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lACC Vol 2. No 2 August 1983 279- 86 279 Salvage of Ischemic Myocardium by Prostacyclin During Experimental Myocardial Infarction JACQUES A. MELIN, MD,* LEWIS C. BECKER, MD, FACC Baltimore, Maryland The effects of prostacyclin (PGI z) on infarct size and regional myocardial blood flow were studied in 28 anesthetized dogs subjected to 5 hours of coronary occlusion. A region of myocardial hypoperfusion was defined by injection of dye into the left atrium just before sacrifice. Infarct size was determined by planimetry of left ventricular slices after incubation in triphenyl tetrazoIium chloride. The animals received either PGI z in Tris 14) or Tris buffer solution (20 to 40 ng/kg per min, n buffer alone (control, n = 14) beginning 10minutes after anterior descending coronary artery occlusion. During = Prostacyclin (PGh), the major active metabolite of arachidonic acid in vascular endothelium (1,2), has potent vasodilating properties in addition to other important biologic effects. Infusion of exogenous PGh has been shown to favorably modify cellular injury produced by a regional or global reduction in myocardial blood flow . After coronary artery ligation, a decrease in enzyme depletion and stabilization of myocardial Iysosomes in ischemic tissue have been observed in the anesthetized cat (3), and a decrease in the extent of myocardial necrosis has been shown to occur in the conscious dog (4). In the latter study, PGI 2 infusion was associated with an increase in collateral blood fl ow to the ischemic myocardium , accounting for at least part of its myocardial salvaging effect. However, some animals demonstrated myocardial salvage despite low levels of collateral flow , suggesting a direct myocardial or nonfl ow-mediated effect. In the study of Araki and Lefer (5), ischemic myocardial injury was diminished in the isolated cat heart perFrom the Department of Medicine. Division of Cardiology. The Johns Hopkins University School of Medicine, Baltimore, Maryland This study was supported by the Ischemic Heart Disease SCOR Grant 2 P 50 HL 17655 from the National Heart, Lung, and Blood Institute . National Institutes of Health, Bethesda, Maryland. Dr. Melin was a Fogarty International Fellow of the National Institutes of Health Manuscript received November 2, 1982; revised manuscriptreceived February9. 1983, accepted February 13, 1983. *Present address: Cliniques Universitaires Saint-Luc, Avenue HlPpocrate 10, 1200 Brussels, Belgium Address for reprints: Lewis C. Becker, MD, Johns Hopkins Hospital, 600 North Wolfe Street. Baltimore. Maryland 21205 . © 1983 by the American College of Cardiology PGIz infusion, mean arterial pressure decreased by 8% , but heart rate was unchanged. Infarct size was significantly less (p<O.OOS) in PGIz-treated dogs compared with the control group , both as percent of left ventricle (8.1 versus 17.7%) and as percent of the hypoperfused zone (39.8 versus 77.3%). No significant changes in regional myocardial blood flow occurred over the 5 hour infusion period in either group . Thus, under the conditions of this study, prostacyclin appeared to protect ischemic myocardium by a direct flow-independent mechanism. fused at constant flow. In addition to its vasodilatory properties, PGh can prevent or reverse in vivo platelet aggregation (6- 8) and stabilize membranes (3). This study was designed to determine whether direct (nonflow-mediated) effects of PGh are important in contributing to myocardial salvage in the intact dog heart after coronary artery ligation. We utilized a model in which a region of myocardial hypoperfusion was defined by injection of dye into the left atrium just before sacrifice. A reduction by PGIz in the extent of infarction within the nondyed , or hypoperfused, myocardial zone was likely to reflect myocardial salvage mediated by direct mechanisms. Methods Surgical preparation. Thirty-two mongrel dogs weighing 18 to 30 kg were anesthetized with intravenous alpha chloralose (1 00 rug/kg) and urethane (I g/kg). Respiration with roomair was maintained by a Harvard pumpand cuffed endotracheal tube. The heart was exposed by a left thoracotomy and suspended in a pericardial cradle. The left anterior descending coronary artery was isolated proximally just beyondits first or second diagonal branch and a ligature was passed underneath. Polyethylene catheters (8 French) were placed in the left atrium for injections of microspheres and dye, and in the common carotid artery for withdrawal of reference blood samples and the recording of systemic 0735-1097/83/$3 ()() 280 lACC Vol. 2. No.2 August 1983:279-86 MELIN AND BECKER PROSTACYCLIN IN EXPERIMENTAL INFARCTION arterial pressure. An additional catheter was placed in the left jugular vein for administration of drugs. Aortic and left atrial pressures and heart rate were continuously monitored on a Gould (Brush 200) recorder. Experimental protocol. After control hemodynamic and electrocardiographic measurements, the left anterior descending coronary artery was ligated. Five minutes after occlusion, 2 X 106 microspheres, 9 ± I JL in diameter, labeled with scandium-46 (3M Company) were injected into the left atrium. A reference arterial sample was drawn at a constant rate of 2.16 mlImin with a calibrated Harvard pump, beginning 30 seconds before microsphere injection and continuing for 2 minutes after injection. After the microsphere injection, a continuous infusion of prostacyclin (PGIz) or drug vehicle (50 roM isotonic Tris hydrochloride buffer adjusted to a pH of 9.4 and infused at room temperature) was started into the left jugular vein. Before each experiment, 500 JLg of PGI z was mixed with the Tris buffer solution to a volume of 150 ml. For PGI z, the infusion rate was adjusted to reduce the mean arterial pressure approximately 10% below the pre-Pfll, (control) level (20 to 40 ng/kg per min). Ifnecessary, the rate was readjusted 2 hours later to return arterial pressure to the desired level. The infusion rate for the drug vehicle was constant at 0.15 ml/ min. Five hours after occlusion, a second injection of microspheres labeled with niobium-95 was made into the left atrium. After the myocardial blood flow determination, monastral blue dye, 1 cc/kg (DuPont Company), was infused into the left atrium over a 1 minute period. Immediately afterward, the heart was arrested by injection of saturated potassium chloride solution. Postmortem tissue preparation. The hearts were excised and the ventricles were sectioned parallel to the atrioventricular groove, forming five slices I to 1.5 em thick. After removing the right ventricle, each slice was weighed and the left ventricular outlines were traced on clear acetate sheets. The outlines of the hypoperfused region (risk region), indicated by the absence of blue staining, were added to the tracing. Each slice was then placed in a solution of triphenyl tetrazolium chloride (ITC) at 37°C for 30 minutes. The TIC solution consisted of 5 mg/ml TTC, 1 M Sorenson's phosphate buffer and water in a 1:1:8 ratio. TTC is reduced by dehydrogenase contained in viable cells, staining noninfarcted myocardium a dark red. Infarcted dehydrogenase-deficient myocardium is a pale grey color and is clearly demarcated from noninfarcted myocardium (9,10). The edges of the infarct were drawn on the same transparent sheets used for the left ventricular and risk region outlines. The tracings of each ring were electronically measured by planimetry to determine the areas of left ventricle, infarct (unstained by TTC) and risk region (not perfused by monastral blue dye). Areas on the top and bottom surfaces were averaged. Masses of infarcts and risk regions for each ring were computed by relating their relative areas to the weights of the rings. Myocardial blood flow calculation. Sampling for regional myocardial blood flow was made transmurally in the center of the infarct (area unstained by TTC), in the lateral risk region (area unstained by monastral blue but stained by TTC), in the region just lateral to the risk region and in the nonischemic posterior papillary muscle. The samples were divided into equal epicardial and endocardial halves, which were weighed (0.5 to I g), placed in vials and counted with the reference blood samples for radioactivity in a well-type gamma scintillation counter (Packard 5986). Regional myocardial bloodfiow (mllmin per g) was calculated as: counts/g in myocardial samples times (reference blood flow/counts in the reference blood sample). The mass of myocardium at risk before treatment (5 minutes after occlusion) was determined by summing the weights of the samples having myocardial blood flow less than 50% of the flow to the nonischemic posterior wall. To assess the accuracy of monastral blue staining as a marker of the hypoperfused or "risk" region, flows were compared in stained and unstainedsamples taken from sites adjacent to the stainedunstained border. For blue stained samples, both endocardial and epicardial halves were used in the analysis. For unstained samples, epicardial halves were excluded because small areas of "contamination" with blue staining were often visible in the lateral subepicardial portion. Flows were expressed as a percent of the posterior wall nonischemic flow. Statistical analysis. Student's t tests for paired and unpaired data were utilized for statistical comparisons between groups. Linear regression analysis was performed by the least-squares fit method. The X axis intercepts and their standard error were obtained by reentering the data with X and Y axes reversed. Probability (p) values of 0.05 or less were considered statistically significant. All results are presented as mean ± standard error of the mean. Results Of the 32 dogs that underwent coronary artery occlusion, 4 were excluded because of ventricular fibrillation during the initial 10 minutes after occlusion. Data in the remaining 28 dogs form the basis for this report: 14 received the drug vehicle and 14 received prostacyclin (PGI z). Hemodynamic changes. Heart rate, mean arterial pressure and left atrial pressure were similar before coronary occlusion in the two groups (Table I). After coronary occlusion, mean arterial pressure decreased and left atrial pressure increased to a similar extent in both groups. During the remainder of the experiment, the control group showed no further significant hemodynamic changes. In the group treated with PGlz, PGlz produced an average 8% decrease in mean arterial pressure at 10 minutes after treatment (from l ACC Vol 2. No 2 August 1983 279-86 PROST ACYCLI N VEHI CLE 25 28 1 MELIN AND BECKER PROST ACYCLI N IN EXPERIM ENT AL INFARCTI ON 25 IIU JE UE 1JI W oJ a. 20 20 15 15 10 10 5 5 ::I: 0( 1JI LL 0 a: w CD ::I: ~ Z 0 o 25 10 20 30 IOf 0 40 50 60 70 80 90 100 1 10 120 25 a.UE 10 20 30 IOf 40 50 60 70 80 90 100 110 120 a.UE 1JI w 20 20 1JI 15 15 oJ a. ::I: 0( LL 0 a: 10 10 5 5 w CD ::I: ~ Z o 0 o 10 20 30 40 50 80 70 80 90 100 110120 MYOCARDI AL BLOOD FLOW (AS PERCENT OF NO ISCHEMI C FLOW) Figure l. Histograms of the flow values in blue and nonblue samples adjacent to the stain border in control and prostacyclin (PGh)-trea ted groups. The blue stained samples (n == 88 in each group) are both endocardial and epicardial and the unstained samples (n == 44 in each group) are endocardial only. Flow is expressed as percent of nonischemic, and groupings are 0 to 10%, II to 20%, 21 to 30% and 3 1 to 40%, and so forth. The data mdicate that staining technique separates myocardium with flow 50% or less and greater than 50% of nonischemic with little overlap and that this fl ow " separation" level is the same m control and PGI!treated groups. 99 to 9 1 mm Hg, p < 0.005). At I hour after treatment, the mean arterial pressure was reduced by 5% (to 94 mm Hg, p < 0.05) and at 5 hours by only 2% (to 97 mm Hg). The mean arterial pressure at 5 hours in the POh group was 5% less than that in the control group (97 versus 102 mm Hg; NS = not significant). After administration of POll , left atrial pressure decreased (p < 0.005) and this change was maintained during the 5 hours of treatment. Heart rate did not change significantly. Thus, hemodynamically, the major difference between control and POh -treated dogs was a decrease in mean arterial pressure and left atrial pressure during POh treatment. Risk region determination. Figure I shows the myocardial blood flow values in the blue and nonblue samples taken adjacent to the stained border in control and POlztreated groups. A lack of staining identified myocardium 0 10 2 0 30 40 50 80 70 80 90 100 1 10 120 MYOCARD IAL BLOOD FLOW (AS PERCENT OF ONISC EMIC FLOW) Table I, Hemodynamic Changes in Open Chest Dogs Treated With Vehicle (control group) and Prostacyclin (PGh) Heart Rate min) Mean Artenal Pressure (mm Hg) Mean Left Atnal Pressure (mm IIg) 151 ± 8 105 ± 4 4 ± 0.3 162 162 161 163 97 ± 99 ± 100 ± 102 ± 47 4 5 ± 0.3* 5 ± 0.3 5 3 5 ± 04 6 ± 0.5 149 ± 6 108 ± 4 3 ± 03 153 152 148 154 99 ± 91 ± 94 ± 97 ± 5 4 4 4 (beats/ Vehicle (n= 14) Preocclusion Postocclusron 5 nu n 20 mm Ih 5h PGl 2 (n= 14) Prcocc lusion ± 8* ± 8 ± 8 ± 7 Postocclusion 5 01 111 20 mm Ih 5h 7 7 ± 8 ± 6 ± ± 4t 4§ 4+ 5 O.4t 0.4§ ± 0.4§ ± 04§ ± ± Valuesareexpressedas mean ± standard error of the mean. Statistical analysis was performed to compare preocclusion With 5 minute postocc1usion values (*p<O 05. t p<0.OO5l. or 5 nunute postocclusron with later postocc lu sron values rj:p<o OS . §p<0.005) Treatment was begun 10 mi nutes after occlusion. h = hourts), mi n = minutes, n = number. 282 lACC Vol. 2, No 2 August 1983:279-86 MELIN AND BECKER PROSTACYCLIN IN EXPERIMENTAL INFARcnON with flow less than 50 % of the nonischemic value in both control and PGh groups, with relatively little overlap between stained and unstained sample s. Of 88 stained samples, 2% (2 of 88) in the control group and 6% (5 of 88) in the group treated with PG h had flows between 41 and 50 % of the nonischemic flow , and in none was flow 40% or less. In all of the remaining samples, flow was greater than 50% of the nonischemic value . The slight difference between control and PGl z-treated groups was not significant. Of 44 nonblue endocardial samples, 7% (3 of 44 ) in both control and PGh groups had flows 51 to 60% of nonischemic flow and none had flows greater than 60% . In all of the remaining samples flow was 50 % or less of the nonischemic value. Effect of PGIz on infarct size. Prostacyclin (P0I 2 ) significantly reduced infarct size, both as a percent of the left ventricle and as a percent of the hypoperfused region (" risk region ") (Table 2) . A wide range of infarct and risk region sizes was seen in both groups. There was no significant difference in risk region mass betwe en control and PGh groups , although risk region mas s was slightly smaller in the PGh group (17 versus 2 1 g; t = I. 32; not significant [NSJ) . Expressed as a percent of the left vent ricle , the mass of the infarct was linearly related to the mass of the risk region in both groups (Fig. 2). The slopes of the linear regres sion were not different for the two groups (control dogs = 0.85; PGh = 0.79) but the horizontal intercept, repre senting the minimal risk region required for infarction , was smaller for the control group than for the PGh group (6 versus 10%; p < 0 .05 ). Regional myocardial blood flow. Regional myocardial blood flow data were available in 22 of the 28 dogs ( l O in the control group and 12 in the PGh group). In all dogs , there was a gradient of collateral flow from lateral to central Figure 2. Infarct size was directly related to the size of hyperperfused region ("risk region" ). Data for the prostacyciin (PGIz)treated dogs were shifted downward, indicating a smaller infarct for given size risk region. PGI2 displaced the horizontal intercept to the right (p < 0.05), although the slope was unchanged. "l ..J 50 Z 40 ....U ....a: "l • PROSTACYCLIN > 0 .111 0 .115 0 .9' 6 VEHICLE .... r- SLOPE 0 .19 c n •• •• e DO' < .00. IL. "l ...J 30 .... Z UJ u a: 20 UJ Q. ..... A til . U ....Z • • A a: lJ.. . A 10 0 0 10 20 30 40 RISK REGION AS PERCENT LEFT VENTRICLE 50 Table 2. Effect of Prostacyclin on Infarct Size in Open Chest Dogs Control Group (vehicle) (n = 14) PGIr Treated Group (n = 14) Infarct mass (g) 16.6 :!: 2.3 (4.4 to 31.4) 7.6 :!: 1.4* (0 to 19.3) Risk region mass (g) 21.2 :t 2.4 (4 4 to 40.3) 17.2 :t 1.8 (7 to 29.9) 92.8 :t 5.1 (57.4 to 137.5) 96.9:!: 3.1 (76.9 to 115.6) InfarctlLV (%) 17.7 :t 2.2 (5.9 to 33.4) 8.1 :t 1.7* (0 to 23) Risk region/LV (g) 22.9 :!: 2.4 (5.9 to 43) 18.3 :!: 2.0 (8 to 31) 77.3 :!: 4. 1 (56.2 to 100) 39.8 :t 4.9* (0 to 76) LV mass (g) Infarctln sk region (%) *p < 0.005, PGIz versus vehicle. Values are expressed as mean :!: standard error of the mean; values in parentheses represent range. portions of the risk region, and epicardial flows exceeded endocard ial flows throughout the risk region . Table 3 shows the time related chan ges in blood flow after coronary occlusion in different zone s of the myocardium. In dogs from the control group, myocardial blood flow did not change significantly in any zone or layer between 5 minutes and 5 hours of coronary occlu sion (paired t test). In dog s from the PGI 2 group, mean flow chan ges were also not statistically significant. Howe ver , in three dogs, there was an increase in collateral flow, which explains the slight increase in mean collat eral flow seen in the risk region of the PGI 2 treated dogs . Figure 3 shows the changes in subepicardial collateral flow after 5 hours of treatment in individual dog s. Only three dogs in the PGI 2 group and one in the vehicle group had an increase of more than 0.05 ml/min per g . The lack of a significant mean effect of PGl 2 on collateral flow was confirmed by the comparable sizes of the ischemic region 5 minutes after occlusion (before treatment) and ju st before sacrifice (after treatment ) (Fig. 4). The extent of initial ischemia was determined by summing the wei ghts of samples with flow less than 50% of nonischemic flow , while after treatment the size of the nond yed area was determined by planimetry. The slope of the linear regression was clo se to the line of identity and similar for the two groups (control = 1.07; PGI 2 = 0.96), suggesting that no significant collateral development occurred in the two groups between 5 minutes and 5 hours after occlu sion. If the PGIz-treated group had shown significant collateral development after treatment , the slope should have been shifted downward . The relation between subepicardial collateralflow 5 minutes after occlusion (bef ore treatment) and infarct size is shown in Figure 5 . Subepicardial collateral flow and infarct 283 MELIN AND BECKER PROSTACYCLIN IN EXPERIM ENTAL INFARCTION JACC Vol. 2. No 2 August 1983 279-86 Table 3. Regional Myocardial Blood Flows in Dogs Given Vehicle or PGh Nonischemic Region Vehicle (n = 10) Before treatment 5 hours after occ lusion PG h (n = 12) Before treatment 5 hours after occlusion Infarct Center Noninfarcted Risk RegIOn Inner Outer Inner Outer Inner Outer 089 ::!: 0 .07 0 .93 ::!: 0 .08 0 .83 ::!: 0.07 0 .87 ::!: 007 03 1 ::!:OO4 0 .32 ::!: 0.03 0 .34 1: 00 5 03 4 ::!: 0 .04 0 .05::!:OO I 0 .04 =: 0 .0 1 0 .1 3 ::!: 0 .02 0 . 12 :!: 0 .02 0 .80 ::!: 0 .08 0 .92 + 0 .09 0 .82 1: 0 . 10 0 .93 ::!: 0. 10 0 .28 ::!: 0 .03 0 .36 ::!: 0 .08 0 .39 1: 0.07 0.4 6 ::!: 0 .09 0 .0 7 ::!: 001 0 .07 ::!: 0 .0 1 0. 13 ::!: 0 .02 0 . 15 ::!: 002 Values for flows are mean z standard error of the mean (mllmin per g) size were inversely related in control and treated groups (linear r = - 0.77, - 0.76, respectively) with larger infarcts for lower flows; at flows above 20% of nonischemic flow, the relation plateaued with infarct size ranging between 5 and 10% of left ventricle . The amount of infarction associated with a given level of initial collateral flow was reduced in the PGl z group , as shown by the downward shift of the curve. The relation between infarct size and posttreatment subepicardial collateral flow 5 hours after occlusion is shown for compari son in Figure 6. Two dogs treated with PGI z had a relatively high post-treatment subepicardial flow greater than 0.25 mllmin per g, and had an infarct involving less than 50% of the nondyed (risk) region. However, the remain ing PGIz-treated and control dogs had comparable flows in the range of 0 .05 to 0. 20 mllmin per g; despite this , the dogs treated with PGIz clearly tended to have a smaller infarct, relative to the size of the risk region , than did dogs in the control group . Figure 3. Relation between initial and 5 hour ~post-tr.eatme.nt) subepicardial collateral flow in the center of the ischemic region in individual dogs . Flow values are in mllmin per g . Only three prostacyclin (PGh)-treated dogs and one control dog had increases of more than 0 .05 mllmin per g. ~ 0 .4 / • 0 ..J • U. / Z UJ lI::X Ill ... Hol a:UJ a: / / / ~..J / Z..J UJO XU 0 .2 ~ ol..J UJol a:H • / . ~o a: IIlU 0 .1 OH ll.ll. UJ ~ol / / . ./ • / / / a:~ UJ ... Ul UJUl 0< I X UJ ~ 96 1.07 n o 95 o 97 12 10 p < .001 V < 001 30 • • 20 10 >z / 00 H 0.0 l!l 0 .0 o UJ~ ZUl HO Xll. . . / / m • PFlOSTACYCLIN A VEHICLE 40 O~ ...... ):. . / / r SLoPE ;:: / • Figure 4. Relation between dye-determined risk region mass (posttreatment) and flow-determined risk region mass (pretreatment) . The mass of myocardium at risk before treatment (5 minutes after occlusion) was determined by summing the weights of the samples with myocardial blood flow less than 50% of the flow to the nonischemic myocardium . The slopes of the linear regression were close to the line of identity and were similar for the two groups (P = prostacyclin; V = vehicle) . The agreement between pretreatment and post-treatment values for ischemic mass suggests that no significant collateral development occurred. / / < Effect of prostacyclin on infarct size. Our results demonstrate that prostacyclin (PGIz) reduces infarct size by about 50% after coronary artery occlu sion in the anesthetized dog . Infarct size was measured relative to the size of the ischem ic region, identified by left atrial injection of blue dye after treatment with PGI2 and ju st before sacrifice of the animal. The fact that salvage occurred within this hypoperfused zone strongly suggests that the beneficial effect of PGIz in this study was mediated by direct cellular. or nonflow effect s of the agent. Microsphere measurements indicated no significant mean change in the level of collateral flow in the group treated with PGI z, and topographic measurements showed no change in the size of the ischemic region as a result of treatment. Analysis of the relat ion between collateral flow and infarct size indicated that dogs treated with / / ~~ III V£HICL£ A t:..J .col 0.3 a: IOUJ ::l PAClSTACYCLIN / Discussion 0.1 0.2 0 .3 0.4 PRETREATMENT SUBEPICARDIAL COLLATERAL FLOW UJ a: 0 0 10 20 30 40 FLOW-DETERMINED RISK REGION MASS (PRETREATMENT) 284 lACC Vol 2. No 2 August 1983 279-86 MELIN AND BECKER PROSTACYCLIN IN EXPERIMENTAL INFARCTI ON uJ .J u H 35 ct ~ • PROSTACYCL!N VEHICLE 30 uJ A > n 12 - 17 - 16 to D < 005 < Ot ... 25 II.. w .J 20 ~ 15 ...z ct uJ Q. . ... . 10 Vl u ct II.. Z 5 oL.---'---_L-_---'-_ _. L - _ - ' - -_ o 00 0 10 _ 0 20 .L-_ --' 0 30 SUBEPICARDIAL COLLATERAL FLOW AS A RATIO OF NONISCHEMIC FLOW (PRETREATMENT ) Figure5. Pretreatment subepicardial collateral flow was inversely related to infarct size in both control and prostacyclin (PGI2 ) treated groups. The downward shift of the relation in the POI 2 group indicates a smaller infarct size for a given level of initial collateral blood flow. PGh had a smaller infarct for a given level of either initial pretreatment or post-treatment flow. However, within the PGh group, three dogs may have had an increase in fl ow which contributed to myocardial protection; the two dogs with the highest flows after treatment (0.27 to 0.35 ml/min per g) were from the PGI group. Identification of region at risk for infarction . The method used in this study for delineation of the region " at risk" was specifically chosen to allow clear interpretation of the results. We used the technique described by Kloner's laboratory (II) of in vivo left atrial injection of dye just Figure 6. Post-treatmentsubepicardial collateral flow (ml/min per g) is compared with mass of infarct normalized to the mass of risk region. In the prostacyclin (PG I2)-t reated group, two dogs had fl ows greater than 0.25 mllmin per g and a small infarct (less than 50% of the risk region) . The remaining PGlz-treated dogs had low fl ow values similar to those of control dogs « 0. 2 mllmin per g) and smaller infarct size than that of control dogs . It> 100 4 A II.. 13 A I- • A Z wz uo CIH Wl!l D.W CI Ul 1- ... 60 A •• • 40 <I: ...z A A UCI CI II.. A • Ul <I:~ o PAOSUcYCLIN AVEHICl.E 60 20 • • • • • It is unclear why an increase in collatera l flo w occurred after administration of PGl z in our earlier study (4) but not in the present one . Other investigators found no change ( (5) 0 0.0 0 .1 0.2 before sacrifice of the animal. The dye distributes to the left ventricle under prevailing perfusion pressure and stains myocardium with flow of more than 50% of nonischemic value. Importantly, this method delineates a zone of ischemic myocardium present at the end of the experiment that may be different in size from the zone that existed early after coronary occlusion and before treatment. Therefore, the method differs from another recently described approach of defin ing the hypoperfused region (12), in which radioactive microspheres are injected early after coronary occlusion and autoradiography is subsequently performed after sacrifice. For the purposes of our study, it was advantageous for the hypoperfused zone to be identifi ed after treatment. We were interested in whether PGlz could reduce ischemic damage within a defined area of low fl ow, indicating that direct cellular effects, independent of fl ow, were important. However, this method may have serious drawbacks in screening for potential myocardial protective agents. The beneficial effects of a treatment that only increased flow but did not possess direct cytoprotective actions might be missed because infarct size is measured relative to the low flow zone existing at the end of the experiment. It is also useful to point out that the in vivo dye method delineates a zone of myocardium smaller than the anatomic occluded bed size. determined by postmortem intracoronary injection of dyes or barium gels ( 13 , 14). Anatomic bed size includes a certain amount of myocardium that is relatively well protected by collateral flow. Values for infarct size, expressed relative to anatomic bed size, are smaller than similar values normalized to ischemic zone size. Comparison with previous studies . Our finding of a direct cytoprotective, nonflow-mediated effect of PGlz is supported by at least two other studies. Araki and Lefer (5) found that PGh is beneficial during myocardial ischemia in the isolated perfused cat heart. Significant improvement of cardiac function and moderation of depletion of tissue creatine kinase activity by PGI2 were observed in this model, independent of effects on platelet aggregation and coronary vasodilation. Previous results from our laboratory (4) showed that a 6 hour infusion of PGh , sufficie nt to reduce mean arterial pressure by 5%, reduced infarct size by 67% and increased mean collateral fl ow by about threefold compared with that in control animals. Analysis of individual responses, however, suggested that PGh could produce considerable myocardial salvage despite low levels of collateral flow after treatment, implying that at least a portion of the effect was direct and not flow-mediated. 0 .3 POSTTREATMENT (5hr) SUBEPICARDIAL COLLATERAL FLOW 0.4 or a decrease (1 6) in collateral fl ow in anesthetized dogs, but in each of these studies, PGh produced a decrease in mean arterial pressure of 15 to 45%, which decreased the lACC Vol. 2. No 2 MELIN AND BECKER PROSTACYCLIN IN EXPERIMENTAL INFARCTION August 1983 279-86 driving pressure for collateral perfusion. In the present study, mean arterial pressure decreased only 8% 10 minutes after beginning the infusion and was decreased only 5% at I hour and 2% at 5 hours, respectively. There were several differences between this study and our earlier one. Previously, we used saline solution to mix PGlz , with frequent changes of fresh solution to avoid loss of activity, but in this study we used Tris buffer with a pH of 9.4. Although unlikely, it is possible that the small amounts of alkaline solution somehow inhibited collateral function in this study. A more important difference may be that the earlier study used conscious dogs and this study used anesthetized animals. The effects of anesthesia on collateral function have not been well explored, but increased sympathetic tone after anesthesia increases heart rate (17) and results in less diastolic time per minute and relatively more extravascular resistance to collateral flow . In addition, anesthesia could cause an increase in coronary vascular resistance, affecting nonocc1uded arteries that provide the source of collateral flow, and interferring with the usual response to vasodilators. Mechanism of direct myocardial salvage by prostacyelin. Our results point to a direct cytoprotective effect of PGlzas the major mechanism for myocardial salvage in this study. Collateral flow was unchanged for the group of animals receiving PGlz, although flow did increase in three individual dogs. A reduction in myocardial oxygen demand was probably not a major factor, because mean arterial pressure decreased only 8% and heart rate did not change. The stabilizing action of PGl z on lysosomal and other membranes may represent an important aspect of the direct protective effect of the drug. During ischemia, leakage of lysosomal enzymes (hydrolases and proteases) is reported to occur before the irreversible damage of myocardium(18) . Release of these enzymes into the cytoplasm could contribute to the degradation of structural proteins and membrane phospholipids. PGlz has been shown to stabilize lysosomes in the isolated cat liver (19) and in ischemic myocardium of intact animals (20). Activation of platelets and fo rmation of aggregates in obstructed coronary vessels (21,22) could decrease coronary flow on a microvascular level . Platelet aggregation causes increases in platelet thromboxane levels and secretion of granular constituents that include coagulation factors, vasoconstrictors and lysosomal enzymes (23). The granular products could lead to a vicious cycle of platelet plugging, vasoconstriction and necrosis, especially in marginal zones (24). PGh has been found to: I) prevent platelets from clumping , 2) disaggregate preformed platelet aggregates (25,26), and 3) inhibit the conversion by platelets of arachidonic acid to the potent coronary vasoconstrictor, thromboxane A z (27). Although these effects on platelets could serve as the basis for the protective action of PGIz , myocardial protection has been demonstrated in the isolated cat heart perfused with platelet free solution (5). Recently, 285 a PGlz mimetic compound, a new carbocyclin derivative (ZK 36 375), has been shown to have significant cardioprotective activity during acute myocardial ischemia in the cat. This action was dissociated from antiplatelet effects because the drug did not reverse ischemia-induced formation of platelet aggregates in vivo (28). A recent study (29) suggested that PG/ z pres erves intraneuronal catecholamines in adrenegic nerves in the ischemic myocardium, presumably by inhibition of catecholamine release . Overflow of catecholamines stimulates myocardial oxygen demand and platelet aggregation and also increases myocardial ischemia. PGlz may antagonize these potentially deleterious effects by stabilizing catecholamine-containing membranes within the nerve terminals. References I. Dustmg GJ, Moncada S, Vane JR. Prostacyclin (POX) is the endogenous metabolite responsible for relaxation of coronary arteries induced by arachidonic acid. Prostaglandins 1977;13:3-15 . 2 Moncada S. Higgs EA, Vane JR. Human arterial and venous tissues generate prostacyclin (prostaglandin X), a potent inhibitor of platelet aggregation. Lancet 1977; I:18-21. 3. Ogletree ML. Lefer AM, Smith JB. Nicolaou KC. Studies on protective effect of prostacyclin In acute myocardial ischemia. Zur J Pharmacol 1979;56.95-1 03. 4. Jugdutt BI, Hutchins GM, Bulkley BH. Becker LC. Dissimilar effects of prostacyclin, prostaglandin E1, and prostaglandin E2 on myocardial infarct size after coronary occlusion in conscious dogs. Circ Res 1981;49:685- 700. 5. Araki H. Lefer AM. Role of prostacyclin In the preservation of ischemic myocardial tissue In the perfused cat heart Cire Res 1980;47:75763. 6. Bayer B, Blass K. Forster W. 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