Free Radical Biology & Medicine, Vol. 13, pp. 137-142, 1992 Printed in the USA. All fights reserved.
0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Original Contribution EFFECTS OF RECOMBINANT HUMAN EXTRACELLULARSUPEROXIDE DISMUTASE TYPE C ON MYOCARDIAL REPERFUSION INJURY IN ISOLATED COLD-ARRESTED RAT HEARTS
NOBUO HATORI,* PER-OVE S J ~ U I S T , ? STEFAN L. M A R K L U N D , ¢ S. K E N N E T H PEHRSSON,* and EARS RYDI~N* *Department of Cardiology, Karolinska Hospital, Stockholm, Sweden; tH/issle Research Laboratories, MOlndal, Sweden; and *Department of Clinical Chemistry, Umefi University, UmeL Sweden (Received 7 October 1991; Revised 21 January 1992; Re-revised and Accepted 9 March 1992) Abstract--The efficacy of recombinant human extracellular-superoxide dismutase type C (EC-SOD C) on myocardial reperfusion injury was explored in hypothermically arrested rat hearts, as was its site of action. Forty isolated working rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The hearts were arrested by the administration of 10 mL of cold perfusate at the onset of ischemia. At the same time, they were randomly assigned to one of five groups; A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate + bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L + heparin 50,000U/L; E: cold perfusate + heparin 50,000 U/L. Heparin was given to prevent binding of EC-SOD C to endothelial cell surfaces. Left ventricular function was studied before ischemia and at the end of reperfusion. Percent recovery of maximal left ventricular dP/dt after reperfusion was more pronounced in group B ( 109 _ 24%; p < .05) than in groups A (42 _+40%), C (47 + 36%), D (44 _+33%) and E (58 _+25%). Likewise, percent recovery of the double product (heart rate × systolic left ventricular pressure) was better in group B ( 104 _+ 18%; p < .05) than in the other groups (A: 47 _+_37%, C: 49 _+ 36%, D: 50 _+ 35%, E: 69 +_ 31%). Compared to the preischemic level, creatine kinase increased significantly in the coronary effluent after reperfusion in groups A, C, D, and E, but not in group B. The results suggest that EC-SOD C, which attaches to the endothelial cell surfaces, might be particularly effective as protection against myocardial reperfusion injury when given together with cardioplegic solution. Keywords--Myocardial ischemia, Reperfusion injury, Superoxide dismutase, Rat heart, Cardioplegia, Free radicals
any protective effect on reperfusion injury if given during ischemia o n l y . 1°-14 Extracellular-superoxide dismutase (EC-SOD) type C has a unique property since it binds strongly to heparan sulfate proteoglycan in the glycocalyx of cell surfaces. 15-17 This binding may, however, be blocked by the administration of heparin. ~s,~9CuZn-SOD, on the other hand, lacks such binding capacity. The aim of this study is to determine the efficacy of SOD in preventing myocardial reperfusion injury when administered together with cardioplegia during the ischemic period and to compare the efficacy of EC-SOD C with that of CuZn-SOD.
INTRODUCrlON
Free oxygen radicals are considered of importance, for post-ischemic myocardial dysfunction. Many studies documented that the free oxygen radical scavenger, superoxide dismutase (SOD), may enhance functional recovery of the reversibly injured myocardium after ischemia.l-5 Free oxygen radical generation studied by electron spin resonance spectroscopy demonstrates the presence of free oxygen radicals very early during myocardial reperfusion.6-8Due to the large molecular weight of SOD (for instance, in the form of CuZn-SOD), this compound does not penetrate cell membranes. Moreover, it does not easily attach to cell surfaces. 9 It is controversial whether CnZn-SOD has
MATERIALS AND METHODS
Forty male Sprague-Dawley rats (300-350 g) were anesthetized with intramuscular fluanisone (1 mg/ 100 g) and fentanyl citrate (0.02 mg/kg). The hearts
Address correspondence to: Lars Ryd6n, MD, Department of Cardiology, Thoracic Clinics, Karolinska Hospital, S- 104 01 Stockholm, Sweden. 137
N. HATORI eta/.
138 LANGENDORFF
15 min
WORKING HEART
ISCHEMIA
10 min
GROUP
Fig. I. Experimental protocol. EC-SOD C bovine C u Z n = superoxide dismutase.
LANGENDORFF
WORKING HEART
10 min
30 min
t
A : COLD PERFUSATE (CP) 10 ml B : CP+EC-SOD C 10.4 mg/L (30,000 U/L) C : CP+CuZn-SOD 7.5 mg/L (30,000U/L) D : CP+EC-SOD C 10.4 mg/L (30,000U/L) +HEPARIN (50,000 U/L) E : CP+HEPARIN (50,000U/L) recombinant h u m a n extracellular-superoxide dismutase type C; C u Z n - S O D -
were rapidly excised and chilled in ice-cold saline. The aorta was cannulated to initiate retrograde perfusion according to the Langendorff technique using Krebs-Hanseleit bicarbonate buffer (pH 7.4) at a hydrostatic pressure of 7.8 kPa (80 cm H20). The buffer is composed of (in mmol/L) NaC1 118, NaHCO3 25, KC1 4.7, KH2PO 4 1.2, MgSO4 1.2, CaCI2 2.5, ethylenediaminetetraacetic acid (EDTA) 0.5, and glucose 11.1. The perfusion medium was equilibrated with 95% oxygen and 5% carbon dioxide. The left atrium was cannulated within 15 min ofperfusion, The Langendorff mode (a nonworking heart preparation) was then converted to a working system by switching the supply of perfusate from the aorta to the left atrial cannula (filling pressure 1.47 kPa = 15 cm H20). Under this condition, the left ventricle spontaneously ejects through the aortic cannula against a hydrostatic pressure of 7.8 kPa (80 cm H20). The entire system was jacketed in water to maintain a temperature of 37°C. A needle cannula (diameter 0.6 mm) was inserted into the left ventricular cavity and connected to a pressure transducer (Statham model P50, Gould Inc., CA). Recordings were made on a multichannel physiologic recorder (Grass Instruments, MA). The left ventricular dP/dt was obtained by electric derivation of the left ventricular pressure. The heart rate was derived from the pressure recordings. Coronary flow rate was measured by timed collection of the perfusate.
Experimental protocol Preischemic data were obtained after a stabilization period of 10 min in the working heart mode (See Fig. 1 for the experimental protocol.) The hearts were then subjected to a 30-min period of global ischemia by clamping the aortic and left atrial cannulas. Infusion of 10 mL of cold perfusate directly into the coronary circulation (through a side arm of the aortic cannula from a reservoir located 80 cm above the heart) ascertained cardiac arrest at the time of clamping.
The hearts were randomly assigned to one of five groups, receiving A (n = 8): cold perfusate only; B (n = 8): cold perfusate containing EC-SOD C (10.4 mg/ L:30,000 U/L); C (n = 8): cold perfusate containing bovine CuZn-SOD (7.5 mg/L:30,000 U/L); D (n = 8): cold perfusate containing EC-SOD C (10.4 mg/ L:30,000 U/L) and heparin (50,000 IU/L); and E (n = 8): cold perfusate containing heparin (50,000 IU/L). Thus, SOD was administered only at the onset ofischemia. During ischemia the heart was still surrounded by the water-jacketed chamber maintaining temperature at 37°C. After 30 min ofischemia, a 10-min period of retrograde perfusion was started before switching the system to the working mode. Hemodynamics were recorded 30 min later.
EC-SOD and CuZn-SOD The specific activities in the xanthine oxidase-cytochrome c assay2° of the EC-SOD C (Symbicom AB, Sweden) and the bovine CuZn-SOD (BoehringerMannheim Gmbh, Germany) preparations were 2885 and 4000 units per milligram, respectively. Thus, the gravimetric concentrations used in the experiment correspond to equal enzymatic activities.
Biochemical analysis The coronary effluent was collected during 1 min just prior to ischemia and after 30 min of reperfusion in the working mode. Creatine kinase (CK) activity in the coronary effluent was measured in a Cobas Bio centrifugal analyzer (Hoffman-La Roche, Switzerland) using a commercially available kit (BoehringerMannheim, Germany, cat. no. 181188).
Data analysis The data are presented as mean + standard deviation. They were analyzed to see whether the assumptions for parametric testing (normal distribution and equal variances) were met. As the data did not satisfy
EC-SOD and reperfusion injury
139
Table 1. Hemodynamic Parameters During the Preischemic Period (n = 8 in each group)
GroupA GroupB Group C Group D Group E
Heart Rate (HR) (beat/min)
Systolic Left Ventricular Pressure (SVP) (mm Hg)
Double Product (HR × SVP) (mm Hg × beat × 10-2/min)
Left Ventricular dP/dt (mm Hg/s)
Coronary Flow (mL/min)
259+_34 271_+67 289 _+ 65 235 _+ 73 239 _+ 75
111+27 100_+19 108 _+ 29 115 _+ 28 106 + 25
292_+ 92 268-+ 67 306 -+ 86 268 _+ 103 254 _+ 100
4062_+ 1159 3250_+ 841 3900 _+ 1021 3462 _+ 643 3400 _+ 938
21_+11 19-+ 4 20 -+ 6 17 _+ 6 18 _+ 6
Group A: Cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate + bovine CuZn-SOD 10.4 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L) + heparin 50,000 U/L; E: cold perfusate + heparin 50,000 U/L.
these assumptions, further testing was performed by nonparametric procedures. 21'22Intergroup differences of the variables were examined using Kruskall-Wallis's test with a 0.05 alpha level. A significant result prompted a multiple comparison test (Fisher's least significant test) for possible pairwise comparisons. Differences within each group were studied by applying the Wilcoxon signed rank test. An alpha level of 0.05 was accepted as significant. All p values are twotailed. RESULTS
Hemodynamics There were no differences between the groups in any of the recorded parameters prior to the induction of ischemia (Table 1). Hemodynamic parameters after reperfusion are presented as the percent recovery of preischemic values (Table 2). After 30 min ofreperfusion in the working mode, the recovery of the systolic left ventricular pressure, the double product, and the left ventricular dp/dt was complete (around 100%) in group B compared to groups A, C, D, and E (p < .05). Coronary flow recovered somewhat better in group B than in the other groups, but this difference did not reach statistical significance (p = .077).
CK release during reperfusion The CK release to the coronary effluent is reported in Table 3. In hearts from groups A, C, D, and E, CK
release increased after 30 min reperfusion compared to the preischemic levels. The release in group B did not differ significantly from preischemia. DISCUSSION
EC-SOD is the major SOD isoenzyme in the extracellular space in vivo.18'23'24 Nevertheless, most investigations regarding the effects of SOD on myocardial ischemia/reperfusion have been studied with the cytosolic CuZn-SOD because this isoenzyme is more easily available. EC-SOD, a secretory tetrameric copper and zinc-containing glycoprotein, may be divided into three fractions, defined by the affinity to hepar i n : z5'26 A, without affinity; B, with weak affinity; and C, with relatively strong affinity. The physiologic ligand of EC-SOD C is the heparin analog heparan sulfate proteoglycan, 15-17 which exists in the glycocalyx of cell surfaces and in the connective tissue matrix. EC-SOD C forms an equilibrium between the fluid phase and endothelial surfaces in the vasculat u r e 15'18'19'24 and cell surfaces and connective tissue matrix in tissues. 17Heparin releases EC-SOD C to the fluid phase by competing efficiently with heparan sulfate for the heparin-binding domain of the enzyme. EC-SOD C bound both to heparin and to heparan sulfate retains essentially all enzymatic activity.27 The physiologic role and importance of EC-SOD still remain unclarified. Johansson 28 reported that the combination of recombinant human EC-SOD C and catalase reduced oxygen free radicals detectable by
Table 2. Percent Recovery of Hemodynamic Parameters in the 30-Min Reperfused Hearts (n = 8 in each group)
Group Group Group Group Group
A B C D E
Heart Rate (HR)
Systolic Left Ventricular Pressure (SVP)
Double Product (HR × SVP)
Left Ventricular dP/dt
Coronary Flow
85 _+ 30% 106 .+ 16% 81 -+ 26% 93+21% 100 + 38%
48 _+ 30% 100 + 22%* 54 _+ 33% 50+31% 66 + 24%
47 _+ 37% 104 _+ 18%* 49 _+ 36% 50+35% 69 + 31%
42 _+ 40% 109 +_ 24%* 47 _+ 36% 44+_33% 58 + 25%
53 _+ 37% 87 _+ 19% 59 _+ 34% 45+_31% 59 +- 22%
Group A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate + bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L) + heparin 50,000 U/L; E: cold perfusate + heparin 50,000 U/L. *p < .05 versus groups A, C, D, and E.
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N. HATORI el a/.
Table 3. Creatine Kinase Activity in the Coronary Effluent (n = 8 in each group)
Group A GroupB Group C Group D Group E
Preischemia (U/L)
30 Min after Reperfusion (U/L)
8+ 8 9_+6 3 +_ 4 3 +_ 3 5+ 5
239 _+ 48* 27+_ 31 99 +_ 198" 139 + 180" 142 + 362*
Group A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate + bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L) + heparin 50,000 U/L; E: cold perfusate + heparin 50,000 U/L. *p < .05 versus preischemia.
spin trap generation more effectively than CuZnSOD in the reperfused isolated rat heart. SOD was administered prior to ischemia and throughout the reperfusion period. The effect of EC-SOD C on the recovery of myocardial function after ischemia and reperfusion has been studied by SjOquist et al. 29 ECSOD C was administered throughout the experiment, and the action site was not detected. The main finding of this study is that recombinant human EC-SOD C, when given together with cardioplegia, improved the recovery of myocardial function and prevented CK release after global ischemia. There were no effects of CuZn-SOD or EC-SOD C administered together with heparin in a dose efficiently preventing binding to endothelial cell surfaces. Is A very likely explanation for the protective effect on hearts in group B receiving only EC-SOD C is that EC-SOD C could adhere to the vascular walls and be active on that site during the reperfusion period. In contrast, CuZn-SOD in group C and EC-SOD C in group D (administered with heparin in a concentration totally inhibiting EC-SOD C binding to vascular walls) were washed away directly at the start ofreperfusion by the reperfusion medium. In these two groups, as in group A (control) and in group E (heparin only), no protective enzyme was present in the vasculature of the heart a few seconds after the start of reperfusion. The rat heart should constitute an adequate model for exploring the efficacy of EC-SOD C. The effect of native EC-SOD C in rats can be eliminated. Unlike other investigated mammals, it lacks the high heparin affinity fraction C of EC-SOD. 18 Nevertheless, ECSOD C binds to the vascular endothelium in the rat because the receptor for EC-SOD C, heparan sulfate, ~5 does not require any specific structure of sulfated polysaccharides to attach. The precise mechanisms of oxygen radical production in the heart are not fully understood. Several sources have been p r o p o s e d , 3°-36 including the xanthine-xanthine oxidase system, oxidized forms of myoglobin, autooxidation of substances reducing isch-
emia, injured mitochondria leaking electrons, activated prostaglandin and leukotriene synthesis with superoxide radicals formed as a byproduct, and activated phagocytic leukocytes. The latter source should be of minor importance since the hearts in this study were perfused with Krebs-Henseleit buffer. However, other cell types, such as endothelial cells, 37-39 fibroblasts, 4° and B-lymphocytes,41 may also be stimulated to secrete significant amounts of superoxide. The large SOD molecules do not penetrate to any significant extent. Interception with the superoxide radicals should therefore take place in the extracellular space. Superoxide radicals are secreted from some of the potential sources. Superoxide generated intracellulary may also cross the plasma membranes through anion c h a n n e l s . 35,42
Superoxide dismutase converts superoxide anions into hydrogen peroxide, which is further reduced to water by catalase or glutathione peroxidase. The excellent myocardial recovery following EC-SOD C without catalase indicates that the ischemic myocardium contains sufficient amounts of catalase and/or glutathione peroxidase. An alternative explanation would be that hydrogen peroxide is less important for induction of the reperfusion injury.43 The hydroxyl radical is the most toxic of the oxygen-generated radicals. 44 In the present context, it may be formed b y a transition metal ion-catalyzed reaction between hydrogen peroxide and superoxide. 45 Since superoxide can release iron in reactive form from ferritin,46 enhanced superoxide dismutase activity may protect b y reducing the amount of superoxide serving as a precursor for hydroxyl radicals and by reducing the availability of the transitional metal ion catalyst. An alternative explanation is that superoxide radicals cause degradation of the endothelium-derived relaxing factor (EDRF). 47'48 Generation of superoxide radicals during the early reperfusion may therefore impair the endothelium-dependent vasorelaxation. An intriguing mechanism of action for EC-SOD C would therefore be protection against loss ofendothelium-dependent coronary vessel relaxation at the start of reperfusion, thereby diminishing a no-reflow phenomenon. The tendency to better coronary flow in group B may be indicative of such a mechanism. The product of the reaction between superoxide and EDRF, which apparently is identical to nitric o x i d e , 49 is the highly cytotoxic peroxynitrite. 5°'5J The protection conferred by SOD may thus partly be due to reduced peroxynitrite formation. Production of free oxygen radicals requires oxygen as a substrate but already occurs at low oxygen tensions. 52 Bolli et al. 7 reported on low-grade oxygen free-radical production in the ischemic canine myocardium, detected by spin trapping. Arroyo et al. 53
EC-SOD and reperfusion injury
described oxygen radical generation in the ischemic rat heart. However, oxygen may be supplied by collateral blood flow in canines, and the low-flow global ischemic preparation used by Arroyo et al. may have provided enough oxygen for free radical production during the period of ischemia. Henry et alJ 4 studied ischemic isolated rat hearts, which were continuously monitored for free radical generation by means of enhanced chemiluminescence. Radicals were already produced during the very early reperfusion period but not during ischemia. Superoxide dismutase attenuated the elevation of free radicals during reperfusion. The lack of effect of SOD without attachment to cell surfaces (CuZn-SOD and EC-SOD with heparin) indicates that radicals formed during the ischemic period were of minor importance in the present experimental model. Beneficial effects of CuZn-SOD have been reported when the administration of this compound includes not only the ischemic but also the reperfusion period. '-5 Some localization of CuZn-SOD to myocardial structures seems necessary to prevent reperfusion injury when the drug is given prior to reperfusion only. 5s Taken together, these findings suggest that SOD present in the interstitium or bound to extracellular surfaces is important for the protective effect. When polylysine is substituted for CuZn-SOD to facilitate association with negatively charged cell membranes, the ability of the enzyme to protect activated polymorphonuclear leukocytes against self-inactivation is highly potentiated, s6 The cell-membrane-associated SOD of the bacterium Nocardia asteroides confers efficient protection against activated polymorphonuclear leukocytes. 57 In the light of these observations, the present findings strongly suggest that binding of SOD to cellular surfaces might be of particular importance for the cellular protection against superoxide radicals. Fixation of SOD to the endothelial surfaces in the ischemic myocardium may be an important tool by enhancing the possibility of protecting jeopardized myocardium from subsequent reperfusion injury. A limitation of the study is that CK release was analyzed only prior to and 30 min after reperfusion. Still, and since a recirculating system was used, the CK release during the reperfusion period should be reasonably well reflected by the data presented although not truly representative of the total release of the enzyme. Furthermore, the results of the CK release are consistent with the functional data. Further investigations may be needed to determine the exact effect of SOD on CK release. Since oxy radicals may interfere with CK when measured by an enzymatic assay, an immunological technique may then be preferred.
141
Acknowledgement - - This work has been supported by grants from the Swedish Heart and Lung Foundation and from Arbetsmarknadens FOrs~kringsaktiebolag, Stockholm, Sweden.
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38. Ratych, R. E.; Chuknyiska, R. S.; Bulkley, G. B. The primary localization of free radical generation after anoxia/reoxygenation in isolated endothelial cells. Surgeo~ 102:122-131 : 1987. 39. G0rOg, P.: Pearson, J. D.: Kakkar, V. V. Generation of reactive oxygen metabolites by phagocyting endothelial cells. Atherosclerosiv 72:19-27: 1988. 40. Meier, B.: Cross, A. R.: Hancock, J. T.: Kaup, F. J.: Jones, O. T. G. Identification ofa superoxide-generating NADPH oxidase system in human fibroblasts. Biochem. J. 275:241-245: 1991. 41. Maly, F. E.: Cross, A. R.; Jones, O. T. G.; Wolf-Vorbeck, G.: Walker, C.; Dahinden, C. A.: De Weck, A. L. The superoxide generating system of B cell lines. Structural homology with the phagocytic oxidase and triggering via surface lg. J. lmmunol. 140:2334-2339: 1988. 42. Lynch, R. E.: Fridovich. 1. Permeation of the erythrocyte stoma by superoxide radical..I. Biol. Chem. 253:4697-4699: 1978. 43. Werns, S. W.: Shea, M. J.; Driscoll, E. M.: Cohen, C.: Abrams, G. D.; Pitt, B.: Lucchesi, B. R. The independent effects of oxygen radical scavengers on canine infarct size. Reduction by superoxide dismutase but not catalase. Circ. Res. 56:895-898: 1985. 44. Del Maestro, R. F. An approach to free radicals m medicine and biology. Acta Physiol. S t a n d 492(Suppl.): 153-168; 1980. 45. McCord, J. M.: Day, E. D. Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEBS Lett. 86:139-153: 1978. 46. Biemond, P.: van Eijk, H. G.: Sweak, A. J. G.: Koster, J. F. Iron mobilization from ferritin by superoxide derived from stimulated polymorphonuclear leukocytes. J. ('/in. Inves't. 73:15761579: 1984. 47. Rubanyi, G. M.: Vanhoutte, P. M. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am. J Phy.s'iol 250:H822-H824: 1986. 48. Gryglewski, R. J.: Palmer, R. M. J.: Moncada, J. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320:454-456; 1986. 49. Palmer, R. M. J.: Ferrige, A. G.: Moncada, S. The release of nitric oxide by vascular endothelial cells accounts for the activity of EDRF. Nature 327:524-526: 1987. 50. Beckman, J. S.; Beckman, T. W.: Chen, J.; Marshall, P. A.: Freeman, B. A. Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc. Nail. ~tcad. Sci. USA 87:1620-1624: 1990. 51. Radi, R.; Beckman, J. S.; Bush, K. M.: Freeman, B. A. Peroxynitrite oxidation of sulfhydryls. J. Biol. ('hem. 266:42444250; 1991. 52. Southorn, P. A.: Powis, G. Free radicals in medicine II. Involvement in human disease. Mayo ~7in. Proc. 63:390-408: 1988. 53. Arroyo, C. M.; Kramer, J. H.: Dickens, B. F.; Weglicki, W. B. Identification of free radicals in myocardial ischemia/reperfusion by spin trapping with nitrone DMPO. FEBS Lelt. 221:101-104: 1987. 54. Henry, T. D.: Archer, S. L.: Nelson, D.: Wet, E. K.: From, A. H. k. Enhanced chemiluminescence as a measure of oxygen-derived free radical generation during ischemia and reperfusion. Cipv. Res. 67:1453-61; 1990. 55. Omar, B. A.: McCord, J. M. Interstitial equilibration of superoxide dismutase correlates with its protective effect in the isolated rabbit heart. ,L Mol. Cell Cardiol. 23:149-159:1991. 56. Salin, M, L.: McCord, J. M. In: Michelson, A. M.: McCord, J. M.: Fridovich, I.; eds. Superoxide andsuperoxide dismutase New York: Academic Press; 1977:257-270. 57. Beaman, B. L.: Black, C. M.; Doughty, F.; Beaman, L. Role of superoxide dismutase and catalase as determinants of pathogenicity of Nocardia asteroids: Importance in resistance to microbicidal activities of human polymorphonuclear neutrophils. ln[~ct, lmmun. 47:135-140; 1985.
0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Original Contribution EFFECTS OF RECOMBINANT HUMAN EXTRACELLULARSUPEROXIDE DISMUTASE TYPE C ON MYOCARDIAL REPERFUSION INJURY IN ISOLATED COLD-ARRESTED RAT HEARTS
NOBUO HATORI,* PER-OVE S J ~ U I S T , ? STEFAN L. M A R K L U N D , ¢ S. K E N N E T H PEHRSSON,* and EARS RYDI~N* *Department of Cardiology, Karolinska Hospital, Stockholm, Sweden; tH/issle Research Laboratories, MOlndal, Sweden; and *Department of Clinical Chemistry, Umefi University, UmeL Sweden (Received 7 October 1991; Revised 21 January 1992; Re-revised and Accepted 9 March 1992) Abstract--The efficacy of recombinant human extracellular-superoxide dismutase type C (EC-SOD C) on myocardial reperfusion injury was explored in hypothermically arrested rat hearts, as was its site of action. Forty isolated working rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The hearts were arrested by the administration of 10 mL of cold perfusate at the onset of ischemia. At the same time, they were randomly assigned to one of five groups; A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate + bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L + heparin 50,000U/L; E: cold perfusate + heparin 50,000 U/L. Heparin was given to prevent binding of EC-SOD C to endothelial cell surfaces. Left ventricular function was studied before ischemia and at the end of reperfusion. Percent recovery of maximal left ventricular dP/dt after reperfusion was more pronounced in group B ( 109 _ 24%; p < .05) than in groups A (42 _+40%), C (47 + 36%), D (44 _+33%) and E (58 _+25%). Likewise, percent recovery of the double product (heart rate × systolic left ventricular pressure) was better in group B ( 104 _+ 18%; p < .05) than in the other groups (A: 47 _+_37%, C: 49 _+ 36%, D: 50 _+ 35%, E: 69 +_ 31%). Compared to the preischemic level, creatine kinase increased significantly in the coronary effluent after reperfusion in groups A, C, D, and E, but not in group B. The results suggest that EC-SOD C, which attaches to the endothelial cell surfaces, might be particularly effective as protection against myocardial reperfusion injury when given together with cardioplegic solution. Keywords--Myocardial ischemia, Reperfusion injury, Superoxide dismutase, Rat heart, Cardioplegia, Free radicals
any protective effect on reperfusion injury if given during ischemia o n l y . 1°-14 Extracellular-superoxide dismutase (EC-SOD) type C has a unique property since it binds strongly to heparan sulfate proteoglycan in the glycocalyx of cell surfaces. 15-17 This binding may, however, be blocked by the administration of heparin. ~s,~9CuZn-SOD, on the other hand, lacks such binding capacity. The aim of this study is to determine the efficacy of SOD in preventing myocardial reperfusion injury when administered together with cardioplegia during the ischemic period and to compare the efficacy of EC-SOD C with that of CuZn-SOD.
INTRODUCrlON
Free oxygen radicals are considered of importance, for post-ischemic myocardial dysfunction. Many studies documented that the free oxygen radical scavenger, superoxide dismutase (SOD), may enhance functional recovery of the reversibly injured myocardium after ischemia.l-5 Free oxygen radical generation studied by electron spin resonance spectroscopy demonstrates the presence of free oxygen radicals very early during myocardial reperfusion.6-8Due to the large molecular weight of SOD (for instance, in the form of CuZn-SOD), this compound does not penetrate cell membranes. Moreover, it does not easily attach to cell surfaces. 9 It is controversial whether CnZn-SOD has
MATERIALS AND METHODS
Forty male Sprague-Dawley rats (300-350 g) were anesthetized with intramuscular fluanisone (1 mg/ 100 g) and fentanyl citrate (0.02 mg/kg). The hearts
Address correspondence to: Lars Ryd6n, MD, Department of Cardiology, Thoracic Clinics, Karolinska Hospital, S- 104 01 Stockholm, Sweden. 137
N. HATORI eta/.
138 LANGENDORFF
15 min
WORKING HEART
ISCHEMIA
10 min
GROUP
Fig. I. Experimental protocol. EC-SOD C bovine C u Z n = superoxide dismutase.
LANGENDORFF
WORKING HEART
10 min
30 min
t
A : COLD PERFUSATE (CP) 10 ml B : CP+EC-SOD C 10.4 mg/L (30,000 U/L) C : CP+CuZn-SOD 7.5 mg/L (30,000U/L) D : CP+EC-SOD C 10.4 mg/L (30,000U/L) +HEPARIN (50,000 U/L) E : CP+HEPARIN (50,000U/L) recombinant h u m a n extracellular-superoxide dismutase type C; C u Z n - S O D -
were rapidly excised and chilled in ice-cold saline. The aorta was cannulated to initiate retrograde perfusion according to the Langendorff technique using Krebs-Hanseleit bicarbonate buffer (pH 7.4) at a hydrostatic pressure of 7.8 kPa (80 cm H20). The buffer is composed of (in mmol/L) NaC1 118, NaHCO3 25, KC1 4.7, KH2PO 4 1.2, MgSO4 1.2, CaCI2 2.5, ethylenediaminetetraacetic acid (EDTA) 0.5, and glucose 11.1. The perfusion medium was equilibrated with 95% oxygen and 5% carbon dioxide. The left atrium was cannulated within 15 min ofperfusion, The Langendorff mode (a nonworking heart preparation) was then converted to a working system by switching the supply of perfusate from the aorta to the left atrial cannula (filling pressure 1.47 kPa = 15 cm H20). Under this condition, the left ventricle spontaneously ejects through the aortic cannula against a hydrostatic pressure of 7.8 kPa (80 cm H20). The entire system was jacketed in water to maintain a temperature of 37°C. A needle cannula (diameter 0.6 mm) was inserted into the left ventricular cavity and connected to a pressure transducer (Statham model P50, Gould Inc., CA). Recordings were made on a multichannel physiologic recorder (Grass Instruments, MA). The left ventricular dP/dt was obtained by electric derivation of the left ventricular pressure. The heart rate was derived from the pressure recordings. Coronary flow rate was measured by timed collection of the perfusate.
Experimental protocol Preischemic data were obtained after a stabilization period of 10 min in the working heart mode (See Fig. 1 for the experimental protocol.) The hearts were then subjected to a 30-min period of global ischemia by clamping the aortic and left atrial cannulas. Infusion of 10 mL of cold perfusate directly into the coronary circulation (through a side arm of the aortic cannula from a reservoir located 80 cm above the heart) ascertained cardiac arrest at the time of clamping.
The hearts were randomly assigned to one of five groups, receiving A (n = 8): cold perfusate only; B (n = 8): cold perfusate containing EC-SOD C (10.4 mg/ L:30,000 U/L); C (n = 8): cold perfusate containing bovine CuZn-SOD (7.5 mg/L:30,000 U/L); D (n = 8): cold perfusate containing EC-SOD C (10.4 mg/ L:30,000 U/L) and heparin (50,000 IU/L); and E (n = 8): cold perfusate containing heparin (50,000 IU/L). Thus, SOD was administered only at the onset ofischemia. During ischemia the heart was still surrounded by the water-jacketed chamber maintaining temperature at 37°C. After 30 min ofischemia, a 10-min period of retrograde perfusion was started before switching the system to the working mode. Hemodynamics were recorded 30 min later.
EC-SOD and CuZn-SOD The specific activities in the xanthine oxidase-cytochrome c assay2° of the EC-SOD C (Symbicom AB, Sweden) and the bovine CuZn-SOD (BoehringerMannheim Gmbh, Germany) preparations were 2885 and 4000 units per milligram, respectively. Thus, the gravimetric concentrations used in the experiment correspond to equal enzymatic activities.
Biochemical analysis The coronary effluent was collected during 1 min just prior to ischemia and after 30 min of reperfusion in the working mode. Creatine kinase (CK) activity in the coronary effluent was measured in a Cobas Bio centrifugal analyzer (Hoffman-La Roche, Switzerland) using a commercially available kit (BoehringerMannheim, Germany, cat. no. 181188).
Data analysis The data are presented as mean + standard deviation. They were analyzed to see whether the assumptions for parametric testing (normal distribution and equal variances) were met. As the data did not satisfy
EC-SOD and reperfusion injury
139
Table 1. Hemodynamic Parameters During the Preischemic Period (n = 8 in each group)
GroupA GroupB Group C Group D Group E
Heart Rate (HR) (beat/min)
Systolic Left Ventricular Pressure (SVP) (mm Hg)
Double Product (HR × SVP) (mm Hg × beat × 10-2/min)
Left Ventricular dP/dt (mm Hg/s)
Coronary Flow (mL/min)
259+_34 271_+67 289 _+ 65 235 _+ 73 239 _+ 75
111+27 100_+19 108 _+ 29 115 _+ 28 106 + 25
292_+ 92 268-+ 67 306 -+ 86 268 _+ 103 254 _+ 100
4062_+ 1159 3250_+ 841 3900 _+ 1021 3462 _+ 643 3400 _+ 938
21_+11 19-+ 4 20 -+ 6 17 _+ 6 18 _+ 6
Group A: Cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate + bovine CuZn-SOD 10.4 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L) + heparin 50,000 U/L; E: cold perfusate + heparin 50,000 U/L.
these assumptions, further testing was performed by nonparametric procedures. 21'22Intergroup differences of the variables were examined using Kruskall-Wallis's test with a 0.05 alpha level. A significant result prompted a multiple comparison test (Fisher's least significant test) for possible pairwise comparisons. Differences within each group were studied by applying the Wilcoxon signed rank test. An alpha level of 0.05 was accepted as significant. All p values are twotailed. RESULTS
Hemodynamics There were no differences between the groups in any of the recorded parameters prior to the induction of ischemia (Table 1). Hemodynamic parameters after reperfusion are presented as the percent recovery of preischemic values (Table 2). After 30 min ofreperfusion in the working mode, the recovery of the systolic left ventricular pressure, the double product, and the left ventricular dp/dt was complete (around 100%) in group B compared to groups A, C, D, and E (p < .05). Coronary flow recovered somewhat better in group B than in the other groups, but this difference did not reach statistical significance (p = .077).
CK release during reperfusion The CK release to the coronary effluent is reported in Table 3. In hearts from groups A, C, D, and E, CK
release increased after 30 min reperfusion compared to the preischemic levels. The release in group B did not differ significantly from preischemia. DISCUSSION
EC-SOD is the major SOD isoenzyme in the extracellular space in vivo.18'23'24 Nevertheless, most investigations regarding the effects of SOD on myocardial ischemia/reperfusion have been studied with the cytosolic CuZn-SOD because this isoenzyme is more easily available. EC-SOD, a secretory tetrameric copper and zinc-containing glycoprotein, may be divided into three fractions, defined by the affinity to hepar i n : z5'26 A, without affinity; B, with weak affinity; and C, with relatively strong affinity. The physiologic ligand of EC-SOD C is the heparin analog heparan sulfate proteoglycan, 15-17 which exists in the glycocalyx of cell surfaces and in the connective tissue matrix. EC-SOD C forms an equilibrium between the fluid phase and endothelial surfaces in the vasculat u r e 15'18'19'24 and cell surfaces and connective tissue matrix in tissues. 17Heparin releases EC-SOD C to the fluid phase by competing efficiently with heparan sulfate for the heparin-binding domain of the enzyme. EC-SOD C bound both to heparin and to heparan sulfate retains essentially all enzymatic activity.27 The physiologic role and importance of EC-SOD still remain unclarified. Johansson 28 reported that the combination of recombinant human EC-SOD C and catalase reduced oxygen free radicals detectable by
Table 2. Percent Recovery of Hemodynamic Parameters in the 30-Min Reperfused Hearts (n = 8 in each group)
Group Group Group Group Group
A B C D E
Heart Rate (HR)
Systolic Left Ventricular Pressure (SVP)
Double Product (HR × SVP)
Left Ventricular dP/dt
Coronary Flow
85 _+ 30% 106 .+ 16% 81 -+ 26% 93+21% 100 + 38%
48 _+ 30% 100 + 22%* 54 _+ 33% 50+31% 66 + 24%
47 _+ 37% 104 _+ 18%* 49 _+ 36% 50+35% 69 + 31%
42 _+ 40% 109 +_ 24%* 47 _+ 36% 44+_33% 58 + 25%
53 _+ 37% 87 _+ 19% 59 _+ 34% 45+_31% 59 +- 22%
Group A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate + bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L) + heparin 50,000 U/L; E: cold perfusate + heparin 50,000 U/L. *p < .05 versus groups A, C, D, and E.
140
N. HATORI el a/.
Table 3. Creatine Kinase Activity in the Coronary Effluent (n = 8 in each group)
Group A GroupB Group C Group D Group E
Preischemia (U/L)
30 Min after Reperfusion (U/L)
8+ 8 9_+6 3 +_ 4 3 +_ 3 5+ 5
239 _+ 48* 27+_ 31 99 +_ 198" 139 + 180" 142 + 362*
Group A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate + bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L) + heparin 50,000 U/L; E: cold perfusate + heparin 50,000 U/L. *p < .05 versus preischemia.
spin trap generation more effectively than CuZnSOD in the reperfused isolated rat heart. SOD was administered prior to ischemia and throughout the reperfusion period. The effect of EC-SOD C on the recovery of myocardial function after ischemia and reperfusion has been studied by SjOquist et al. 29 ECSOD C was administered throughout the experiment, and the action site was not detected. The main finding of this study is that recombinant human EC-SOD C, when given together with cardioplegia, improved the recovery of myocardial function and prevented CK release after global ischemia. There were no effects of CuZn-SOD or EC-SOD C administered together with heparin in a dose efficiently preventing binding to endothelial cell surfaces. Is A very likely explanation for the protective effect on hearts in group B receiving only EC-SOD C is that EC-SOD C could adhere to the vascular walls and be active on that site during the reperfusion period. In contrast, CuZn-SOD in group C and EC-SOD C in group D (administered with heparin in a concentration totally inhibiting EC-SOD C binding to vascular walls) were washed away directly at the start ofreperfusion by the reperfusion medium. In these two groups, as in group A (control) and in group E (heparin only), no protective enzyme was present in the vasculature of the heart a few seconds after the start of reperfusion. The rat heart should constitute an adequate model for exploring the efficacy of EC-SOD C. The effect of native EC-SOD C in rats can be eliminated. Unlike other investigated mammals, it lacks the high heparin affinity fraction C of EC-SOD. 18 Nevertheless, ECSOD C binds to the vascular endothelium in the rat because the receptor for EC-SOD C, heparan sulfate, ~5 does not require any specific structure of sulfated polysaccharides to attach. The precise mechanisms of oxygen radical production in the heart are not fully understood. Several sources have been p r o p o s e d , 3°-36 including the xanthine-xanthine oxidase system, oxidized forms of myoglobin, autooxidation of substances reducing isch-
emia, injured mitochondria leaking electrons, activated prostaglandin and leukotriene synthesis with superoxide radicals formed as a byproduct, and activated phagocytic leukocytes. The latter source should be of minor importance since the hearts in this study were perfused with Krebs-Henseleit buffer. However, other cell types, such as endothelial cells, 37-39 fibroblasts, 4° and B-lymphocytes,41 may also be stimulated to secrete significant amounts of superoxide. The large SOD molecules do not penetrate to any significant extent. Interception with the superoxide radicals should therefore take place in the extracellular space. Superoxide radicals are secreted from some of the potential sources. Superoxide generated intracellulary may also cross the plasma membranes through anion c h a n n e l s . 35,42
Superoxide dismutase converts superoxide anions into hydrogen peroxide, which is further reduced to water by catalase or glutathione peroxidase. The excellent myocardial recovery following EC-SOD C without catalase indicates that the ischemic myocardium contains sufficient amounts of catalase and/or glutathione peroxidase. An alternative explanation would be that hydrogen peroxide is less important for induction of the reperfusion injury.43 The hydroxyl radical is the most toxic of the oxygen-generated radicals. 44 In the present context, it may be formed b y a transition metal ion-catalyzed reaction between hydrogen peroxide and superoxide. 45 Since superoxide can release iron in reactive form from ferritin,46 enhanced superoxide dismutase activity may protect b y reducing the amount of superoxide serving as a precursor for hydroxyl radicals and by reducing the availability of the transitional metal ion catalyst. An alternative explanation is that superoxide radicals cause degradation of the endothelium-derived relaxing factor (EDRF). 47'48 Generation of superoxide radicals during the early reperfusion may therefore impair the endothelium-dependent vasorelaxation. An intriguing mechanism of action for EC-SOD C would therefore be protection against loss ofendothelium-dependent coronary vessel relaxation at the start of reperfusion, thereby diminishing a no-reflow phenomenon. The tendency to better coronary flow in group B may be indicative of such a mechanism. The product of the reaction between superoxide and EDRF, which apparently is identical to nitric o x i d e , 49 is the highly cytotoxic peroxynitrite. 5°'5J The protection conferred by SOD may thus partly be due to reduced peroxynitrite formation. Production of free oxygen radicals requires oxygen as a substrate but already occurs at low oxygen tensions. 52 Bolli et al. 7 reported on low-grade oxygen free-radical production in the ischemic canine myocardium, detected by spin trapping. Arroyo et al. 53
EC-SOD and reperfusion injury
described oxygen radical generation in the ischemic rat heart. However, oxygen may be supplied by collateral blood flow in canines, and the low-flow global ischemic preparation used by Arroyo et al. may have provided enough oxygen for free radical production during the period of ischemia. Henry et alJ 4 studied ischemic isolated rat hearts, which were continuously monitored for free radical generation by means of enhanced chemiluminescence. Radicals were already produced during the very early reperfusion period but not during ischemia. Superoxide dismutase attenuated the elevation of free radicals during reperfusion. The lack of effect of SOD without attachment to cell surfaces (CuZn-SOD and EC-SOD with heparin) indicates that radicals formed during the ischemic period were of minor importance in the present experimental model. Beneficial effects of CuZn-SOD have been reported when the administration of this compound includes not only the ischemic but also the reperfusion period. '-5 Some localization of CuZn-SOD to myocardial structures seems necessary to prevent reperfusion injury when the drug is given prior to reperfusion only. 5s Taken together, these findings suggest that SOD present in the interstitium or bound to extracellular surfaces is important for the protective effect. When polylysine is substituted for CuZn-SOD to facilitate association with negatively charged cell membranes, the ability of the enzyme to protect activated polymorphonuclear leukocytes against self-inactivation is highly potentiated, s6 The cell-membrane-associated SOD of the bacterium Nocardia asteroides confers efficient protection against activated polymorphonuclear leukocytes. 57 In the light of these observations, the present findings strongly suggest that binding of SOD to cellular surfaces might be of particular importance for the cellular protection against superoxide radicals. Fixation of SOD to the endothelial surfaces in the ischemic myocardium may be an important tool by enhancing the possibility of protecting jeopardized myocardium from subsequent reperfusion injury. A limitation of the study is that CK release was analyzed only prior to and 30 min after reperfusion. Still, and since a recirculating system was used, the CK release during the reperfusion period should be reasonably well reflected by the data presented although not truly representative of the total release of the enzyme. Furthermore, the results of the CK release are consistent with the functional data. Further investigations may be needed to determine the exact effect of SOD on CK release. Since oxy radicals may interfere with CK when measured by an enzymatic assay, an immunological technique may then be preferred.
141
Acknowledgement - - This work has been supported by grants from the Swedish Heart and Lung Foundation and from Arbetsmarknadens FOrs~kringsaktiebolag, Stockholm, Sweden.
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