studies in open-chest dogs given RSR13. When given before regional low-flow ischemia (accomplished by perfusing the LAD coronary artery at a constant pressure of 35 mmHg), an RSR13 dose sufficient to raise P50 from 33 to 51 Torr attenuated the regional decrease in high-energy compounds and intracellular pH observed in controls. When given after onset of low-flow ischemia, the RSR13 resulted in significantly improved high-energy PCr-to-ATP ratios and contractility in the LAD bed. Our demonstration of improved function and O2 uptake in the isolated heart with a P50 shift due to IHP is in agreement with these P50 results and furnishes strong evidence that the increase in P50 per se is the likely explanation for the benefit observed with RSR13.

Wagner et al. (reviewed in Refs. 43 and 44) systematically examined numerous perturbations of O2 delivery to working skeletal muscle, in both isolated canine muscle and normal human subjects, to clarify which factor or factors in the O2 transport pathway limit maximal VO2 (VO2 max). In brief, they concluded that O2 uptake is limited in various settings not only by altered total O2 transport to the microvasculature but also by diffusion from the microvascular network to mitochondria. The most compelling data in support of the latter are from studies of isolated canine skeletal muscle during electrical stimulation that produces VO2 max as defined in this model (18, 32). In these studies, a left ODC shift due to carbamylation decreased VO2 max (18), whereas a right shift due to RSR13 increased VO2 max (improvement observed in five of eight animals) (32). Meanwhile Kohzuki et al. (22) showed decreased O2 uptake in perfused dog gracilis muscle with a left ODC shift due to carbamylation during submaximal work. Because total O2 transport to the microvasculature is unchanged in these studies with altered P50, these findings argue strongly that O2 diffusion does limit O2 delivery in certain settings and that O2 uptake can be modulated by ODC shifts. This conclusion is supported by the recent demonstration of adaptations that would improve muscle O2 diffusion in animals without myoglobin (16), a protein shown to facilitate O2 diffusion (37).

The above results with altered O2 affinity in the heart and in working skeletal muscle are of note because they differ completely from results in resting skeletal muscle during ischemia and in whole animals with decreased cardiac output. Thus Ross and Hlastala (33) and Kohzuki et al. (23) found no effect of a left ODC shift on O2 uptake in resting canine skeletal muscle at basal perfusion and no change in the relationship between O2 delivery and VO2, as perfusion was lowered to and below the critical point (defined as the point at which VO2 begins to decrease), despite appreciable changes in venous PO2. Curtis et al. (10) also recently examined the effect of a large right shift due to RSR13 in isolated resting skeletal muscle and found no change in the O2 delivery-VO2 relationship as O2 delivery was reduced, despite higher muscle surface PO2 and higher venous PO2 in the high P50 (RSR13) animals. Likewise, there appears to be no effect of a leftward ODC shift in whole animals on VO2 or on the O2 delivery-VO2 relationship as cardiac output is lowered to and below the critical point (34). Such results have at times been broadly interpreted as indicating that changes in the microvascular-to-cell O2 diffusion gradient do not ordinarily limit O2 diffusion to cells, with the necessary implication that changes in P50 are unlikely to be of much practical importance. However, the present results suggest that the heart, like brain (48) and working skeletal muscle, is critically dependent on O2 diffusion. This is particularly so during cardiac ischemia and is consistent with our earlier observation that coronary flow in intact animals rises with an acute left shift of the ODC (47).

A detailed understanding of how improved diffusion counteracts the absolute reduction in total O2 transport due to ischemia is not available, as measurements of O2 pressure, flux density, and VO2 throughout the microvasculature are not locally accessible. In general, however, O2 diffusion is known to depend on path length and conductance as well as on the PO2 gradient (30). Well-established or possible changes affecting these three parameters during low-flow ischemia include 1) longer residence time for RBCs within the microvasculature, thus allowing more complete O2 extraction, 2) increased time for O2 diffusive shunting from arterioles to venules, 3) increased perfusion heterogeneity (9, 24), and, at least with time, 4) tissue edema and other anatomic changes. A shift of the ODC to the right should have little impact if mechanism 1 is of major importance for O2 delivery during ischemia (given the convergence of ODCs at low O2 saturation) and might actually impair VO2 if mechanism 2 is of major importance (given longer arteriolar residence times for RBCs). The fact that a right shift improves function and VO2 thus suggests that mechanisms 3 and 4 are of importance in this model, inasmuch as both should increase the average path length and/or decrease conductance, which an increase in P50 would tend to counteract.

These observations may have clinical significance, as changes in P50 of this magnitude are observed in clinical medicine. For example, a P50 of ~17 Torr is normally observed after storage of human blood for more than 10 days (8, 40); a P50 of 33 Torr is only ~6 Torr higher than the human mean normal and well within the range observed in low cardiac output states in humans (49). Drugs and techniques that avoid such P50 changes could be of benefit when organ function is threatened by ischemia, as could methods that increase P50 above normal in a number of clinical conditions (1, 6, 14). However, the current results, obtained in isolated hearts subjected to global ischemia, although provocative, cannot be translated directly to clinical settings.


    ACKNOWLEDGEMENTS

This work was supported in part by the US Army Medical Research and Development Command under Grant NoDAMD17-89-Z-9004. G. Berlin acknowledges a grant from the Swedish Medical Research Council (Project B90-19F-8947-01), and R. D. Woodson acknowledges a grant from the University Hospital of Linköping, Sweden and sabbatical support from the University of Wisconsin-Madison, Madison, WI.


    FOOTNOTES

Present address of K. E. Challoner: Clinical Laboratory, University Hospital and Clinics, Madison, WI 53792.

Address for reprint requests and other correspondence: R. D. Woodson, H4/534 Clinical Science Center, Madison, WI 53792 (E-mail: woodson{at}medicine.wisc.edu).

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. Section 1734 solely to indicate this fact.

10.1152/japplphysiol.00194.2001

Received 26 February 2001; accepted in final form 14 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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