Chemotactic activity in the coronary sinus after experimental myocardial infarction: Effects of pharmacologic interventions on ischemic injury

Chemotactic Activity in the Coronary Sinus After Experimental Myocardial Infarction : Effects of Pharmacologic Interventions on Ischemic Injury

JOSEPH R . HARTMANN, MD JOHN A. ROBINSON, MD ROLF M . GUNNAR . MD, FACC Maywood, Illinois

Various pharmacologic interventions that suppressed chemotactic factor activity in the coronary sinus after acute Ischemla were analyzed for protective effects on myocardium . Ischemic injury was determined by comparing the slopes of the regression lines derived from 24 hour myocardial creatine klnase content versus S-T segment elevation 15 minutes after coronary ligation . Dogs treated 30 minutes after ligation with cobra venom factor, hydrocortisone or Trasylol showed a marked decrease in chemotactic activity in the coronary sinus . These agents also showed a protective effect on ischemic injury when compared to control . Myocardial biopsy specimens from areas of significant ischemia defined by S-T segment elevations in dogs treated with cobra venom factor were essentially devoid of an inflammatory response whereas those from dogs treated with Trasylol or hydrocortisone showed moderate neutrophil Infiltration and minimal tissue exudate.

The current interest in pharmacologic alteration of myocardial responses to ischemic injuryl 2 has been generated by the introduction of a reproducible technique of quantitating ischemic myocardial injury and the availability of an increasing array of inhibitors of inflammation . The ability of cobra venom factor, hydrocortisone and Trasylol®, a protease inhibitor, to suppress the canine inflammatory response to myocardial ischemia has already been reported . 3-5 The specific mechanisms by which the inflammatory response is suppressed by these agents are unknown . Experiments were designed to explore the possibility that the common denominator of these agents' ability to reduce infarct size is suppression of chemotactic factors that amplify neutrophil infiltration and secondary release of destructive hydrolytic lysosomal enzymes in ischemic myocardium. Pharmacologic interventions that acted on different steps of the inflammatory sequence all reduced the appearance of chemotactic factors in the coronary sinus after infarction and provided significant protection against ischemic injury . Methods

From the Department of Medicine, Loyola University Medical Center, Maywood, Illinois . Manuscript received January 21, 1977 ; revised manuscript received April 5, 1977, accepted April 6, 1977 .

Address for reprints : John A, Robinson, MD, Loyola University Medical Center, 2160 South First Avenue, Maywood, Illinois 60153. 550

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Experiments were performed according to the method of Maroko and CarpenterR in four groups of mongrel dogs weighing 15 to 30 kg : Group A, 16 control dogs given no treatment ; Group B, 8 dogs treated with cobra venom factor (20 units/kg body weight) ; Group C, 8 dogs treated with Trasylol (50,000 units every 6 hours) ; and Group D, 8 dogs treated with hydrocortisone (50 mg/kg) . To eliminate the possibility that zero time pharmacologic alterations might alter S-T measurements, all interventions were made 30 minutes after coronary ligation . Surgical procedure : The dogs were anesthetized with sodium pentobarbital, and respiration was maintained with a Harvard respirator . Additional small doses of pentobarbital were given as needed to maintain sedation . The blood pressure was monitored through a carotid arterial catheter . A catheter was placed into the coronary sinus from a jugular vein . Through a left thoracotomy, the heart was exposed by suspension in a pericardial cradle . The left anterior descending coronary artery or one of its branches was isolated and later ligated . A salineCARDIOLOGY

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soaked cotton wick electrode was used to obtain unipolar epicardial electrocardiograms from numerous sites on the anterior surface of the left ventricle . The sites chosen were close to anatomic landmarks so that they could be easily located . Analysis of ischemic injury : In each dog, control areas far from the occluded artery were selected along with areas adjacent to, and in the zone of, myocardium supplied by the occluded artery . Epicardial electrocardiograms were recorded before and 5,10, 15 and 20 minutes after occlusion . The chest was then closed, a chest tube left in place for auctioning . The dogs were allowed to recover from anesthesia . Twenty-four hours after occlusion they were killed and their hearts removed . Transmural biopsy specimens from areas where epicardial electrograms had been recorded were taken for analysis of myocardial creatine kinase (CK) content and histologic examination. Myocardial CK content at 24 hours was then compared with S-T segment elevation 15 minutes after ligation . The slope of this regression line was used to relate initial ischemia to resultant myocardial injury. Myocardial CK analysis : Myocardial biopsy specimens of approximately 400 mg were homogenized in a 25 ml/g solution of cold 0 .001 molar ethylenediamine-tetraacetic acid, 0 .25 molar sucrose and 0 .001 molar mercaptoethanol, in a Virtis "45" for 2 to 15 second bursts . The homogenate was twice clarified by centrifugation at 16,000 revolutions/min for 10 minutes, and 0-02 ml of the supernatant was added to 7 .5 ml Tris buffer containing 0 .2 percent bovine serum albumin . CK analysis was performed spectrophotometrically . 4 Protein concentration in the supernatant was measured with the biuret method. Myocardial CK, calculated by dividing CK content per ml by the protein concentration per ml, was expressed as myocardial CK content in IU per mg of supernatant protein . Complement analysis : Simultaneous sequential samples of carotid arterial and coronary sinus blood were collected 0, 0 .5, 1, 2, 4, 6, 12 and 24 hours after ligation . Samples were allowed to clot and serum was harvested and frozen at -70° C until tested . A procedure modified for optimal titration of canine hemolytic complement was used . 6 Thirty serum donors were used to establish normal canine complement values (range 25 to 55 hemolytic units) . There was no significant difference between parallel arterial and coronary sinus complement values in normal dogs . Detection of chemotactic activity in vitro : A . Responding leukocytes: Canine or human plasma was separated from heparinized blood by dextran sedimentation at 37° for 30 minutes. Harvested leukocyte suspensions, predominantly neutrophils, were washed twice with Hanks' balanced salt solution, pH 7 .4, and resuspended to between 1 and 5 X 106 cells/ml. After confirmation that normal canine and human leukocytes gave identical results, leukocytes from a single human donor were used for all experiments . B. Chernotactic attractants : Endotoxin-activated human serum was the positive chemotactic control for all samples assayed. Canine peripheral blood and coronary sinus sera were diluted 1 :10 with Hanks' balanced salt solution in plastic tubes, and a complete set of sequential samples from a single dog were assayed the same day. C . Chernotactic assay : 0 .2 ml of leukocytes was placed in the upper chamber of a modified Boyden blind-well chamber and separated from chemotactic stimulants by a 5 .0 µ polycarbonate filter (Nuclepore Inc) . This technique measures the positive movement of an indicator cell toward a chemotactic signal diffusing through a restricting filter. A responding cell must actively migrate to appear on the opposite side of the filter . Duplicate assayed samples were incubated for 2 to 4 hours at 37° C . After incubation, the filters were rinsed gently October

in Hanks' balanced salt solution, fixed in absolute methanol for 15 seconds and stained with hematoxylin and aqueous Wright's solution . D . Quantification of chernotaxis : This was done by counting all responding cells, always 90 percent or greater neutrophils, in 20 oil immersion fields (X 1,100) that had migrated completely through the filter . Proportional directional changes in chemotaxis were expressed by comparing chemotactic activity in the timed samples after an intervention with the background chemotactic activity in the control coronary sinus and peripheral sera from the same dog . Samples were quantitated without knowledge of interventions . Histology: Portions of each myocardial biopsy specimen were stained with hematoxylin-eosin and studied for evidence of necrosis and inflammatory response . Inflammatory cell response was estimated by the relative amount of cell infiltration, and the presence or absence of necrosis was noted . Data analysis : Myocardial CK content 24 hours after ligation was compared with the S-T segment elevation 15 minutes after ligation . The relation between the two could be expressed as a line and the slope of the line indicated the resultant ischemic injury as it related to the initial ischemic response to coronary ligation . The line established for the control group was compared with the lines for Groups B through D . A significant difference in the slopes of the lines would indicate either a deleterious or protective effect of the intervention on ischemic injury . The significance of changes in coronary sinus complement and chemotaxis values in all groups was assessed by standard t test calculation . Results

There were no significant differences among any of the groups with respect to heart rate and arterial pressure during the study period . Peripheral complement values tended to rise within 6 to 12 hours after ligation and then returned to control values in Groups A, C and D . The changes in complement were not significant . Group A (control) : The relation between CK and S-T segment elevation is given by the line CK = 32 3 .3 S-T (r = 0 .76) (Fig . 1) . Coronary sinus complement

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I I I I 1 t 123456769101112131415 ST SEGMENT ELEVATION (MV) A=CONTROL B=CVF C=TRASYLOL 0 = HYDROCORTISONE

S=33 r= .78 S=2 .3 r= .76,p<,0I S=1 .3, r= .6S,p< .OI 5=1 .6, r= .71, p < . 01

FIGURE 1 . Relations between myocardial creatine kinase (CK) measured 24 hours after ligation and ST segment elevation 15 minutes after ligation . The slope of the line is an indication of the degree of ischemic injury. Slopes, correlation coefficients and P values are given for each group of dogs . CVF = cobra venom factor . 1977

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12 HOURS A .-CONTROL 8--CVF

Co .TRASYLOL CA . HYDROCORTISONE

decreased slightly but not significantly, 0 .5 and 1 hour after ligation (P >0 .10) and then returned to control levels (Fig. 2) . Chemotaxis in the coronary sinus began increasing within 30 minutes, peaked at 1 hour and remained elevated throughout the 24 hour study period (P <0.001) (Fig. 3) . The inflammatory response on the epicardial surface of a control dog is shown in Figure 4 . The biopsy specimen was taken from a site with 7 mm S-T segment elevation. Specimens from areas with greater than 2 mm S-T elevation were always characterized by neutrophilic infiltration and interstitial edema . Ninety percent of samples taken from sites with S-T elevation greater than 2 mm 15 minutes after ligation showed evidence of necrosis .

FIGURE 2. Sequential percent changes after Ilgatlon compared with prellgation values of complement levels in the coronary sinus of Groups A to D . Each point of measurement represents a mean value with 1 standard deviation . CVF = cobra venom factor .

Group B (cobra venom factor) : The relation between myocardial CK and S-T segment elevation in the decomplemented dogs is expressed by the line CK = 31 - 2.3 S-T (r = 0.76) . There was a significant difference in the slopes of the regression lines for Groups A and B (P <0 .01), thus indicating that decomplementation with cobra venom factor protects myocardium against acute ischemic injury (Fig . 1) . Both total coronary sinus and peripheral serum complement decreased within 2 hours to less than 25 percent of the control value. Decomplementation was maintained throughout the 24 hour study period (Fig . 2) . Chemotaxis in the coronary sinus decreased significantly (P <0.001) by 2 hours and remained decreased during the study period (Fig . 3) .

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FIGURE 3 . Sequential percent changes after ligation compared with preligation chemotactic activity in the coronary sinus of Groups A to D . Measurements represent mean values with 1 standard deviation . CVF = cobra venom factor .



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A significant reduction in inflammatory cell response was seen . Biopsy specimens from areas with S-T segments greater than 6 mm showed little or no evidence of necrosis or inflammatory cell invasion (Fig . 4B) . The inflammatory cell response was definitely less in the

decomplemented dogs in Group B than in the control dogs in Group A . Group C (Trasylol) : The relation between CK and S-T segment elevation in dogs treated with Trasylol is given by the regression line CK = 31 - 1.3 S-T (r =

FIGURE 4 . Epicardial biopsy sections . A, from an area with 7 mm S-T segment elevation in a control dog (Group A) . A marked exudative response with numerous polymorphonuclear leukocytes is present at the epicardial surface . B, from an area with greater than 6 mm S-T segment elevation in a dog previously treated with cobra venom factor (Group B) . There is almost complete inhibition of inflammatory response at the epicardial surface . C, from an area with greater than 6 mm S-T segment elevation in a dog previously treated with Trasylol (Group C) . There is a moderate inflammatory response with mild neutrophilia and tissue exudation .

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0.65) . There is a significant difference between the slope of the line in Groups A and C (P <0 .01), indicating that Trasylol also decreases the extent of ischemic myocardial injury (Fig. 1) . Throughout the study, serum complement levels in this group were not different from those of control animals (Fig. 2) . Chemotaxis in the coronary sinus increased briefly but not significantly (P >0 .10), decreased below control levels after 2 hours and slowly returned to normal by 6 hours (Fig . 3) . There was definitive and prolonged suppression of chemotaxis with this dose of Trasylol . The direct addition of Trasylol to the in vitro chemotaxis assay did not inhibit leukocyte migration. Figure 4C shows a typical inflammatory response in the acutely infarcted myocardium of a dog treated with Trasylol. There is only moderate neutrophil infiltration and tissue exudate in an area with S-T segment elevation greater than 6 mm . Group D (hydrocortisone) : The relation between CK and S-T segment elevation is given by the line CK = 32 - 1.6 S-T (r = 0 .71) . There is a significant difference between the slope of the lines in Groups A and D (P <0.01), indicating that hydrocortisone protects myocardium against ischemic injury (Fig . 1) . Throughout the study, serum complement levels in this group were not different from those of control animals (Fig . 2) . Chemotaxis in the coronary sinus also increased initially, then was significantly suppressed (P <0.00 1) to an extent seen in the dogs given Trasylol (Fig. 3) . Myocardial samples from sites with 6 mm S-T elevation had an amount of neutrophil infiltration similar to that of Trasylol-treated dogs . Dogs in neither Group C nor Group D had suppression of inflammation comparable with that found in Group B (cobra venom factor) . Although Groups B, C and D were significantly protected from ischemic injury, there was no significant difference among the various interventions . Discussion The acute inflammatory response is characterized by the chemotactic recruitment of large numbers of neutrophils . In infarcted rat myocardium, the accumulation of neutrophils can be accounted for, at least in part, by the leukotactic activity of fragments of the third component of complement (C3) . Cleavage of C3 was shown to be mediated by a protease that appeared in acutely infarcted heart muscle . 7,8 Multiple tissue proteases, as well as trypsin, thrombin and plasmin, have been shown to be capable of this type of nonimmunologic activation of C3. Our study documented the appearance of chemotactic activity in the canine coronary sinus after acute myocardial ischemia and provided circumstantial data on the possible activation mechanism . Cobra venom factor: This factor combines with C3 proactivator and mediates inactivation of C3 . 9 The effective depletion of C3 by cobra venom factor prevents further activation of the complement system through either normal or alternate pathways or direct C3 cleavage . There may be very transient appearance of 55 4

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chemotactic fragments during cobra venom factor degradation of C3 . The clearance of these chemotactic fragments is very rapid, and chemotactic activity is absent within 1 minute after administration of cobra venom factor . Cobra venom factor has no effect on other organ systems, platelets or neutrophils . The marked suppression of neutrophil migration in dogs treated with cobra venom factor is strong evidence that C3 activation is a critical component of the inflammatory response during myocardial ischemia . The significant reduction of inflammatory response and improved CK/S-T segment ratios suggests that administration of a cobra venom factor has a protective effect on myocardial ischemic injury. The dogs were not followed up for more than 24 hours and it was not possible to study the development of myocardial fibrosis in those infarcts that had little or no neutrophil response . In this regard, it has been observed that fibrous scarring developed in infarcted myocardium of C3-depleted rats even though these rats were unable to generate an inflammatory response similar to that of control rats .8 Although cobra venom factor decomplementation provides direct evidence of complement participation in ischemia-related inflammation, its antigenicity and ablation of an adequate inflammatory response to infection negates any potential use in human myocardial infarction . Less antigenic, reversible chemotactic inhibitors were then tested in the same model . Trasylol : This agent is a nonspecific inhibitor of proteolysis . Kallikrein, plasmin and trypsin are a few of the identified proteases inhibited by this drug . Trasylol produces no hemodynamic alteration and has no effect on platelet or leukocyte function . Trasylol's antiinflammatory properties are probably related to inhibition of kallikrein and plasmin. A vicious cycle may be present during ischemic injury . The initial trauma generates a tissue protease that activates C3 with subsequent chemotaxis of neutrophils into myocardium . Neutrophil infiltration leads to further tissue destruction, release of kininogenase, kinin activation and perpetuation of cell necrosis, thereby restarting the cycle . Direct inhibition of kinin could also be effective in modifying ischemic tissue injury . We have shown activation of plasminogen in the coronary sinus of control animals after infarction .' 0 Trasylol inhibits this activation, and this action may account in part for its effect on chemotaxis because plasmin is capable of nonimmunologic C3 activation . 1 t Hydrocortisone: The antiinflammatory effects of hydrocortisone are widely recognized but not well understood . Stabilization of lysosomal membranes by hydrocortisone may prevent extension of ischemic injury, edema and cell death, thereby reducing the release of chemotactic factor . Hydrocortisone may also affect leukocyte receptors, preventing a normal response to chemotactic stimuli . Neutropenia : Phagocytizing or dying neutrophils, or both, at inflammatory sites release lysosomal hydrolytic enzymes that perpetuate chemotaxis and cell death . 12 Whether the neutrophil itself plays some part

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in the perpetuation of chemotaxis during a myocardial inflammatory response is unknown . We have been unable to evaluate the effect of severe neutropenia on infarct size because we have not been able to maintain a neutropenic animal for 24 hours after coronary ligation .

Although the long-term advantage of myocardial protection as defined by a high level of CK myocardial content is not known, further delineation of specific chemotactic signals that mediate tissue necrosis may provide insight for rational therapeutic attempts to inhibit the inflammatory response .

References 1 . Maroko PR, Kjekshus JK, Sobel BE, et al : Factors influencing infarct size following experimental coronary artery occlusions . Circulation 43 :67-82, 1971 2. Maroko PR, Libby P, Sobel BE, et al : Effect of glucose-insulinpotassium infusion on myocardlal infarction following experimental coronary artery occlusion . Circulation 45 :1160-1175, 1972 3 . Maroko PR, Carpenter CB : Reduction in infarct size following acute coronary occlusion by the administration of cobra venom factor (abstr) . Olin Res 22 :289, 1974 4 . Libby P, Maroko PR, Bloor CM, et al : Reduction of experimental myocardial infarct size by corticosteroid administration . J Olin Invest 52:599-607, 1973 5 . Dlaz PE, Maroko PR: The effects of aprotinin on myocardial ischemic injury following experimental coronary artery occlusion (abstr) . Clin Res 23 :180, 1975 6 . Barta 0, Berta V : Canine hemolytic complement : optimal condi-

tions for its titration . Am J Vet Res 34 :653-657, 1973 7 . Hill JH, Ward PA: The phlogistic role of C3 leukotactic fragments in myocardial infarcts of rats . J Exp Med 133 :885-900, 1971 8 . Hill JH, Ward PA : C3 leukotactic factors produced by a tissue protease . J Exp Med 130 :505-518, 1969 9 . Gotze 0, Muller-Eberhard HJ : The C3-activator system : an alternate pathway of complement activation, J Exp Med 134 : 90s-108s,1971 10 . Hartmam J, Fareed J, Mesemore H, et al : Post-infarct fibrinolytic activity : inhibition by Trasylol (abstr) . Am J Cardiol 37 :141, 1976 11 . Ruddy S, Gigli I, Austen KF: The complement system of man . I . Activation, control and products of the reaction sequence . N Engi J Med 287 :489-495, 1972 12 . Weissmann G: Lysosomal mechanisms of tissue injury in arthritis . N Engl J Med 286 :141-147, 1972

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