Introduction

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RSR13 {2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxyl]2-methylproprionic acid} (fig. 1) is a member of a new class of drugs that mimic the natural physiological effects of 2,3-diphosphoglycerate by allosterically modifying O₂-hemoglobin affinity. A derivative of the antilipidemic agent bezafibrate, RSR13 has been shown to bind selectively to and stabilize deoxyhemoglobin in vitro (Abraham et al., 1992a, b; Randad et al., 1991; Wireko et al., 1991). These actions cause a rightward shift of the O₂-hemoglobin dissociation curve and directly increase tissue O₂ delivery in vivo independent of changes in regional blood flow, metabolism or O₂ content (Khandelwal et al., 1993; Kunert et al., 1996a; Liard and Kunert, 1993). Recent data have indicated that RSR13 preserves intracellular pH and high-energy phosphate concentrations during low-flow myocardial ischemia, presumably by improving O₂ delivery and reducing the adverse metabolic consequences of critically diminished coronary blood flow (Mejia et al., 1996). These preliminary findings suggest that allosteric modification of hemoglobin affinity for O₂ with new compounds designed to stabilize the deoxy form of the molecule may represent a unique therapeutic approach to the treatment of tissue hypoxemia including ischemic heart disease. The effect of specific allosteric modifiers of hemoglobin on the functional recovery of postischemic, reperfused myocardium has yet to be evaluated. Although improved O₂ release to myocardium during ischemia and reperfusion may directly enhance contractile function by favorably affecting metabolism, the presence of additional O₂ theoretically also may contribute to increased production of O₂-derived free radicals, molecular species that have been strongly implicated in the pathogenesis of myocardial stunning (Bolli, 1990). The present investigation tested the hypothesis that RSR13 enhances the recovery of contractile function of stunned myocardium generated with repetitive, brief episodes of ischemia and reperfusion in barbiturate-anesthetized, acutely instrumented dogs. This experimental model has been demonstrated to sensitively elicit profound contractile dysfunction after ischemia and reperfusion in vivo (Nicklas et al., 1985; Yao et al., 1993).

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Chemical structure of RSR13.

	Materials and Methods

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All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin. All procedures conformed to the "Guiding Principles in the Care and Use of Animals" of the American Physiological Society and were performed in accordance with the "Guide for the Care and Use of Laboratory Animals" [DHEW(DHHS) publication (NIH) no. 85-23, revised 1996].

Surgical instrumentation. Conditioned mongrel dogs (n = 28) of either sex weighing between 24 and 33 kg fasted overnight. Anesthesia was induced with an intravenous bolus of sodium barbital (200 mg·kg¹) and sodium pentobarbital (15 mg·kg¹). Fluid deficits were replaced before experimentation with 0.9% saline (500 ml). Intravenous fluids were continued at 3 ml·kg¹·hr¹ for the duration of the experiment. After tracheal intubation, the lungs of each dog were ventilated using positive pressure with oxygen (100% at 1 l·min¹). Respiratory rate and tidal volume were adjusted to maintain acid-base status and carbon dioxide partial pressure within physiologic limits. The right femoral vein was isolated through a small incision, and a catheter was placed in this vessel for fluid and drug administration. A 7F, dual micromanometer-tipped catheter (Millar Instruments, Houston, TX) was inserted through the left carotid artery and positioned across the aortic valve with the distal transducer in the LV and the proximal transducer in the ascending aorta for measurement of continuous LV and arterial pressures, respectively. The +dP/dt_max was determined by electronic differentiation of the LV pressure waveform.

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Regional myocardial blood flow. Carbonized plastic microspheres (15 ± 2 µm [SD] in diameter; New England Nuclear, Boston, MA) labeled with ¹⁴¹Ce, ¹⁰³Ru, ⁵¹Cr or ⁹⁵Nb were used to measure regional myocardial perfusion (Domenech et al., 1969). The microsphere suspension was ultrasonicated (model B-3, Branson Company, Shelton, CN) for 15 min and agitated in a vortex mixer (model K-500-2, Scientific Instruments, Bohemia, NY) immediately before injection. The injection consisted of 2 to 3 million microspheres administered into the left atrium as a bolus for 10 s and flushed with 10 ml warm (37°C) saline. A timed collection of reference arterial blood was initiated a few seconds before the microsphere injection and maintained at a constant rate of 7 ml·min¹ for 3 min (precalibrated Harvard infusion-withdrawal pump, model 1941, Natick, MA). Transmural tissue samples were selected for mapping tissue flow in the myocardium at the end of each experiment. The samples were obtained from two regions of the LV: (1) normal zone (myocardium supplied by the LCCA) and (2) ischemic zone (distal to the site of LAD occlusion). At the conclusion of each experiment, India Ink was injected into the coronary circulation immediately distal to the site of LAD occlusion at a pressure of 100 mm Hg to identify the ischemic zone of the LV. The heart was fibrillated immediately, removed and fixed in formalin for 24 hr. On the next day, myocardial tissue samples were divided into subepicardial, midmyocardial and subendocardial layers of approximately equal thickness. Samples were weighed and placed in scintillation vials, and the activity of each isotope was determined. Similarly, the activity of each isotope in the reference blood sample was assessed. Tissue blood flow (ml·min¹·g¹) was calculated as Q_r·C_m·C_r¹, where Q_r = rate of withdrawal of the reference blood sample (ml·min¹); C_m = activity (counts·min¹·g¹) of the myocardial tissue sample; and C_r = activity (counts·min¹·g¹) of the reference blood sample. Transmural blood flow was determined as the average of subepicardial, midmyocardial and subendocardial blood flows.

Experimental protocol. The experimental design is illustrated in figure 2. RSR13 (Allos Therapeutics, Denver, CO) was dissolved in 0.45% saline to a concentration of 20 mg·ml¹ and prepared fresh on each experimental day. Dogs were randomly assigned to receive intravenous vehicle (0.45% saline), low-dose RSR13 (100 mg·kg¹ bolus followed by a 0.50 mg·kg¹·min¹ infusion; total cumulative dose = 228 mg·kg¹) or high dose RSR13 (150 mg·kg¹ bolus followed by a 0.75 mg·kg¹·min¹ infusion; total cumulative dose = 341 mg·kg¹) continued for the duration of each experiment. Pilot experiments demonstrated that these bolus and infusion doses of RSR13 produced stable increases in the partial pressure of O₂ and the Hill coefficient at 50% hemoglobin saturation (P₅₀ and n₅₀, respectively) for the duration of each experiment. Dogs received equal volumes of 0.45% saline with or without RSR13 in all three experimental groups. Thirty minutes after instrumentation was completed and again 30 min after drug vehicle or RSR13 had been initiated, systemic hemodynamics and regional contractile function were recorded. Arterial pH, O₂ tension (PO₂), carbon dioxide tension (PCO₂) and hemoglobin concentration were measured with a blood gas analyzer (model ABL3, Radiometer, Copenhagen, Denmark) that was calibrated with known standards before and during experimentation. The O₂-hemoglobin dissociation curve was calculated by multiple point tonometry (model IL 237 Tonometer, Instrumentation Laboratories, Lexington, MA) followed by nonlinear regression analysis. P₅₀ and n₅₀ were then determined from the O₂-hemoglobin dissociation curve. Thirty minutes after the start of drug or vehicle infusion, all dogs were subjected to five 5-min periods of LAD occlusion separated by 5-min periods of reperfusion and followed by a final 180 min of reperfusion during which hemodynamics, contractile function, arterial blood gases, P₅₀ and n₅₀ were determined at selected intervals. Regional myocardial blood flow was measured at base line, during the fifth LAD occlusion and after 60 and 180 min of reperfusion.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Schematic diagram of the experimental protocol used to study the effect of RSR13 or drug vehicle on the functional recovery of postischemic, reperfused myocardium produced by multiple LAD occlusions and reperfusion. RSR13 or vehicle was initiated 30 min after instrumentation had been completed and continued throughout each experiment. After 30 min of drug or vehicle infusion, dogs were subjected to five 5-min periods of LAD occlusion (OCC), interspersed with 5-min periods of reperfusion and followed by a final 180 min of reperfusion. %SS, systemic and coronary hemodynamics, arterial blood gases, P50, n50 and regional myocardial blood flow were measured at the indicated intervals.

Statistical analysis. Statistical analysis of the data within and between groups was performed by analysis of variance (ANOVA) with repeated measures, followed by use of Student's t test with Duncan's adjustment for multiplicity (Wallenstein et al., 1980). Two-way ANOVA was used for group-time comparisons. Changes were considered to be statistically significant when the probability (P) value was < .05. All data are expressed as mean ± standard error of the mean (S.E.M.).

	Results

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Twenty-eight dogs were instrumented to obtain 21 successful experiments. Four dogs (1 vehicle, 2 low-dose RSR13 and 1 high-dose RSR13) were excluded from data analysis because transmural coronary collateral blood flow during the fifth LAD occlusion was greater than 50% of the base-line value. Two dogs were excluded because of problems with instrumentation, and another dog developed ventricular fibrillation immediately after the fifth LAD occlusion.

Hemodynamic effects of ischemia and reperfusion in dogs receiving drug vehicle. No differences in systemic hemodynamics, contractile function, arterial blood gases, P₅₀, n₅₀, regional myocardial perfusion (tables 1 to 4) and the ratio of myocardial area at risk to total LV mass (fig. 3) were observed across groups after completion of instrumentation and before experimental intervention. Vehicle produced no hemodynamic effects (table 1). LAD occlusions caused a significant (P < .05) increase in LV end-diastolic pressure and decreases in cardiac output and stroke volume (table 1). Heart rate, mean arterial and LV systolic pressures, LV +dP/dt_max, systemic vascular resistance and pressure-work index remained unchanged. Systolic aneurysmal bulging of ischemic myocardium occurred during each 5-min LAD occlusion (fig. 4). Percent segment shortening in the LAD perfusion territory also was decreased from base line during each 5-min reperfusion and during the entire 180 min of final reperfusion. No changes in %SS were observed in the normal (LCCA) zone during LAD occlusions and reperfusions (fig. 5). Increases in heart rate and systemic vascular resistance and decreases in LV +dP/dt_max, cardiac output and stroke volume were observed during the final reperfusion period. Arterial blood gases, acid-base status, P₅₀ and n₅₀ were unchanged during LAD occlusions and reperfusions in dogs receiving vehicle.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Ratio of the area at risk (AAR) to total LV mass in dogs receiving drug vehicle or two doses of RSR13.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Segment shortening in the ischemic and reperfused LAD region. Percent segment shortening was significantly (P < .05) decreased from base line during each 5-min LAD occlusion in dogs treated with vehicle. Significant decreases in %SS during each 5-min reperfusion and throughout the final 180 min of reperfusion were observed in dogs receiving vehicle and low-dose RSR13. In contrast, dogs receiving high-dose RSR13 maintained %SS at baseline values during the first LAD reperfusion and regional contractile function partially recovered toward baseline values 60, 120 and 180 min after final reperfusion. *Significantly (P < .05) different from vehicle.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Segment shortening in the normal LCCA region during multiple brief LAD occlusions and reperfusions and 180 min of final LAD reperfusion.

Hemodynamic effects of ischemia and reperfusion in dogs receiving RSR13. No changes in systemic hemodynamics and regional contractile function were observed in dogs receiving either dose of RSR13 (tables 2 and 3 and figs. 4 and 5, respectively) before LAD occlusions and reperfusion. RSR13 caused dose-related increases in P₅₀ (e.g., 33 ± 1 during base line to 46 ± 1 mm Hg during high-dose RSR13; table 3) and decreases in n₅₀ (e.g., 2.77 ± 0.08 during base line to 1.91 ± 0.03 during high-dose RSR13; table 3). A significant increase in LV end-diastolic pressure occurred during the fifth LAD occlusion in dogs receiving low- and high-dose RSR13. Modest decreases in mean arterial and LV systolic pressures, cardiac output, stroke volume and pressure-work index were observed during the fifth LAD occlusion in dogs treated with high-dose, but not low-dose RSR13.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Relation between %SS in the ischemic LAD region and the partial pressure of oxygen at 50% hemoglobin saturation (P50) 180 min after final reperfusion for all dogs (n = 21) receiving vehicle, low-dose RSR13 and high-dose RSR13.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Relation between %SS in the ischemic LAD region and the partial pressure of oxygen at 50% hemoglobin saturation (P50) during the fifth LAD occlusion for all dogs (n = 21) receiving vehicle, low-dose RSR13 and high-dose RSR13.

Regional myocardial perfusion. Regional myocardial blood flow in the ischemic (LAD) and normal (LCCA) zones is summarized in tables 4 and 5, respectively. Blood flow to ischemic myocardium decreased in the subepicardium, midmyocardium and subendocardium during LAD occlusion to equivalent degrees in each experimental group (table 4). These findings indicate that a similar degree of coronary collateral blood flow was present in dogs receiving drug vehicle and the two doses of RSR13. Blood flow to normal myocardium remained constant during LAD occlusion and reperfusion in all three experimental groups. There were no differences among groups in blood flow to ischemic and normal myocardium during reperfusion. Blood flow recovered to base-line values at 180 min after reperfusion in all three experimental groups.

View this table: [in this window] [in a new window]   TABLE 4 Effects of myocardial stunning on regional myocardial perfusion (ml · min1 · g1) in the ischemic (LAD) regiona

View this table: [in this window] [in a new window]   TABLE 5 Effects of myocardial stunning on regional myocardial perfusion (ml · min1 · g1) in the normal (LCCA) regiona

	Discussion

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A new series of allosteric modifiers of hemoglobin, including RSR13 and several other 2-(aryloxy)-2-methylproprionic acid derivatives, were synthesized after two antilipidemic drugs, clofibrate and bezafibrate, were found to shift the allosteric equilibrium of hemoglobin toward the deoxy conformation of the molecule (Poyart et al., 1994). RSR13 stabilizes deoxyhemoglobin by binding to specific sites in the hemoglobin central water cavity via multiple polar interactions (Wireko et al., 1991). This effect causes a rightward shift of the O₂-hemoglobin dissociation curve (Abraham et al., 1992a, b; Randad et al., 1991) and enhances the transfer of O₂ from the saturated hemoglobin moiety to peripheral tissue (Khandelwal et al., 1993; Kunert et al., 1996a). RSR13 and structurally related derivatives are more potent allosteric effectors of hemoglobin than the fibrates because these new compounds exhibit minimal binding to serum albumin (Randad et al., 1991). Unlike inositol hexaphosphate (a structurally unrelated allosteric modifier of hemoglobin), RSR13 does not alter serum catecholamine, renin and vasopressin concentrations nor produce hemolysis and hemoglobinuria in vivo (Kunert et al., 1996b). These characteristics make RSR13 useful for the study of enhanced O₂ release in experimental models of tissue hypoxemia, including myocardial ischemia and reperfusion injury.

The present results indicate that RSR13 causes dose-related increases in P₅₀ (+40 ± 4% from base line for the high dose) and decreases in n₅₀ (31 ± 2% from base line for the high dose). These findings are consistent with a shift of the O₂-hemoglobin dissociation curve to the right and confirm the observations of previous studies in mice (Khandelwal et al., 1993), rats (Kunert et al., 1996a), cats (Wei et al., 1993) and dogs (Mejia et al., 1996). The increases in P₅₀ observed during the administration of high-dose RSR13 in the present study are similar to those reported to increase tissue O₂ delivery and exert physiological effects (Kunert et al., 1996a; Mejia et al., 1996), presumably by enhancing the O₂ pressure gradient from the capillary to the tissue itself (Kunert et al., 1996a; Liard and Kunert; 1993; Woodson, 1988). Although increases in tissue PO₂ measured with a microelectrode technique have been observed during administration of RSR13 in vivo (Kunert et al., 1996a), myocardial PO₂ in the area at risk was not specifically measured during ischemia and reperfusion in the present investigation. The high dose of RSR13 enhanced the functional recovery of stunned myocardium (47 ± 9% of base line at 180 min after the final reperfusion) produced by repetitive, brief LAD occlusions and reperfusions, in contrast to the persistent contractile dysfunction present 180 min after the final reperfusion observed with low-dose RSR13 (10 ± 18% of base line) or vehicle alone (2 ± 16% of base line). It appears likely that the relatively small shift of the O₂-hemoglobin dissociation curve (increase in P₅₀ of 8 mm Hg from base line) produced by low-dose RSR13 did not sufficiently improve O₂ delivery to the myocardium at risk or significantly enhance the recovery of contractile function in the ischemic zone. Nevertheless, recovery of contractile function in the LAD perfusion territory was correlated positively with P₅₀ 180 min after the final reperfusion when data from all three groups of experiments were pooled (fig. 6).

RSR13 did not alter systemic hemodynamics before coronary artery occlusion. These results confirm and extend the findings of a previous study (Kunert et al., 1996b), which indicates that intravenous bolus administration of this drug (200 mg·kg¹) does not acutely affect heart rate, arterial pressure, cardiac output or systemic vascular resistance in conscious rats. In addition, systemic hemodynamics, determinants of myocardial oxygen consumption, regional myocardial perfusion and coronary collateral blood flow were similar in dogs receiving vehicle or RSR13 during ischemia and reperfusion. These results suggest that the enhanced functional recovery of postischemic, reperfused myocardium was determined by RSR13-induced increases in O₂ delivery independent of hemodynamic effects or alterations of coronary perfusion. There was no correlation between the degree of contractile dysfunction and P₅₀ during the fifth LAD occlusion when data from all three groups were pooled (fig. 7). In addition, ischemic zone contractile function 180 min after final reperfusion was not correlated with transmural coronary blood flow during ischemia. These data further suggest that RSR13-induced increases in O₂ delivery does not improve the functional recovery of stunned myocardium by effectively reducing the magnitude of the ischemic stress or improving coronary collateral blood flow during brief LAD occlusions and reperfusion, respectively. A significant decrease in pH was observed 180 min after final reperfusion in dogs treated with low- and high-dose RSR13. Acidosis may exert a cardioprotective effect in stunned myocardium (Bolli, 1990), and this reduction in pH that occurred in the presence of RSR13 may have contributed the enhanced functional recovery of postischemic, reperfused myocardium observed in the present investigation. However, LAD %SS recovered in dogs treated with high- but not low-dose RSR13 despite a similar magnitude of acidosis between groups. In addition, pH was unchanged from base line and recovery of contractile function was observed before 180 min after final reperfusion in dogs receiving high-dose RSR13. These findings indicate that the modest acidosis observed 180 min after reperfusion was not singularly responsible for observed results with high-dose RSR13.

The present findings in a canine model of stunned myocardium produced by multiple, brief LAD occlusions and reperfusion are consistent with recent observations which indicate that RSR13 preserves several indices of myocardial metabolism during supply ischemia caused by a flow-limiting coronary artery stenosis in open-chest dogs (Mejia et al., 1996). When extrapolated to the present observations, the results of this previous study (Mejia et al., 1996) suggest that RSR13 may improve the functional recovery of stunned myocardium by favorably protecting vital cellular processes and conserving high-energy phosphates required for contraction during ischemia and reperfusion. This contention has yet to be tested, however. Oxygen-derived free radicals produced by myocardial xanthine oxidase or infiltrating polymorphonuclear leukocytes are important factors that contribute to the pathophysiology of reperfusion injury (Bolli, 1990). The previous (Mejia et al., 1996) and present results also suggest indirectly that RSR13-induced increases in O₂ release from hemoglobin to the cardiac myocyte during ischemia and reperfusion do not appear to exacerbate the metabolic consequences of critically reduced coronary blood flow or worsen the recovery of stunned myocardium during reperfusion, respectively, by stimulating the formation of additional O₂-derived free radicals. These findings may be somewhat surprising because beneficial increases in tissue O₂ delivery produced by RSR13 have been shown to directly enhance the radiosensitivity of hypoxic tumor cells (Khandelwal et al., 1996) and reduce the number and size of metastases (Ikebe et al., 1996) by stimulation of radiation-induced O₂-derived free radical production and the subsequent destruction of tumor DNA by these reactive intermediates (Coleman, 1988). However, the oxidative stress imposed by large doses of external irradiation is probably more profound than the endogenous production of O₂-derived free radicals immediately after reperfusion. This apparent paradox will require additional investigation to clarify.

Rightward shifts of the O₂-hemoglobin dissociation curve produced by RSR13 may have important implications for O₂ loading onto the desaturated hemoglobin molecule. In the present investigation conducted with 100% inspired O₂, arterial O₂ tensions exceeded 250 mm Hg (tables 2 and 3), and hemoglobin saturation approached unity (fig. 8) during administration of both RSR13 doses. However, use of inspired O₂ concentrations less than 100% resulting in arterial O₂ tensions less than 200 mm Hg may compromise O₂ loading onto deoxyhemoglobin to some degree during RSR13-induced rightward shifts of the O₂-hemoglobin dissociation curve.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Theoretical O2-hemoglobin dissociation curves obtained during administration of vehicle and both doses of RSR13 calculated with the average P50 and n50 values in each group by the equation: Hemoglobin saturation = (PO2)n50·[(PO2)n50 + (P50)n50]1.

In summary, the results of the present investigation indicate that RSR13 causes dose-related rightward shifts of the oxygen-hemoglobin dissociation curve as indicated by dose-dependent increases in P₅₀ and decreases in n₅₀. RSR13 enhanced the recovery of stunned myocardium independent of systemic hemodynamic effects and myocardial perfusion in barbiturate-anesthetized dogs, presumably by increasing O₂ availability during ischemia and reperfusion. The present findings suggest that decreases in hemoglobin-O₂ affinity and augmented O₂ unloading produced by RSR13 may represent a new therapeutic strategy in the management of ischemic heart disease that differ from traditional approaches that alter myocardial O₂ supply or demand.