JACC Vol. 2, No 4October 1983:681-8

Role of Cellular Proteinases in Acute Myocardial InfarctionII. Influence of in Vivo Suppression of Myocardial Proteolysis by Antipain,Leupeptin and Pepstatin on Myocardial Infarct Size in the Rat

ROBERTO BOLLI, MD,* RICHARD O. CANNON, MD, EDITH SPEIR, BS,

ROBERT E. GOLDSTEIN, MD, FACC, STEPHEN E. EPSTEIN, MD, FACC

Bethesda, Maryland

681

Proteinases present in myocytesand leukocytes have beenthought to contribute to ischemic myoceUular death. Totest this concept, the effects of three potent proteinaseinhibitors (Ieupeptin, antipain and pepstatin) on myo­cardial infarct size were investigated. In the first exper­iment, rats received leupeptin intravenously (in either a"low" [10 mg/kg] or a "high" [40 mg/kg] dose) 10 min­utes before coronary artery occlusion; additional doseswere given 2, 4, 6 and 24 hours after occlusion. Lowand high doses were previously shown to reduce pro­teolysis in ischemic myocardium by 49 and 72%, re­spectively. Infarct size, measured histologically at 72hours, did not differ significantly among control (40 ±2% of left ventricular surface, n =30), low dose (37 ±5%, n = 21) and high dose (41 ± 3%, n = 23) groups.

In the second experiment, rats received a combina­tion of leupeptin (40 mg/kg), antipain (20 mg/kg) and

Several studies (1-7) seem to support the concept that cel­lular proteinases (both those present in myocytes and thosecarried by leukocytes) increase ischemic myocellular dam­age. This hypothesis could be tested by inhibiting the ac­tivity of cellular proteinases during the development of acutemyocardial infarction and by evaluating the effect of suchinhibition on the extent of ischemic damage, In a previousstudy (8), we noted that proteolysis in ischemic rat myo­cardium can be almost completely suppressed by in vivoadministration of leupeptin, antipain and pepstatin, three

From the Section on Experimental Physiology and Pharmacology, Car­diology Branch, National Heart, Lung, and Blood Instttute, Nattonal In­stitutes of Health. Bethesda, Maryland. This paper was presented in partat the 54th Scientific Sessions of the Amencan Heart Association Mcenng.Dallas, Texas, November 1981 Manuscript received December 28, 1982;revised manuscnpt received May 9, 1983, accepted May 13, 1983

*Present address and address for repnnts: Roberto Bolli, MD. Sectionof Cardiology, Baylor College of Medicme and The Methodist Hospital.6535 Fannin M.S. F-905, Houston, Texas 77030.

© 1983 by the Amencan College of Cardiology

pepstatin (5 mg/kg) intravenously before and 2 and 4hours after coronary occlusion. This treatment was pre­viously shown to decrease proteolysis in ischemic regionsby 88% at 15 minutes and by 72% at 6 hours of occlu­sion. Infarct size, determined at 6 hours by incubatingheart slices in triphenyl-tetrazolium-chloride, was notsignificantly different between control (44 ± 3% of leftventricular weight, n = 23) and treated (48 ± 2%, n= 30) animals. Thus, despite almost complete suppres­sion of proteolysis throughout the development of irre­versible injury, the ultimate extent of ischemic damagewas not reduced.

These results indicate that proteolysis occurring dur­ing the early phase of acute myocardial infarction doesnot in itself cause ischemic myocellular death. Accord­ingly, proteinase inhibition soon after coronary occlusiondoes not appear to be an infarct-sparing mechanism.

recently developed proteinase inhibitors (9). The presentstudy was undertaken to investigate whether in vivo phar­macologically induced suppression of proteolysis reducesthe size of acute myocardial infarction in rats.

MethodsExperiments were performed in male Sprague-Dawley

rats (230 to 270 g).Evaluation of the hemodynamic effects of proteinase

inhibitors. Preliminary experiments were carried out to as­sess whether the doses of ieupeptin, antipain and pepstatin,which suppress proteolysis in ischemic myocardium, sig­nificantly alter heart rate and systemic arterial pressure, twoimportant determinants of infarct size. To evaluate thehemodynamic effects of leupeptin, five rats were anesthe­tized with ether. The femoral artery was cannulated with aPE 10 catheter and mean arterial pressure measured with a

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682 BOLLI ET ALSUPPRESSION OF PROTEOLYSIS AND INFARCT SIZE

JACC Vol. 2. No.4October 1983:681 -8

ANT 12 milk. + ANTImilk. + ll'T 20milk.. PEP 1.25 milk.PEP 125 milk, PEP 161milk. + PEP 0.13milk. t•• •

tn03 mil.' . lPT 20milk.I.' I.'

+ + + +

TIME (min)

Figure 2. Response of heart rate (HR, open squares) and systolic(open circles), diastolic (closed squares) and mean (closed cir­cles) systemic arterial pressure (SAP) to four intravenous (i.v.)injections of proteinase inhibitors . Values are mean ± standarderror of the mean . ANT = antipain; LPT = leupeptin; PEP =pepstatin.

P23DbStatham transducer. Lead IIof the electrocardiogramwas continuously monitored. Heart rate and mean arterialpressure were recorded before and for 5 minutes after in­travenous injection of leupeptin (40 mg/kg in 0.25 ml ofnormal saline solution) (Fig. I).

A second experiment was performed to evaluate thehemodynamic effects of the combination of leupeptin, an­tipain and pepstatin . In this experiment, rats were studiedin the conscious state. Under ether anesthesia, a PE 50cannula was introduced into the femora l artery in six rats.Phasic and mean arterial pressures were measured with aP23Db Statham transducer. After recovery from anesthesia,recordings were taken under basal conditions and I and 10minutes after each intravenous injection of proteinase in­hibitors (Fig. 2). Each injection was performed under etheranesthesia, from which the animals recovered within 2 to3 minutes.

Evaluation of the effect of leupeptin on myocardialinfarct size. Before coronary artery occlusion, rats wererandomly given either 0.9% saline solution (control group),leupeptin in " low" dose or leupeptin in "high" dose. Be­cause the effect of leupeptin on proteolysis lasts at least 2hours (8), this agent was readministered at 2, 4 and 6 hoursafter coronary occlusion to achieve continuous inhibition of

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proteolysis during the early phase of myocardial infarctionwhen irreversible damage develops. Additional doses weregiven at 24 hours to prevent possible rebound increases inproteolysis. The details of the treatment are illustrated inFigure 3. The technique to produce coronary artery occlu­sion in rats has been described previously (8,10).

Seventy-two hours after coronary occlusion, animals werereanesthetized with ether. The heart was excised and fixedin [0% phosphate-buffered formalin for 24 hours. The ven­tricle was sectioned into four slices, each 2 to 2.5 mm thick,from apex to base in a plane parallel to the atrioventriculargroove. The slices were then embedded in paraffin. Sec­tions, 6p. thick, were cut from the middle of each slice (sothat they were evenly distributed along the apical-basal axis),mounted on 3 X 2 inch (7.6 x 5. [ ern) glass slides andstained with hematoxylin-eosin. The histologic sections ofall four slices from each heart were projected onto a screenat a lfl- fold magnification and the following measurementswere taken in each section: I) the area of normal and in­farcted left ventricular myocardium (determined by plani­metry), 2) the length of the arcs of the endocardial andepicardialcircumference of the left ventricle underlying thenormal and the infarcted myocardium, and 3) the averagethickness of normal and infarcted left ventricle. This lattervariable was determined by measuring thickness at six points,obtained by dividing the left ventricular cavity into six 600

angles (usuallythree pointswerewithin the normal andthreewithin the infarcted myocardium) .

l ACC Vol 2. No 4October 1983"68 1-8

BOLLI ET ALSUPPRESSION OF PROTEOLYSIS AND INFARCT SIZE

683

Saline 0.25 ml orleupeptin

40 mglkg10 0.25 rnl r,v.

Saline 0.25 ml orleupeptin 40 mg/kgin 0.25 ml i.m.

Saline 0.25ml orleupeptm 40 mg/kgin 0.25 ml i.m.

Saline 0.125ml orleupeptln 20 mg/kgIn 0.125 ml i.m,

1oCT'ON 1 1 1 SACr

CE

I ~ ~.-10 min 0 2 hrs 4 hrs 24 hrs 72 hrs

Saline 0.25ml orleupepnn10 mg/~g

in 0.25 ml i.v.

Saline 0.25 ml orleupeptin 10 mg/kgin 0.25 ml i.m.

Saline 0.25ml or Saline 0.25ml or Saline 0.125mlorleupeptin 10 mg/kg leupeptin 10 mg/kg leupeptln 5 mglkgIn 0.25ml i.m, In 0.25ml i.m. in 0.125 ml i.m.

!oCT'ON 1 1 1 1 SATcE

I ~ ~~-10 min 0 2 hrs 4 hrs 6 hrs 24 hrs 72 hrs

From these measurements . the size of the infarct wascalculated as percent of the total left ventricular mass andof the total left ventricular surface. The fanner detenni­nation was made by averaging the percent infarcted area ineach of the four slices. The latterwasestimatedby averagingthe lengths of the endocardial and epicardial arcs corre­sponding to infarcted myocardium. The resulting value wasexpressed as percent of the average between total endocar­dial and epicardialcircumference of each slice. The percentsof the four slices were then averaged to give the fraction ofthe left ventricular surface that was infarcted. Because thethickness of the infarct in the rat decreases after the firstday of coronaryocclusionwhilethe surface remains constant(10), infarct size at 3 days is underestimated whenexpressedas percent of left ventricular mass and is more accuratelyestimated when related to left ventricular surface (0). Forty­one hearts were analyzed by two independent observers; theinterobserver variability was less than 5% (correlation coef­ficient = 0.93).

The degree of thinning of the infarct was measured bytwo methods: I) in each slice the average thickness of normaland infarcted ventricle was obtained (see earlier) and theratio of normal to infarct was calculated; the ratios of thefour slices were then averaged; and 2) the thickness of the

Figure 3. Protocols of the experiment on the effect of leupeptinon myocardial infarct size. Upper panel. High dose of leupeptin;Lower panel. Low dose of leupeptin. i.m. = intramuscularly;i.v. = intravenously .

infarct was measured at its thinnest point in any of the foursections.

The only rats excluded from the final analysis were thosewith either no histologic evidence of necrosis or a smallsubepicardial area of necrosis at the level of the coronaryligature not extending to either of the adjacent slices (thusthe necrotic area was < 2.5 mm in the apical-basal axis).Lack of infarct in these animals was assumed to result fromfailure to occlude the coronary artery. Failure to occludethe coronary artery (as indicatedby lack of infarct) occurredin 9% of the control group, 17% of the low dose group and12% of the high dose group. These values were not statis­tically different.

Evaluation of the effect of leupeptin, antipain andpepstatin on myocardial infarct size. Because the com­bination of leupeptin, antipain and pepstatin appeared moreeffective than leupeptin alone in inhibiting proteolysis in

684 BOLLI ET AL.SUPPRESSION OF PROTEOLYSIS AND INFARCT SIZE

lACC Vol. 2. No 4October 1983 681-8

ischemic myocardium (8), we performed an additional ex­periment to evaluate the influence of this combination oninfarct size. Rats were randomized to control or treatedgroups. The treatment is illustrated in Figure 4. Six hoursafter coronary occlusion, rats were reanesthetized with ether.The heart was excised and cut into four slices in a planeparallel to the atrioventriculargroove. To delineateinfarctedmyocardium, slices were incubated for 45 minutes in 1%triphenyl-tetrazolium-chloride at 37°C . This agent stains vi­able dehydrogenase-containing myocardium dark red (II) ,while the necrotic myocardium remains unstained and ap­pears as a white-gray zone (Fig. 5).

Ultrastructural (12-14) and histochemical (15) studiesstrongly suggest that, in the rat model, the infarct virtuallyachieves its eventual size by 6 hours of coronary occlusion.To confirm this conclusion, in nine rats (five control andfour treated) the size of the infarcted region (determined bytriphenyl-tetrazolium-chloride) was compared with that ofthe ischemic region. The latter was delineated by intrave­nous injection of 1.0 ml of black ink just before sacrifice.Perfused myocardium was stained black while the ischemicregion remained pink. After the heart was cut into four

Figure 4. Protocol of the experiment on the effect of antipain(ANT), leupeptin (LPT) and pepstatin (PEP) on myocardial infarctsize. i.p. = intrapentoneaIly; i.v. = intravenously; TIC = tri­phenyl-tetrazolium-chloride.

transverse slices, the border between stained and unstainedmyocardium was marked with shallow cuts on each slice(Fig. 5). Slices were then incubated in triphenyl-tetrazo­lium-chloride as described previously.

After staining with triphenyl-tetrazolium-chloride, the rightventricle and the atria were excised and the slices weighed.Only rats that had no tetrazolium-negativemyocardiumwereexcluded from the final analysis. In these animals, failure toocclude the coronary artery was assumed to have occurred.The percent of control and treated rats excluded accordingto such criteria was similar (15 and 20%, respectively).

Infarct size was estimated by three techniques. In thefirst technique, slices were photographed, transparencieswere projected onto paper and necrotic and surviving por­tions were traced. Each area was measured by planimetry,and the infarct mass weight was expressed as a fraction ofthe weightof each slice. Total infarct weight was calculatedas percent of the left ventricle. In the animals in which theischemic region was delineated by ink injection, the weightof the ischemic region was calculated as percent of the leftventricle in a similar manner. In these animals, infarct weightwas also expressed as percent of the weight of the ischemicregion. In the second technique, infarct size was expressedas percent of the left ventricular surface by a procedureanalogous to that described for the leupeptin experiment.Finally, a third measure of infarct size was obtained byexcising the necrotic portion of each slice. Both infarctedand normal myocardium were weighed and infarct wet weightwas expressed as percentof total left ventricular wet weight.

SALINE 1. 2 ml orTOTAL: ANT 20 mg/kg + PEP 5 mg/kg + LPT 40 mg/kg

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I.V. In 0.4 ml tv.

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Incubation inTTC

lACC Vol. 2. No 4October 1983 681-8

BOLLI ET ALSUPPRESSION OF PROTEOLYSIS AND INFARCT SIZE

685

The reproducibility of planimetric estimate s of infarct sizewas excellent: the difference between values obtained bytwo observers was less than 5% (r = 0.95 , n = 2\).

Statistical analysis. All values are expre ssed as mean± standard error of the mean. Difference s between meanvalues of two groups were evaluated by unpaired Student's( tests. One way analysis of variance was used for com­parisons involving more than two groups. Incidence of eventswas analyzed by chi-square test using Yates' correction forcontinuity. Probability (p) values were considered signifi­cant if they were less than 0 .05 .

ResultsHemodynamic effects of proteinase inhibitors. Leu­

peptin in the high dose (40 mg/kg intravenously) produceda transient drop in blood pressure lasting less than 5 minutes(Fig . I) . Heart rate was not affected. Thu s, this agent didnot produce any significant sustained hemodynamic effect.Figure 2 depicts the hemodynamic effects of the combi­nation of leupeptin , antipain and pepstatin given in dosesidentical to those emplo yed in the infarct size study . Ratswere studied in the conscious state . The first set of mea­surements was taken under basal conditions and the secondunder ether anesthesia before the first injection . Ether low­ered blood pressure , an effect that might have contributed

to the fall in blood pressure observed I minute after eachinjection , but pressure returned to baseline levels within 10minutes after the injection. Heart rate did not change sig­nificantly . No cumul ative effect was produced by subse­quent injections, so that at the end of the experiment hemo­dynamic values were unchanged from the baseline levels.

Effects of leupeptin on mortality and infarct size (Ta­ble I). Overall mortality dur ing the 3 days of coronaryocclusion was not significantly differe nt among con trol, leu­peptin low dose and leupeptin high dose groups. Only onesubendocardial infarct was observed (in the control group) .Incidence of small infarcts ( < 20% of left ventricular sur­face) was also not significantly different in the three groups,excluding a selective beneficial effect of lcupeptin on smallerinfarcts. Infarct size, expressed as percent of either leftventricular weight or left ventricular surface, was similar in

Figure 5. A typicalexampleof myocardial infarctionasdelineatedby triphcnyl-tetrazo lium-chloride at 6 hours of occlusion. Heartsections are numbered 1 to 4 from the apex (a = apical aspect)to the base (b = basal aspect) of the heart. The white region istetrazolium-negative and, therefore, infarcted, while the dark reogion is tetrazolium-positive and viable. Pins indicate the bordersof the ischemic region, demarcated by intravenous injection ofblack ink immediately before sacrifice. Note that virtually all ofthe ischemic region is infarcted.

686 BOLLI ET AL.SUPPRESSION OF PROTEOLYSIS AND INFARCT SIZE

lACC Vo l 2. No 4October 1983:68J-8

contro l rats and in rats given low and high doses of leu­peptin . Even when rats that exhibited no infarct were in­cluded in the final analysis, no significant difference wasobserved between control and treated groups.

Leupeptin did not affect the degree of thinning of theinfarct, The ratio of the average thickness of normal tissueto that of the infarct was 1.35 ± 0.04 in the control group,1.29 ± 0.06 in the low dose group and 1.34 ± 0 .04 inthe high dose group. The thinnest point of the infarct mea­sured 0.70 ± 0.02 mm in control rats, 0.80 ± 0.05 mmin the low dose group and 0.75 ± 0.03 mm in the highdose group. Thus, suppressio n of proteolysis early in thecourse of myocardial infarction did not result in significantthinning or expansion of the infarct 3 days after occlusion.The degree of thinning, estim ated by measuring the thinnestpoint of the infarct , was significantly correlated with infarctsize exp ressed as percent of left ventricular surface (r =

0.63, p<O .OO I); this relation could be described by theequation: infarct size = 1.9 1 - 0.012 x thinnestmeasurement.

Effects of leupeptin, antipa in and pepstatin on mor­tality and infarct size. Mortality was similar in control andtreated groups (Table I). Infarct size , expressed either aspercent of left ventricular weight measured directly. as per-

cent of left ventricular weight estimated by planimetry or aspercent of left ventricular surface estimated by planimetry ,was not significantly differe nt between control and treatedgroups (Table I). Even when rats exhibiti ng no tetrazolium­negative (that is, infarcted) myocardium were included inthe final analysis , the results were similar in the two groups.

Infa rc t size vers us ischemic region after 6 hours ofcorona ry ar tery occlusion. In nine rats (five control andfour treated) sacrificed at 6 hours of ischemia, the infarc tedregion was determined by triphenyl-tetrazolium-chloride andthe ischemic region was delineated by intrave nous ink in­jection. The weight of these two regions, dete rmined byplanimetry, was almost identical in both control and treatedanimals (Fig . 5, Tabl e 2).

DiscussionModeling considerations. Two variations of the rat model

of acute myocardial infarct ion were used in this study . Inthe experiment in which leupeptin alone was administered,infarct size was determined by planimetry of histologic slides3 days after coronary artery occlusion. This technique, usedat 2 days of occlusion , has been able to detect infarct sizereduction by a variety of interventions, including reserpine

Table 1. Effects of Proteinase Inhibitors on Mortality and Myocardial Infarct Size

Mortality rate

Small infarcts«20 % of LV surface)

Infarct size(% of LV mass)

Infarct size(% of LV surface)

Leupeptm Alone

Leupeptin

Control "Low" Dose " High" Dose(n = 30) (n = 21) (n = 23)

10 (33 %) 7 (33'k) 9 (39%)

3 (10%) 4 ( 19%) 2 (9%)

34 ± 2 33 ± 4 36 ± 2

40 ± 2 37 ± 5 41 ± 3

p

NS

NS

NS

NS

Combination of Lcupcptm, Antipain and Pepstann

Control LPT + ANT + PEP(n = 23) (n = 30) p

Mortality rate 5 (22%) 7 (23%)

Infarct size 44 ± 3 48 ± 2 NS(% of LV mass, direct measurement)

Infarct size 48 ± 3 51 ± 2 NS(% of LV mass, planimetry)

Infarct size 46 ± 3 48 ± 2 NS(% of LV surface)

Values are mean ± standard error of the mean.ANT = antipain; LPT = leupeptin; LV = left ventricular; NS = not significant; p = probability (versus control); PEP = pepstatin.

JACC Vol 2. No 4October 1983'681- 8

BOLLI ET ALSUPPRESSION OF PROTEOLYSIS AND INFARCT SIZE

687

Table 2. Infarct Size Versus Ischemic Region After 6 Hours ofCoronary Artery Occlusion

Control LPT + ANT + PEP(n = 5) (n = 4) P

Infarct size 45 ± 2 44 ± 4 NS(% of LV mass)

Ischemic region 47 ± 4 46 ± 4 NS(% of LV mass)

Infarct size 95 ± 3 96 ± 3 NS(% of ischemic region )

Values are mean ± stand ard error of the mean Abbrevianons as inTable I .

(16), methylprednisolone (16), hydrocortisone (16), cobravenom factor (16), hyaluronidase (16), ibuprofen (17), ex­ercise (18) and labetalol (19) . We determined infarct sizeat 3 days to have an even better histologic demarcation ofthe necrotic region without the excessive thinning that hasbeen reported to occur at a later stage (10 ).

In the experiment with the combination of proteinaseinhibitors. infarct size was measured at 6 hours of coronaryartery occlusion, The rationale for assessing the extent ofnecrosis after 6 hours of ischemia was to rule out the pos­sibility that an early beneficialeffect of proteinase inhibitorson ischemic damage might have been missed at 3 days aftercoronary occlusion because of the progressive decrease intissue concentration of these agents. Such a possibility mustbe considered in view of the fact that leupeptin, antipainand pepstatin have short plasma half-lives (approximately2 hours) (20). We have previously shown (8) that the re­peated treatments used in this experiment result in contin­uous suppression of proteolysis, which is already evidentat 15 minutes and is still present at 6 hours of occlusion(time of sacrifice in this protocol). These experimental con­ditions appear extremely favorable for the detection of anybeneficial effect of proteolysis inhibition on ischemic dam­age, even if such an effect consisted of a mere delay ofnecrosis.

The validity of determining infarct size at 6 hours ofischemia requires that the process of myocellular death becomplete, or almost complete, at this time. Ultrastructural(12-14 ) and histochemical (15 ) studies indicate that theevolution of the infarct is very rapid in the rat model, andstrongly suggest that it is virtually complete by 6 hours ofcoronary occlusion. This speculation is substantiated by twoobservations in the present study: I) virtually all of theischemic region (determined by intravenous ink injection)was unstained by triphenyl-tetrazolium-chloride (and thuswas necrotic) at 6 hours both in control and in treated rats;and 2) in the control groups, the surface of the infarct, asdetermined by planimetry, was similar at 6 hours (46 ±

3% of the left ventricle) and at 3 days (40 ± 2%). Therefore,the lack of an infarct-sparing action of proteinase inhibitors

at 6 hours of ischemia indicates that these agents failed toaffect the eventual extent of ischemic damage.

The virtual identity between ischemic region and infarctin the rat model implies that infarct size should be relativelyuniform if coronary occlusion is performed at similar an­atomic sites. Indeed, the variability in infarct size was smallin both of our experiments, as indicated by the narrowstandard error of the mean in the control groups (± 2% inthe 3 day experiment and ± 3% in the 6 hour experiment).These considerations emphasize that even small reductionsin ischemic necrosis would have been detected in the presentstudy. The likelihood of a type II error in our conclusionsis minimal also because consistently negative results wereobtained with the two methods used to express infarct sizein the 3 day experiment (percent of left ventricular massand of left ventricular surface) and with the three methodsused in the 6 hour experiment (percent of left ventricularweight measured directly, of left ventricular weight esti­matedby planimetryand of left ventricularsurfaceestimatedby planimetry).

Significance of proteo lysis in acute myocardial in­farction. If cellular proteinases are biologically importantin the production of ischemic cell death, even by selectivedigestion of key substrates, inhibition of proteolysis in vivoduring the development of irreversible damage would beexpected to limit the extent of such damage. In the presentstudy, the administration of proteinase inhibitors in dosesthat markedly suppress proteolysis in ischemic myocardiumconsistently failed to reduce infarct size. Thus, no beneficialeffect on ischemic damage was produced by either low orhigh doses of leupeptin, which reduce proteolysis in isch­emic regions by 49 and 72%, respectively (8). Likewise,no appreciable reduction of ischemic necrosis was observedwith the combination of leupeptin, antipain and pepstatin,which inhibits protein breakdown almost completelythroughout the phase of acute coronary occlusion duringwhich myocellular death develops (- 88% at 15 minutesand - 72% at 6 hours [8]). The lack of any beneficialeffectof proteinase inhibitors cannot be ascribed to deleteriouschanges in heart rate or blood pressure, or both, becausethese agents did not significantly affect hemodynamic val­ues. The similarity in mortality rates between control andtreated groups excludes the possibility that suppression ofproteolysis improved survival after acute coronary occlu­sion, an effect that theoretically might interfere with thedetection of an infarct-sparing action.

The results of this study and those of our previous in­vestigations (8.21) indicate that the activity of cellular pro­teinases during acute ischemia docs not contribute in a bio­logicallyimportantwayto Irreversibledamage. Accordingly.the current concepts regarding the mechanisms of ischemicmyocellular death need to be reassessed. Activation of pro­teolytic enzymes (preexisting in the myocytes or carried byleukocytes accumulating in the ischemic zone) is unlikely

688 BOLLI ET ALSUPPRESSION OF PROTEOLYSIS AND INFARCT SIZE

lACC Vol 2, No 4October 1983:681-8

to be a significant causal factor in this process. The earlyrelease of lysosomal proteases whose significance for cel­lular integrity has been thus far debated (6), appears to bea consequence, rather than a cause , of ischemic damage.In light of our results , it seems important that future studiesof the mechanisms of ischemic myocellular death focus onother phenomena, such as mitochondrial and sarcolemmaldamage (5).

Implications for the mechanisms of action of infarct­sparing interventions. Our data have implications for themechanisms of action of several infarct-sparing interven­tions. The salutary effect s on ischemic myocardium of pro­teinase inhibitors, such as aprotinin (22), and of " lysoso­mal-stabili zing" agents, such as corticosteroids (23,24) andibuprofen (17,25), have been generally attributed to reduc­tion in the activit y of cellular, and particularly lysosomal ,proteinases (22-27). Because leukocytes contain abundantproteinases, a similar mechanism has been recently invokedto explain the observation that infarct size is markedly re­duced by depletion of white blood cells (7). The failure ofnear complete suppression of proteolys is to reduce ischemicdamage in the present study, however, implies that pro­teinase inhibition soon after coronary occlusion does notrepresent an important infarct-sparing mechanism. Accord­ingly, the beneficial effects of these interventions are likelyto depend on other actions (for example, inhibition of com­plement, reduced activity of phospholipases or reduced re­lease of activated oxygen products).

We gratefully acknowledge the technical assistance of Mary Slego wski ,

Shannon Smith, I. C Loggms, Joseph Larkin. Jr. and Nancy Nowlin andthe expert secretarial assistance of Carolyn M. Ferrante.

ReferencesI. Decker RS, Poole AR, Griffin EE. Dingle JT. Wildenthal K. Altered

distribution of lysosomal cathepsin D in ischemic myocardium. J ClinInvest 1977;59:911- 21.

2 Decker RS. Poole AR, Wildenthal K. Distribution of lysosomalcath­epsin D In normal. ischemic. and starved rabbit cardiac myocytesCirc Res 1980;46.485- 94.

3. Decker RS. Wildenthal K. Sequential lysosomal alterallons dunngcardiac ischemia II Ultrastructural and cytochemical changes. LabInvest 1978:38:662-73.

4. Lefer AM. Spath JA. Protective effects of protease inhibition in myo­cardial Ischemia. In: Cantin M, Harberland GL, Shells G, Selye B,ed. New Aspects of Trasylol Therapy: Experimental Myocardial In­farcnon. Stuttgart: Shattauer Verlag, 1975:289-331

5 Fozzard HA. How do cells die'? Circulation 1976;53(suppl 1)1-40-1.

6. Wildenthal K. Lysosomal alterations in ischemic myocardium: resultor cause of myocellular damage?J Mol Cell Cardiol 1978;I0:595-603.

7. Romson JL. Hook BG. Kunkel SL, Abrams GD, Schork MA. Luc-

chesi BR. Rcducnon of the extent of ischemic myocardial injury byneutrophil depletion in the dog. Circulation 1983;67:1016-23.

8. Bolli R, Cannon RO, Speir E, Goldstein RE, Epstein SE. Role ofcellular proteinases In acute myocardial infarction. I. Myocardialpro­teolysis In nonischemic and ischemic rat myocardium and the effectsof antipam, leupeptm, pepstatin and chymostatin administered In VIVO.

9. UmezawaH. Structure and activities of protease inhibitorsof microbialongm. Methods Enzymol 1976;45:678-95,

10. Fishbein MC. Maclean D, Maroko PRoExperimental Infarctionin therat. Am J Pathol 1978;90:57- 66.

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