Because of their ability to perfuse remote regions and deliver oxygen, hemoglobin-based oxygen carriers (HBOCs) may be considered in the treatment of several ischemic conditions such as acute coronary syndromes or high-risk percutaneous intervention. Here we studied the effects of intracoronary infusion of ex vivo preoxygenated HBOC-201 during brief total coronary artery occlusion (CAOs) on myocardial oxygenation and left ventricular (LV) function in a large animal model and investigated the influence of HBOC-201 temperature and infusion rate on these effects. Thirteen open-chest anesthetized swine were instrumented for measurement of global and regional LV function and metabolism. CAOs were induced by inflating an intracoronary balloon catheter; preoxygenated HBOC-201 (12 g/dL) was infused distally through the central lumen of the balloon catheter. Animals underwent consecutive 3-min CAOs interspersed by 30 min of reperfusion, accompanied by different HBOC-201 infusion rates (0, 15, 23, 30, 40, and 50 ml/min) and/or two infusion temperatures (18°C or 37°C) in random order. CAO elicited immediate loss of systolic shortening (SS) in the ischemic region (19 ± 1% at baseline vs. −3 ± 2% at end of CAO), resulting in decreases in maximum rate of rise in LV pressure (15 ± 5%) and stroke volume (12 ± 4%; all P < 0.05). Balloon deflation resulted in marked coronary reactive hyperemia (to 472 ± 74% of baseline), increases in coronary venous concentrations of adenosine + inosine (to 218 ± 26% of baseline; both P < 0.05) and rapid restoration of SS toward baseline. HBOC-201 ameliorated the CAO-induced changes in SS, stroke volume, reactive hyperemia, and coronary venous adenosine + inosine. The effects were temperature and flow dependent with full preservation of SS at 50 ml/min HBOC-201 of 37°C. In conclusion, intracoronary preoxygenated HBOC-201 preserved myocardial oxygenation and LV function in swine during CAO in a dose- and temperature-dependent manner. In our study setting, preoxygenated HBOC-201 can match the oxygen delivery role of endogenous blood in the heart on an almost equivalent-volume basis.
- coronary blood flow
- intracoronary infusion
- left ventricular function
- myocardial metabolism
hemoglobin-based oxygen carrier (HBOC)-201 is a cell- and endotoxin-free, glutaraldehyde-polymerized hemoglobin solution produced by chemical modification of hemoglobin extracted from isolated bovine red blood cells (RBCs). In an era during which the blood supplies of developed countries were characterized by HIV and other blood-borne infectious agents, HBOC-201 was initially developed as an alternative to RBCs to treat anemia in surgical patients. HBOC-201 has been approved for the treatment of acute surgical anemia in South Africa since 2001. Improvement in some aspects of stored blood safety (10, 11) over the past 20 years, a room-temperature shelf life of three years and elimination of the need to cross-match blood type reoriented interests in HBOC-201 toward applications in military casualties, prehospital civilian hemorrhagic trauma, and other indications in which blood is not an option or immediately available. Of late, needs beyond trauma and hemorrhagic shock have evolved to include ischemic rescue, applicable to cardiology and vascular surgery.
HBOC-201 has the ability to restore tissue oxygenation in persistently ischemic tissue. By facilitating oxygen diffusion and convective oxygen delivery, HBOC-201 may act as a direct oxygen donor and increase oxygen transfer between RBCs and between RBCs and tissues (22, 23). These mechanisms could improve tissue oxygenation (26), especially in poststenotic areas that plasma, but not RBCs, may be capable of reaching. Because of their ability to perfuse remote regions and deliver oxygen, HBOCs may be considered in the treatment of several ischemic conditions such as acute coronary syndromes (ACS) or high-risk percutaneous intervention (PCI). Studies conducted in animal models demonstrated that both the prophylactic (before induction of ischemia) and late (after ischemia onset) HBOC infusion are well tolerated and effective (2, 4, 12, 14). Elective PCI induces transient myocardial ischemia due to reduction of coronary flow during balloon inflation in the coronary artery (19, 20), thus simulating in a controlled setting the occurrence of an ACS. Preliminary evidence of HBOC-201 efficacy in preserving global left ventricular (LV) function, as a surrogate for myocardial oxygenation, using the angioplasty balloon inflation model of brief ischemia in humans, has recently been established in a small patient set (20).
The aim of the present study was to investigate in more detail the effects of intracoronary infusions of preoxygenated HBOC-201 on regional myocardial oxygen delivery and utilization, global and regional LV function, coronary hemodynamics, and ventricular arrhythmias in a swine model of brief total coronary artery occlusions.
MATERIAL AND METHODS
Experiments were performed in a total of 16 Yorkshire × Landrace pigs (55–60 kg) of either sex. Experimental procedures were performed in accordance with the Guiding Principles in the Care and Use of Animals as approved by the Council of the American Physiological Society and under the regulations of the Animal Care Committee of the Erasmus Medical Center Rotterdam.
Experimental Design and Protocol
The first aim of the study was to investigate the effects of moderate doses of preoxygenated HBOC-201 (in doses of 5.4 g up to 10.8 g; Biopure) on global LV function and on regional myocardial function and metabolism during brief coronary artery occlusions (CAOs) and to determine the influence of HBOC-201 infusion temperature. For this purpose, six pigs were subjected to seven consecutive episodes of 3-min CAO, separated by 30 min of reperfusion. During each CAO, animals received either no treatment (balloon occlusion only, i.e., without any infusion) or preoxygenated HBOC-201 (12.0 ± 0.1 g%; O2 saturation 90.5 ± 0.2%) infused directly into the coronary artery via the central lumen of the balloon catheter at a rate of 15, 23, or 30 ml/min, either at room temperature (18°C) or at 37°C. These seven treatments were applied in random order.
The second aim of the study was to determine whether higher doses (10.8 up to 18 g) of intracoronary preoxygenated HBOC-201 could fully restore regional myocardial function and metabolism during brief, transient CAOs. For this purpose, seven pigs were subjected to four consecutive episodes of 3-min CAOs, separated by 30 min of reperfusion, and simultaneously received in random order either no treatment or preoxygenated HBOC-201 (12.8 ± 0.1 g%; O2 saturation 90.0 ± 0.1%) at a rate of 30, 40, or 50 ml/min at 37°C. Finally, to determine whether high HBOC-201 intracoronary infusion rates were associated with elevated coronary artery pressures, a small 22-gauge angiocatheter was inserted into the distal left anterior descending coronary artery (LAD) of six of these pigs. Animals were then subjected in random order to three 2-min CAOs separated by 30 min of reperfusion and simultaneously received preoxygenated HBOC-201 (12.8 ± 0.1 g%; O2 saturation 90.0 ± 0.1%) at a rate of 50, 60, or 70 ml/min at 37°C. The maximum volume of 150 ml of the syringe necessitated a limitation of CAO duration to 2-min for this part of the protocol.
In three additional pigs, we investigated the contribution of enhanced metabolite washout to the cardioprotective effects of intracoronary preoxygenated HBOC-201. For this purpose, pigs were subjected to five consecutive episodes of 3-min CAOs, separated by 30 min of reperfusion, and simultaneously received no treatment, nonoxygenated HBOC-201 (13.3 ± 0.2 g%; O2 saturation 3.2 ± 1.1%) at a rate of 30, 40, and 50 ml/min at 37°C, or Ringer's solution at a rate of 50 ml/min at 37°C.
Pigs were sedated with ketamine (20 mg/kg im) and midazolam (1 mg/kg im), anesthetized with pentobarbital sodium (15 mg/kg iv), and intubated for ventilation with O2 and N2 (1:3 vol/vol) (7, 15). Catheters were inserted into the superior caval vein for infusion of pentobarbital sodium (10–15 mg·kg −1·h −1) to maintain anesthesia and infusion of saline and drugs. A fluid-filled catheter was inserted in the left femoral artery for measuring aortic blood pressure and for arterial blood sampling. A micromanometer-tipped catheter (Millar Instruments, Houston, TX) was inserted into the right carotid artery and advanced into the left ventricle for measurement of LV pressure (LVP) and its first derivative (LV dP/dt).
The chest was opened via the sternum, an electromagnetic flow probe was placed on the ascending aorta for the measurement of cardiac output, and a transit-time flow probe (Transonic Systems, Ithaca, NY) was placed around the LAD coronary artery for measuring coronary blood flow. Regional LV wall function was measured using two pairs of ultrasonic crystals placed in the midmyocardium ∼10 mm apart in the LAD and left circumflex coronary artery (LCx) regions (6, 7). The anterior interventicular vein was cannulated with a 20-gauge angiocatheter for coronary venous blood sampling. Finally, a 4Fr compliant, short (4 mm), low-pressure balloon catheter (Helios; Lightlab Imaging) was positioned in the proximal LAD. For CAO, the balloon was inflated up to 0.5 atm in order to achieve complete artery occlusion as documented by the flow probe. A compliant, low-pressure balloon catheter was selected to allow for complete coronary occlusion without the potential for mechanical vessel wall damage as typically involved in standard PCI balloons. During the 3-min duration of the balloon occlusion, preoxygenated HBOC-201 was simultaneously administered distally into the target coronary artery through the central lumen of the balloon catheter at a constant infusion rate by a clinical syringe pump (Mark V ProVis; Medrad, Warrendale, PA).
After completion of instrumentation, a stabilization period of ∼30 min was allowed before the animals were subjected to the experimental protocol. Rectal temperature was continuously monitored with an electronic thermometer and was maintained at 37.0°-38.0°C (9). To prevent blood clotting, heparin was administered (200 U·kg −1·h −1 iv) throughout the experimental protocol and aspirin (250 mg iv) was infused just before the induction of the first CAO. Systemic and coronary hemodynamics as well as regional myocardial function were continuously recorded throughout, whereas arterial and coronary venous blood samples were collected at selected time points during the experimental protocol. Pigs that experienced ventricular fibrillation during occlusion or reperfusion were allowed to complete the protocol when conversion to normal sinus rhythm could be reinstated by direct current defibrillation (30–50 J) within 1 min after fibrillation.
Regional Myocardial Function
All segment length data were normalized to an end-diastolic length (EDL) of 10 mm at baseline to correct for variability in the implantation distance between the crystals (6, 7). Systolic shortening was computed as 100%·EDL − end-systolic length (ESL)/EDL, in which EDL and ESL are the segment length at the onset of the rapid increase in LVP (LV dP/dt = 250 mmHg s −1) and at the end of LV ejection, respectively. The area inside the LV pressure-segment length loop was taken as an index of stroke work (7). Because stroke work reflects mechanical work but does not have the dimensions of work, the changes in mechanical efficiency have been expressed as percentage of baseline.
Myocardial Oxygen Balance
Blood samples were maintained in iced syringes until the end of each occlusion. Measurements of Po2 (in mmHg), Pco2 (in mmHg), and pH were then immediately performed with a blood gas analyzer (Acid-Base Laboratory Model 510; Radiometer, Copenhagen, Denmark). Oxygen saturation (O2-sat), hemoglobin concentration (Hb; expressed in grams per 100 ml), and met-Hb concentration were measured with a hemoximeter (OSM3; Radiometer). Blood oxygen content (in micromoles per milliliters) was computed as (Hb·0.621·O2-sat) + (0.00131·Po2). Myocardial oxygen delivery was computed as the product of LAD coronary blood flow and arterial blood oxygen content; myocardial oxygen consumption in the region of myocardium perfused by the LAD coronary artery was calculated as the product of coronary blood flow and the difference in oxygen content between arterial and coronary venous blood. Myocardial oxygen extraction was computed as the ratio of the arteriovenous oxygen content difference and the arterial oxygen content.
Myocardial Purine Production
The adenosine and inosine concentrations in blood samples were determined by reversed-phase high-performance liquid chromatography using a C18 column (Hypersil ODS 3 μm, 150 × 4.6 mm; Alltech, Deerfield, IL) combined with a C18 guard column (Hypersil ODS 5 μm, 7.5 × 4.6 mm). We used an AS 3000 cooled auto sampler, a SCM 1000 vacuum membrane degasser, a P2000 gradient pump, a 50-μl sample loop, and PC 1000 software from Thermo Separation Products (Riviera Beach, FL) in combination with a Spectra Focus forward optical scanning detector (Spectra-Physics, San Jose, CA). Peaks were detected at 254 nm. Purines were identified and concentrations were determined based on external standards in plasma and retention times (17, 18, 25).
HBOC-201 was oxygenated under aseptic conditions 1 to 2 days before each experiment using a proprietary, closed circuit, oxygenation device (Fig. 1) designed and validated for that purpose (Biopure Corporation). Oxygenated product was stored at 4°C until the day of the experiment. Oxygen saturation of hemoglobin attained using this method was 90.3 ± 0.1%; oxygen tension of the solution was 487 ± 19 mmHg.
Data Analysis and Presentation
All hemodynamic and LV global and regional function data were recorded and digitized online using an eight-channel data acquisition program ATCODAS (Dataq Instruments, Akron OH) and stored on a computer for offline analysis with a program written in MatLab (The Mathworks, Natick, MA). A minimum of 15 consecutive beats were selected for analysis of the digitized hemodynamic signals.
All data have been expressed as means ± SE. Statistical significance (P < 0.05) for changes in hemodynamics and regional myocardial function was determined by three-way (Study I, time point in protocol × flow rate × temperature) and two-way (Study II, time point in protocol × flow rate) ANOVA for repeated measures, followed by post hoc testing with Student-Newman-Keuls multiple comparison test.
Systemic Hemodynamics and Global LV Function
The treatment conditions were performed in a random order per study, and there were no differences between the baseline values of systemic hemodynamics and LV global and regional function before each 3-min CAO in either Study I (Table 1) or Study II (Table 2).
Effects of ischemia-reperfusion.
The 3-min CAO alone had negligible effects on heart rate, but resulted in a 15 ± 1% (Study I) and 18 ± 2% (Study II) decrease in mean aortic blood pressure, due to a 12 ± 4% (Study I) and 18 ± 3% (Study II) decrease in stroke volume and hence cardiac output (Figs. 2 and 3). The decrease in stroke volume was due to a decrease in LV function, as reflected by decreases in maximum rate of rise of LVP and maximum rate of fall of LVP (LV dP/dtmax and LV dP/dtmin, respectively) and a modest elevation of LV end-diastolic pressure. All variables recovered toward baseline values during subsequent reperfusion. The total number of premature ventricular contractions (PVCs) during the 3-min CAO amounted to 3.8 ± 2.3 in Study I and 3.9 ± 1.3 in Study II, with even fewer PVCs during the subsequent first 3-min of reperfusion (0.3 ± 0.2 for Study I and 1.4 ± 0.6 for Study II). In one animal ventricular tachycardia (VT) occurred during the 3-min CAO, whereas another animal encountered ventricular fibrillation (VF) during reperfusion (Table 3).
Effects of preoxygenated HBOC-201.
Intracoronary infusion of preoxygenated HBOC-201, starting immediately after the onset of CAO, prevented the CAO-induced decrease in mean aortic blood pressure, stroke volume, cardiac output, LV systolic pressure, and LV dP/dtmax and LV dP/dtmin but did not affect the increase in LV end-diastolic pressure (Figs. 2 and 3). The effects of HBOC-201 on several of these variables were flow dependent and/or temperature dependent. HBOC-201 had no effect on the occurrence of PVCs, although there was a trend toward fewer PVCs during the 3-min CAO. Furthermore, in none of the HBOC-201-treated animals was an episode of VT or VF observed (Table 3).
Regional Myocardial Function and Metabolism
There were no differences between the baseline values of regional myocardial function, coronary hemodynamics, and myocardial metabolism before each 3-min CAO in either Study I (Table 1) or Study II (Table 2).
Effects of ischemia-reperfusion.
CAO abolished coronary blood flow, resulting in rapid decreases in stroke work and systolic shortening in the ischemic LAD region, with no effect on shortening in the remote LCx region (Figs. 4, 5, and 6). Deflation of the balloon resulted in marked reactive hyperaemia and washout of adenosine + inosine, reflecting net myocardial ATP breakdown during the coronary occlusion. Regional myocardial oxygen consumption and contractile function in the LAD area were quickly restored to baseline levels. Reactive hyperemia resulted in an increase in myocardial oxygen delivery that was in marked excess of myocardial oxygen consumption, thereby allowing an increase in coronary venous oxygen tension (Figs. 5 and 6).
Effects of preoxygenated HBOC-201.
Intracoronary administration of preoxygenated HBOC-201, starting immediately after the onset of CAO, prevented the CAO-induced alterations in regional wall motion in a flow-dependent and/or temperature-dependent manner (Figs. 4, 5E, and 6E). LAD regional systolic shortening reached a plateau at a preoxygenated HBOC-201 infusion rate of 50 ml/min when infused at 37°C (Table 4). Interestingly, at room temperature, HBOC-201 resulted in perturbed regional diastolic dysfunction reflected in the counterclockwise rotation of the diastolic phase of the pressure-segment length relation, particularly at the highest flow rate (30 ml/min) at which room-temperature HBOC-201 was evaluated (Fig. 4, Study I, top). Consistent with the pressure-segment length loops, systolic shortening failed to improve further at 30 ml/min relative to systolic shortening at 23 ml/min (Fig. 5E). In contrast, intracoronary infusion of preoxygenated HBOC-201 at 37°C restored regional wall function to baseline values in a flow-dependent manner (Figs. 4 and 6). At 30 ml/min preoxygenated HBOC-201 restored systolic shortening to ∼80% of baseline, whereas higher infusion rates (40 and 50 ml/min) fully preserved regional systolic work (Fig. 4, Study II) and systolic shortening (Fig. 6E). The coronary flow probe confirmed incremental blood flow associated with increasing intracoronary HBOC-201 infusion rates (Fig. 6), resulting in full restoration of baseline coronary flow levels at 50 ml/min HBOC-201 and myocardial oxygen delivery at 30 ml/min HBOC-201. These effects on oxygen delivery were accompanied by a marked attenuation of reactive hyperemia and purine (adenosine + inosine) washout during early reperfusion (Figs. 5 and 6), confirming improved oxygenation during CAO and alleviation of anaerobic metabolism.
The infusion rate of preoxygenated HBOC-201 at 50 ml/min was associated with a normal coronary artery pressure (Table 4). In contrast, infusion rates of 60 and 70 ml/min resulted in elevations of coronary pressures well beyond baseline levels.
Effects of nonoxygenated HBOC-201.
Regional myocardial systolic shortening was reduced to −49 ± 20% of baseline during the 3-min CAO with no treatment, which was not affected (P = 0.83 by 1-way ANOVA) by intracoronary infusions of either nonoxygenated HBOC-201 in a dose of 30 (−45 ± 16% of baseline), 40 (−31 ± 24% of baseline), or 50 (−28 ± 17% of baseline) ml/min or 50 ml/min Ringer's solution (−60 ± 27% of baseline). Similarly, regional stroke work at 3-min CAO with no treatment amounted 7 ± 3% of baseline, which was not affected by intracoronary infusions of either nonoxygenated HBOC-201 in a dose of 30 (16 ± 4% of baseline), 40 (19 ± 8%), or 50 (13 ± 3%) ml/min or 50 ml/min Ringer's solution (11 ± 3% of baseline). These values are in stark contrast with the values of systolic shortening (115 ± 13% of baseline) and regional stroke work (145 ± 9%) observed at 3-min CAO in the presence of 50 ml/min of preoxygenated HBOC-201 (Study II; Figs. 4 and 6). In line with these observations, reactive hyperemia was also not influenced by any of the treatments. Thus coronary blood flow at 1 min of reperfusion amounted 631 ± 154%, 725 ± 234%, and 595 ± 80% of baseline following 30, 40, and 50 ml/min nonoxygenated HBOC-201, respectively, and amounted 486 ± 137% of baseline following 50 ml/min Ringer's solution. These values were not different (P = 0.86 by 1-way ANOVA) from the no-treatment group (609 ± 102%), but were well above the values observed following 50 ml/min of preoxygenated HBOC-201 (190 ± 25%) in Study II (Fig. 6). These findings indicate that the beneficial effects of preoxygenated HBOC-201 were the consequence of an improved oxygen delivery rather than an increased washout of metabolites.
The present study demonstrates that intracoronary infusion of preoxygenated HBOC-201 during total coronary artery occlusion can preserve regional oxygen delivery and utilization, thereby negating anaerobic metabolism and preserving regional myocardial contractile function.
Several HBOCs have been studied in clinical trials for various indications. HBOCs were derived from human or bovine blood and have been chemically modified to minimize extravasation from the circulatory space and modify oxygen binding properties, resulting in a range of products that differ in size, molecular mass, oxygen affinity, viscosity, and oncotic activity. Every formulation should be considered a unique drug with its own physical characteristics, pattern of biological activity, and side effect profile. HBOC-201 is a glutaraldehyde cross-linked bovine polyhemoglobulin in solution with an average molecular mass of 250 kDa and a viscosity less than that of plasma (2.4 cp at 37°C). HBOC-201 has an oxygen dissociation curve that is right-shifted with a P50 of 40 mmHg, compared with 27 mmHg for human hemoglobin and a P50 of ∼40 mmHg for swine. These features provide excellent oxygen transport properties. However, vasoactivity putatively due to the binding of nitric oxide by hemoglobin is considered by some to be a significant liability of HBOCs in general and remains a topic of ongoing research and debate (20, 21).
Safety and efficacy of HBOC-201.
In the present study, a volume ranging from 45 ml up to 150 ml of preoxygenated HBOC-201, equivalent to a maximum of 18 g hemoglobin glutamer-250 bovine, was infused over 3 min. The dose infused changed with the infusion rates, keeping the time of infusion fixed. The maximum total dose per animal was 49 g. No specific clinical signs of toxicity were detected. No animals were lost in the experiment, likely attributable to the brevity of the coronary occlusions.
In Study I, we observed that intracoronary preoxygenated HBOC-201 infusion produced a dose-dependent protection of systolic shortening in the ischemic region served by the LAD coronary artery with 80% preservation of baseline function at an infusion rate of 30 ml/min, the highest infusion rate evaluated in Study I. HBOC-201 infusion rates above 30 ml/min in Study II extended the dose-dependent protection of systolic shortening in the LAD region with full protection (referenced to preocclusion baseline) observed at 50 ml/min. Coronary blood flow also increased progressively with higher preoxygenated HBOC-201 infusion rates, reaching 100% of baseline flow at an infusion rate of 50 ml/min. Convective oxygen delivery at this infusion rate, however, was approximately 65% higher than baseline oxygen delivery. Therefore, preoxygenated HBOC-201 can match the role of blood on an equivalent volume basis as reflected by myocardial systolic shortening, but with a higher total oxygen delivery. That a preoxygenated HBOC-201 infusion rate yielding a coronary blood flow equivalent to that at baseline delivers oxygen at a rate higher than baseline is attributable to the higher Hb concentration in the HBOC-201 solution compared with baseline Hb concentration in swine blood. Our study does not allow us to definitively distinguish which element (coronary flow or myocardial oxygen delivery) is rate limiting in fully preserving regional systolic shortening. It is noteworthy, however, that both coronary flow and myocardial oxygen consumption increased commensurate with increasing preoxygenated HBOC-201 infusion rate and reached baseline levels at an infusion rate of 50 ml/min, suggesting that replacement of coronary blood flow with an equivalent coronary flow of HBOC-201 played a more important role than matching oxygen delivery, which exceeded the baseline level under these conditions. Consistent with full preservation of LAD regional systolic shortening at a preoxygenated HBOC-201 infusion rate of 50 ml/min, purine concentrations, a marker of anaerobic metabolism, were at or below baseline levels under these conditions. In fact purine concentrations in coronary sinus blood were restored to baseline values during preoxygenated HBOC-201 infusion rates as low as 30 ml/min, a condition during which systolic shortening was preserved at only 80% of baseline. These findings suggest that full preservation of LAD regional systolic shortening requires oxygen delivery beyond that which merely restores normal aerobic metabolism. Heart rate and LV end-diastolic pressure, cardiac parameters that could potentially influence systolic shortening, were similar over the range of preoxygenated HBOC-201 infusion rates (15–50 ml/min) driving dose-dependent change in systolic shortening and are, therefore, unlikely to have operated as determinants of systolic shortening in these studies. An explanation for the lack of effect of HBOC-201 infusion on LV end-diastolic pressure, despite a normalization of regional systolic function, is not readily found, but it could be speculated that normalization of LV end-diastolic pressure was, at least in part, opposed by an established volume-loading effect of HBOC-201 (8).
Although LAD regional systolic shortening was fully preserved during coronary occlusion only at a preoxygenated HBOC-201 infusion rate of 50 ml/min, LV regional stroke work during intracoronary infusion of preoxygenated HBOC-201 at 23 ml/min was essentially equivalent to baseline work and became elevated above baseline at higher infusion rates in a dose-dependent manner, reaching a plateau at 50 ml/min. The basis for increased regional stroke work appears to be primarily an elevation in systolic blood pressure over baseline (which likely opposed full restoration of systolic shortening at 30 and 40 ml/min), probably secondary to recirculation of HBOC-201 to the systemic resistance vessels (20). Infusion rate-dependent segment lengthening in the LAD region during diastolic filling was associated with an increase in pressure-segment loop width, contributing to the observed increase in regional stroke work during preoxygenated HBOC-201 infusion at 37°C. The mechanism for this increase in regional segment length during diastolic filling by HBOC-201 is unknown, but is unlikely due to the influence of LV filling pressures, which, although elevated relative to baseline, remained unchanged across all infusion conditions. Although regional stroke work as well as myocardial oxygen delivery were up by as much as 45% above baseline at a preoxygenated HBOC-201 infusion rate of 50 ml/min (Fig. 6), oxygen consumption did not exceed baseline values, suggesting an improvement of either mechanical efficiency (stroke work/myocardial oxygen consumption) and/or metabolic efficiency (increased P:O ratio).
The exact mechanisms underlying possible improvements in mechanical and metabolic efficiency by preoxygenated HBOC-201 cannot be derived from the present study. However, the observation that intracoronary infusions of Ringer's solution or nonoxygenated HBOC-201 did not result in detectable improvements of regional systolic shortening strongly suggests that oxygen delivery rather than enhanced washout of metabolites was the principal mechanism by which preoxygenated HBOC-201 improved systolic function. HBOC-201 has been shown to improve diffusive oxygen delivery to tissues independently of convective oxygen delivery (DO2) (20, 21). This capability is a function of the relatively high P50 for HBOC-201 (8) and a reduction in oxygen diffusion distances between HBOC-201 molecules and RBCs/tissues (13). These biochemical and physical properties of HBOC-201 also translate to improved tissue oxygenation in vivo (26). Future studies will be required to further understand how preoxygenated HBOC-201 may have modulated myocardial mechanical and metabolic efficiency.
Effects of temperature.
Interestingly, systolic shortening was unexpectedly reduced when preoxygenated HBOC-201 infusion at room temperature was increased from 23 to 30 ml/min compared with a progressive, flow-dependent increase in systolic shortening over the same infusion rates of preoxygenated HBOC-201 at 37°C. In fact, systolic shortening was depressed at all preoxygenated HBOC-201 infusion rates performed at room temperature compared with infusions at 37°C. Consistent with these results, dP/dtmax tended to be lower when room-temperature preoxygenated HBOC-201 was infused at 30 ml/min compared with 23 ml/min; the opposite relationship existed for the effect of these two infusion rates on dP/dtmax when infusion was performed at 37°C. In addition, dP/dtmin tended to be greater during infusions at 37°C compared with infusions at room temperature. These observations may signal greater regional cardiac stiffness during diastole when infusing preoxygenated HBOC-201 at the lower temperature, potentially contributing to less myocardial stretch during LV filling. These temperature-dependent effects on systolic and diastolic function likely contributed to a narrower LV pressure-segment length loop and were associated with lower regional stroke work index during preoxygenated HBOC-201 infusions conducted at room temperature. Consistent with regional systolic shortening and LV dP/dtmax, LAD regional work index was impaired when intracoronary HBOC-201 infusion at room temperature was increased from 23 to 30 ml/min. In contrast, LAD regional work index was fully preserved during preoxygenated HBOC-201 infusion at 30 ml/min and 37°C and essentially fully preserved at 23 ml/min and 37°C.
Our study did not assess the mechanism by which infusate temperature influenced myocardial function. Cytosolic calcium concentrations, cytosolic calcium transients, and tension development may be altered at subnormal tissue temperatures (1, 5), and these effects, if operable in our study, could potentially explain the reduced systolic shortening and global pump function during infusion of preoxygenated HBOC-201 at the lower temperature.
Plateau effect of reoxygenation by HBOC-201.
In Study II, we assessed the effects of higher preoxygenated HBOC-201 infusion rates at 37°C. LAD regional systolic shortening increased dose dependently in response to increasing infusion rates, reaching a maximum at 50 ml/min. It is of interest to note that increasing the HBOC-201 infusion rate beyond 50 ml/min resulted in a dose-dependent decrease in systolic shortening. Preoxygenated HBOC-201 infusion at 30 and 50 ml/min delivered oxygen to the myocardium at a rate approximately equal to 100% and 170%, respectively, of oxygen delivery during baseline coronary blood flow. Thus exceeding normal oxygen delivery through higher HBOC infusion rates does not induce an inotropic effect as defined by regional systolic shortening. Preoxygenated HBOC-201 infusion rates exceeding 50 ml/min were associated with intra-coronary pressures well above baseline. Abnormally high intracoronary pressure will act to oppose the normal emptying of coronary conduit vasculature during systole, thereby creating higher interstitial pressures that may inhibit cardiomyocyte thickening, an obligatory consequence of cellular shortening during systole (27). Consequently, systolic shortening may be partially inhibited by high intracoronary pressures.
The results of the present study in swine are in line with a recent pilot study employing intracoronary preoxygenated HBOC-201 in five patients undergoing elective PCI in our center (20) and with earlier work demonstrating myocardial protection with oxygenated autologous blood perfusion at rates of 60 ml/min in a comparable model (16). Our results may be of importance to clinical practice since preoxygenated HBOC-201 can match the oxygen delivery role of endogenous blood on an equivalent-volume basis. This finding is a first and important step in exploring the efficacy of a pharmacoinvasive strategy using HBOC-201 in acute coronary syndromes and high-risk PCI. There have been claims by some that HBOC-201 and other HBOCs may have long-term detrimental effects, including an increase in the risk of myocardial infarction in patients undergoing major surgery or treatment of hemorrhagic shock (3, 21, 24). In contrast, the present study, using repeated short-term intracoronary infusions that resulted in cumulative systemic concentrations below 1 g%, did not reveal any adverse coronary or myocardial effects. Rather, full preservation by intracoronary preoxygenated HBOC-201 of myocardial aerobic metabolism and function in the absence of blood provides a profile of cardiac benefit and the absence of any intrinsic cardiac toxicity. These findings also allow us to speculate on the potential of preoxygenated HBOC-201 as an organ preservation fluid in the organ transplant setting. In conclusion, the present study demonstrates that intracoronary infusion of preoxygenated HBOC-201 during total CAO preserves regional myocardial contractile function and aerobic metabolism, by preserving myocardial oxygen delivery.
G. P. Dubé was employed by Biopure Corp. at the time these studies were conducted.
We acknowledge expert technical assistance of Inge M. Lankhuizen.
- Copyright © 2010 the American Physiological Society