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Ischemic preconditioning is not mediated by free radicals in the isolated rabbit heart
Free Radical Biology & Medicine, Voi. 11, pp. 517-520, 1991 Printed in the USA. All rishts reserved.
0891-5849/91 $3.00 + .00 Copyright © 1991 PergamonPress pie
Brief Communication ISCHEMIC PRECONDITIONING IS NOT MEDIATED BY FREE RADICALS IN THE ISOLATED RABBIT HEART BASSAMA. OMAR, AN'mONYK. HANSON,SWAPANK. BOSE, and JOE M. McCoP.D Webb-Waring Lung Institute, University of Colorado, 4200 E. 9th Avenue, Box C-321, Denver, Colorado 80262, U.S.A. (Received 11 June 1991; Accepted 1 August 1991) Allatraet - - Preconditioning has been implicated to involve a free radical mechanism in the in vivo infarct size model. We have demonstrated preconditioning in the isolated rabbit heart and studied the effect of Mn-superoxide dismutase (SOD) on preconditioning. Buffer perfused control hearts (C) were subjected to 1-h global ischemia followed by l-h reperfusion, and the percent recovery of developed tension (%DT; relative to preischemic) was raeasored via a left ventricular balloon connected to a polygraph recorder. The coronary effluent was assayed for lactate dehydrogenase release CLDH; IU/kg BW). Preconditioned hearts (P) received a single episode of 5-min ischemia and 5-min reperfusion before 1 h of iscbemia and mperfusion. SOD treated hearts received SOD at 5 mg/L in the buffers at all times. The results show that while SOD attenuated enzyme release and functional loss in control hearts, it had no effect on preconditioned hearts. Therefore, preconditioning is unlikely to be mediated by superoxide anion in the isolated rabbit heart. Keywords--Myocardial ischemia, Free radicals, Preconditioning, Superoxide dismutase, Rabbit
INTRODUCTION
attenuates enzyme release, Mn-SOD had no effect on this protection.
Ischemic preconditioning of the myocardium is a phenomenon whereby a short episode of ischemia and repeffusion increases the tolm~nce of the heart to subsequent ischemia. Several laboratories have documented that preconditioning reduces infarct size in the dog and in the rabbit heart. 1-4 Others have shown preconditioning to decrease the incidence of arrhythmias in the rat heart. 5'6 The concept became popular due to the extent of protection observed and the consistency in the f'mdings among several laboratories. Although the mechanism of ischemic preconditioning is unknown, several possibilities have been examined in the literature. Among these possibilities is myocardial smnnir~g, whereby the heart undergoes a reversible depression of function following short episodes of i s c h e m i a . 3"7 Another possibility is that preconditioning slows down energy u"tflizafion such that ATP is consumed at a much lower rate and is not depleted during ischemia, s'9 A third possibility is that free radicals may be responsible for preconditioning the heart, io In this study we examine the effect of Mn-superoxide dismutase (Mn-SOD) on ischemic preconditioning in the isolated rabbit heart. Our results demonstrate that while ischemic preconditioning significantly improves the postiscbemic recovery of contractile function and
MErHODS
Enzyme ana/ys/s Lactate dehydrogenase (LDH) was assayed by monitoring the rate of oxidation of NADH according to Sigma procedure #340-UV (Sigma Chemicals, St. Louis, MO). Human recombinant (hr) Mn-SOD was provided by Biotechnology General (New York, NY) and possessed 4000 standard units per milligram.
Ischemia/reperfusion experiments Healthy New Zealand white rabbits (2-3 kg) were killed with 60 mg/kg sodium pentobarbital. The hearts were quickly excised and mounted via the ascending aorta on a nonrecirculating perfusion apparatus. Retrograde aortic perfusion under gravity at 80 mmHg with modified, oxygenated, glucose-containing Krebs-Henseleit buffer was initiated. A saline-filled latex balloon connected through a pressure transducer to a polygraph recorder (Grass Insu-aments Co., Quincy, MA), together with a I mm caliber polyethylene tube, was inserted into the left ventricle through a left atriotomy and se517
518
B.A. ~
cured by a suture to the mitral annulus. The polyethylene tube drained any fluid that accumulated between the ventricular wall and the balloon. After an initial 15-rain equilibration period, the developed pressure was measured, the balloon was deflated, and the hearts were subjected to 1 h of global ischemia. This was followed by another hour of reperfusion, at the end of which the developed pressure was measured. The results are expressed as percent recovery in the developed tension. The hearts were not paced. The coronary flows were measured at regular intervals and assayed for lactate dehydrogenase (LDH) release by monitoring the rate of oxidation of NADH in the presence of pyruvate at 340 nm. 11 The Krebs-Henseleit buffer employed in these studies was composed of (in miUimolar concentration): NaC1, 118; NaHCO3, 24.8; KCI, 4.7; KH2PO4, 1.2; CaC12, 1.25; MgSO4, 1.2; and glucose, 10. Buffers were bubbled continuously with a mixture of 95% 0 2 and 5% CO 2. The CO2 helped maintain the buffers at a physiological pH of 7.4. The apparatus had extensive water jacketing connected to a water heater that maintained the buffers and the hearts at 37°C (verified by temperature probes) throughout the experiment. All buffers were filtered through a Gelman GA-4, 0.8-1xm metrical membrane before use. Two sets of experiments were completed. In the first set, hearts were subjected to 1 h of ischemia followed by 1 h of reperfusion. The fwst group served as controls, receiving no treatment. The second group received 5 mg/L Mn-SOD in the buffers at all times. In the second set of experiments, hearts were subjected to a single preconditioning episode of 5-min ischemia and 5-rain reperfusion before the 1 h of ischernia and reperfusion. The first group received no treatment in the buffers. The second group received 5 rag/1 MnSOD in the buffers at all times. Statistics
Data are presented as the mean plus or minus standard error in the results section and mean plus or minus standard deviation in the figures. Tests for significance were made using a one-way analysis of variance and the differences between specific groups were determined by the Newman-Keuls test. All experiments were contemporary and randomized. RF.SULTS
L D H release data
Fig. 1 shows the LDH release data for control and preconditioned hearts with and without Mn-SOD. Control hearts had a total LDH release of 170 -+ 12 IU/kg BW. Preconditioned hearts had a significantly lower LDH release of 37 -- 5 IU/kg BW (P<.001). Mn-
et al. a)
25o
,=
I
zoo
P
,
l~P
/ / / /
/ / / /
/ / / /
/ / / /
/ / / /
/ / / .
/ / / .
/ / / .
/ i / .
/ / / .
/ / / .
~/////, "0
l
i i i i i I i i / / / i
o
0
~/////, C
C+SOD
P
P+80D
Fig. 1. Lactate dehydrogenaterelease 0U/kg BW) from control (C) and preconditioned(P) hearts in the absence and presence of MnSOD. Note that preconditionedhearts (n = 10) had a much lower enzyme release compared to controls (n = 14). M.n-SODtreated, nonpreconditionedhearts (C+ SOD, n = 6) had a significantlylower LDH release comparedto controls, but was significantlylower than preconditioned hearts. Mn-SOD treatted preconditionedhearts (P+ SOD. n = 5) had also a significantly lower LDH release, which was not differentfrom preconditioningalone.
SOD treated hearts had also a lower LDH release than controls (80 _+ 7 IU/kg BW, P < .01). Mn-SOD treated preconditioned hearts, however, had an LDH release of 31 +_ 9 IU/kg BW, which was not significantly different from nontreated preconditioned hearts.
Functional recovery data
Fig. 2 shows the percent recovery in developed tension for control and preconditioned hearts with and without Mn-SOD. Control hearts recovered by 16 1% of their preischemic developed tension. Preconditioned hearts had a significantly better recovery of 74 +- 3% (P < .001). Mn-SOD treated hearts also had a better recovery of developed tension compared to controis (37 -+ 4, P < .05). Mn-SOD treated preconditioned hearts, however, had a recovery of 72 -+ 4%, which was not significantly different from nontreated preconditioned hearts. The average preischemic developed tension for all hearts was 98 -+ 6 mmHg, and the average developed tension of preconditioned hearts after a 5-rain ischemic preconditioning episode was 97 + 8 mmHG (nonsignificant compared to preischemic). The average preischemic heart rate was 216 + 17 beats/rain, and the average postischemic heart rate was 179 -- 11 beats/ rain, with no significant difference among the various groups. The average flow rates for all hearts were 61 -- 9 ml/min, 40 __. 7 ml/min, and 34 -- 10 ml/min during preischemia, at reperfusion and at the end of 1 h of reperfusion, respectively. There was no significant difference among the various groups with regard to flow.
lschemic ~ e c o n d i f i o ~ not mediated by fzee radicals 100 ,
P
.
n.J. - - - = - I
i" ]~'2o
/ / / / / /
//////
C
C+SOD
P
//////
P+SOD
Fig. 2. Percent n~overy of developed tension (relative to preischemic) in control (C) and preconditioned (P) hemls with or without Mn-SOD. Preconditioned hearts (n = 10) had a much better functional recovezy compmcd to controls (n = 14). Mn-SOD treated, n o n ~ h e a ~ (C + SOD, n = 6) had a better recovery cempemd to coatmls, but was si~ificanfly lower than preconditioned hearts. Mn-SOD Ueated preconditioned hearts (P + SOD, n = 5) had alao a significant recovery of developed tension, which was not different from lXecondilioning alone.
DISCUSSION
The current study demonstrates that iscbemic preconditioning may be achieved in the isolated rabbit heart and that it results in improved developed tension and lower enzyme release. A beneficial effect of preconditioning on infarct size and arrhythmias has been reported in the literature. 1-4'6 In this study we have chosen a single 5-min episode of ischemia followed by 5 min of reperfusion as the preconditioning event. Preconditioning protocols vary among the different studies. While some investigators use several episodes of ischemia and reperfusion, 1's'9 others find a single episode to be as p r o t e c t i v e . 2'4'12 Although the minimum ischemic time for a preconditioning episode is unknown, a dual 2-min episode of ischemia and reperfusion was reported to be nonprotective, while a single or double 5-min ischemic episode was found to be protective in the same study. 4 The mechanism of ischemic preconditioning is unknown. Some investigators suggest that it could be mediated by sturming, 3'7 or by free radicals, which cause stunning, m Our results demonstrate that there was no significant decrease in developed tension in the hearts due to the 5 rain of ischemia since there was almost complete recovery of developed tension at the end of the 5 min of reperfusion. This is important because it rules out stunning as a prerequisite for preconditioning in this model. Myocardial stnnning has been shown to be mediated by free radicals, and to be ameliorated by radical scavengers in several systems. 13-16 Therefore, the absence of stunning in our preconditioning protocol could mean that no sufficient free radicals
519
were generated to cause stunning of the myocardium. To clarify this point further, we used Mn-SOD at 5 mg/L, a dose that we have previously shown to be optimal in this protocol. ~7 bin-SOD si£mificantly ameliorated stunning in nonpreconditioned hearts as revealed by the improved recovery of developed tension and lower LDH release. If free radicals were to play a role in the preconditioning event, one would expect that M_n-SOD should attenuate the recovery of developed tension in preconditioned hearts, at least to the level of SOD-treated, nonpreconditioned hearts. Nevertheless, as our results show, SOD did not cause any decrease in the recovery of developed tension or rise in enzyme release in preconditioned hearts. Except for the postischemic recovery of developed tension, the hemodynamic data in our study do not show any significant difference between control and preconditioned hearts and, therefore, cannot account for the beneficial effects of preconditioning observed. A recent study in the literature showed improved biochemical and ultrastructural end-points of ischemic preconditioning in the canine heart in vivo during ischemia alone, s In the current study we addressed the effect of ischemic preconditioning on the ability of the myocardium to recover from 1 h of ischemia by measuring developed tension and enzyme release at the end of 1 h of reperfusion. The effect of ischemic preconditioning on the postischemic stunned myocardinm has not been examined previously in the literature, while its effect on infarct size and tissue necrosis has been clearly documented. 1-4 The amelioration of stunning by preconditioning in our study could be due to attenuation of free radical generation at repeffnsion. Therefore, ischemic preconditioning may actually be modulating reperfusion injury as well as influencing the ischemic insult. However, the exact mechanism by which it may decrease either form of injury is not known yet. The fact that free radical scavenging alone cannot attenuate ischemia/reperfusion injury to the same extent as preconditioning implies that other components of injury, such as calcium entry, could be important. The marked attenuation of enzyme release by preconditioning implies that preconditioning caused less cell death and tissue necrosis, in addition to decreasing stunning, in our study. In conclusion, we have demonstrated a protective effect of ischemic preconditioning in the isolated rabbit heart, which precludes the involvenmnt of neutrophils or other blood elements, against enzyme release and functional loss. We have shown that the preconditioning episode does not cause stunning and that Mn-SOD does not attenuate the protective effect of preconditioning, therefore excluding free radicals as causative agents in preconditioning. Finally, the fact that preconditioning attenuates postischemic stunning implies that its
520
B. A. OM~ et al.
action is partially mediated by amelioration of free radical generation. Acknowledgement -- This work was supported by a Cardiovascular
Discovery Grant from Glaxo.
REFERENCES
1. Murry, C.E.; Jennings, R.B.; l~im~, K.A. Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 74:1124-1136; 1986. 2. Li, G.C.; Vasqnez, J.A.; Gallagher, K.P.; Lucchesi, B.R. Preconditioning with single or multiple brief coronary artery occlusions limit infarct size. 3. Mol. Cell. Cardiol. 21(~qspi ID:161 abstr. (1989) 3. Schott, R.J.; Rohmann, S.; Braun, E.R.; Schaper, W. Ischemic preconditioning reduces infarct size in swine myocardium. Circ. Res. 66:1133-1142; 1990. 4. Downey, J.M.; Van Winkle, D.M.; Pollard, J.D. Preconditioning in rabbit hearts: Effect of varying number and duration of transient ischemic events. FASEB Y. 4(3):A852 abstr. (1990). 5. Meerson, F.Z.; Malyshev, I.Y. Adaptation to stress increases the heart resistance to ischemic and reperfusion arrbyflunias. J. Mol. Cell. Cardiol. 21:299-303; 1989. 6. Shiki, K.; Hearse, D.J. Preconditioning of ischemic myocardium: reperfesion-induced arrbythmias. Am. J. Physiol. 253(6 pt 2):HI470-HI476; 1987. 7. Schott, R.J.; Schaper, W. Effects of transient coronary occlusion: Experience with myocardial stunning and preconditioning. lsr. J. Med. Sci. 25:479-482; 1989. 8. Murry, C.E.; Richard, V.J.; Reimer, K.A.; Jennings, R.B. Ischemic preconditioning slows energy metabolism and delays ul-
tras~ damage during a sustmned ischemic episode.Circ. Res. 66(4):913-931; 1990.
9. Murry, C.E.; Reimer, K.A.; Jennings, R.B. Preconditioning with ischemia delays ATP depletion and limits lactate accumulation in severely ischemic canine myocardium. Circulation 76(Suppl IV):IV-228 abstr. (1987). 10. Murry, C.E.; Richard, V.J.; Jennings, R.B.; Reimer, K.A. Preconditioning with ischemia: Is the effect mediated by free radical-induced myocardial stunning? Circulation 78(Suppl II):H-77 abstr. (1988). 11. Amador, E.; Dorfman, L.E.; Wacker, W.E.C. Serum lactic dehydrogenase activity: An analytical assessment of current assays. Clin. Chem. 9:391; 1963. 12. Li, G.C.; Vasquez, J.A.; Gallagher, K.P.; Lucchesi, B.R. Myocardial protection with preconditioning. Circulation 82:609-619; 1990. 13. Przyklenk, K.; Kloner, R.A. Superoxide dismutase plus catalase improve contractile function in the canine model of the "stunned myocardium." Circ. Res. $8:148-156; 1986. 14. Gross, G.J.; Farber, N.E.; Hardman, H.F.; Warltier, D.C. Beneficial actions of superoxide dismutase and catalase in stunned myecardium of dogs. Am. J. Physiol. Z~.H372-H377, 1986. 15. Bolli, R., Patel, B.S.; Jeroudi, M.O.; Lai, E.K.; McCay, P.B. Demonstration of free radical generation in "stunn~" myocardium of intact dogs with the use of the spin trap ¢x-phenyl N-tert-butyl nitrone. J. Clin. Invest. 82:476--485; 1988. 16. Bolli, R.; Jeroudi, M.O.; Patel, B.S.; Aruoma, O.I.; HaUiwell, B.; Lai, E.K.; McCay, P.B. Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion: Evidence that myocardial "stunning" is a manifestation of reperfusion injury. Circ. Res. 65:607-622; 1989. 17. Omar, B.A.; McCord, J.M. The cardioprotective effect of Mnsuperoxide dismutase is lost at high doses in the isolated rabbit heart. Free Radic. Biol. Med. 1990. (In press.)
0891-5849/91 $3.00 + .00 Copyright © 1991 PergamonPress pie
Brief Communication ISCHEMIC PRECONDITIONING IS NOT MEDIATED BY FREE RADICALS IN THE ISOLATED RABBIT HEART BASSAMA. OMAR, AN'mONYK. HANSON,SWAPANK. BOSE, and JOE M. McCoP.D Webb-Waring Lung Institute, University of Colorado, 4200 E. 9th Avenue, Box C-321, Denver, Colorado 80262, U.S.A. (Received 11 June 1991; Accepted 1 August 1991) Allatraet - - Preconditioning has been implicated to involve a free radical mechanism in the in vivo infarct size model. We have demonstrated preconditioning in the isolated rabbit heart and studied the effect of Mn-superoxide dismutase (SOD) on preconditioning. Buffer perfused control hearts (C) were subjected to 1-h global ischemia followed by l-h reperfusion, and the percent recovery of developed tension (%DT; relative to preischemic) was raeasored via a left ventricular balloon connected to a polygraph recorder. The coronary effluent was assayed for lactate dehydrogenase release CLDH; IU/kg BW). Preconditioned hearts (P) received a single episode of 5-min ischemia and 5-min reperfusion before 1 h of iscbemia and mperfusion. SOD treated hearts received SOD at 5 mg/L in the buffers at all times. The results show that while SOD attenuated enzyme release and functional loss in control hearts, it had no effect on preconditioned hearts. Therefore, preconditioning is unlikely to be mediated by superoxide anion in the isolated rabbit heart. Keywords--Myocardial ischemia, Free radicals, Preconditioning, Superoxide dismutase, Rabbit
INTRODUCTION
attenuates enzyme release, Mn-SOD had no effect on this protection.
Ischemic preconditioning of the myocardium is a phenomenon whereby a short episode of ischemia and repeffusion increases the tolm~nce of the heart to subsequent ischemia. Several laboratories have documented that preconditioning reduces infarct size in the dog and in the rabbit heart. 1-4 Others have shown preconditioning to decrease the incidence of arrhythmias in the rat heart. 5'6 The concept became popular due to the extent of protection observed and the consistency in the f'mdings among several laboratories. Although the mechanism of ischemic preconditioning is unknown, several possibilities have been examined in the literature. Among these possibilities is myocardial smnnir~g, whereby the heart undergoes a reversible depression of function following short episodes of i s c h e m i a . 3"7 Another possibility is that preconditioning slows down energy u"tflizafion such that ATP is consumed at a much lower rate and is not depleted during ischemia, s'9 A third possibility is that free radicals may be responsible for preconditioning the heart, io In this study we examine the effect of Mn-superoxide dismutase (Mn-SOD) on ischemic preconditioning in the isolated rabbit heart. Our results demonstrate that while ischemic preconditioning significantly improves the postiscbemic recovery of contractile function and
MErHODS
Enzyme ana/ys/s Lactate dehydrogenase (LDH) was assayed by monitoring the rate of oxidation of NADH according to Sigma procedure #340-UV (Sigma Chemicals, St. Louis, MO). Human recombinant (hr) Mn-SOD was provided by Biotechnology General (New York, NY) and possessed 4000 standard units per milligram.
Ischemia/reperfusion experiments Healthy New Zealand white rabbits (2-3 kg) were killed with 60 mg/kg sodium pentobarbital. The hearts were quickly excised and mounted via the ascending aorta on a nonrecirculating perfusion apparatus. Retrograde aortic perfusion under gravity at 80 mmHg with modified, oxygenated, glucose-containing Krebs-Henseleit buffer was initiated. A saline-filled latex balloon connected through a pressure transducer to a polygraph recorder (Grass Insu-aments Co., Quincy, MA), together with a I mm caliber polyethylene tube, was inserted into the left ventricle through a left atriotomy and se517
518
B.A. ~
cured by a suture to the mitral annulus. The polyethylene tube drained any fluid that accumulated between the ventricular wall and the balloon. After an initial 15-rain equilibration period, the developed pressure was measured, the balloon was deflated, and the hearts were subjected to 1 h of global ischemia. This was followed by another hour of reperfusion, at the end of which the developed pressure was measured. The results are expressed as percent recovery in the developed tension. The hearts were not paced. The coronary flows were measured at regular intervals and assayed for lactate dehydrogenase (LDH) release by monitoring the rate of oxidation of NADH in the presence of pyruvate at 340 nm. 11 The Krebs-Henseleit buffer employed in these studies was composed of (in miUimolar concentration): NaC1, 118; NaHCO3, 24.8; KCI, 4.7; KH2PO4, 1.2; CaC12, 1.25; MgSO4, 1.2; and glucose, 10. Buffers were bubbled continuously with a mixture of 95% 0 2 and 5% CO 2. The CO2 helped maintain the buffers at a physiological pH of 7.4. The apparatus had extensive water jacketing connected to a water heater that maintained the buffers and the hearts at 37°C (verified by temperature probes) throughout the experiment. All buffers were filtered through a Gelman GA-4, 0.8-1xm metrical membrane before use. Two sets of experiments were completed. In the first set, hearts were subjected to 1 h of ischemia followed by 1 h of reperfusion. The fwst group served as controls, receiving no treatment. The second group received 5 mg/L Mn-SOD in the buffers at all times. In the second set of experiments, hearts were subjected to a single preconditioning episode of 5-min ischemia and 5-rain reperfusion before the 1 h of ischernia and reperfusion. The first group received no treatment in the buffers. The second group received 5 rag/1 MnSOD in the buffers at all times. Statistics
Data are presented as the mean plus or minus standard error in the results section and mean plus or minus standard deviation in the figures. Tests for significance were made using a one-way analysis of variance and the differences between specific groups were determined by the Newman-Keuls test. All experiments were contemporary and randomized. RF.SULTS
L D H release data
Fig. 1 shows the LDH release data for control and preconditioned hearts with and without Mn-SOD. Control hearts had a total LDH release of 170 -+ 12 IU/kg BW. Preconditioned hearts had a significantly lower LDH release of 37 -- 5 IU/kg BW (P<.001). Mn-
et al. a)
25o
,=
I
zoo
P
,
l~P
/ / / /
/ / / /
/ / / /
/ / / /
/ / / /
/ / / .
/ / / .
/ / / .
/ i / .
/ / / .
/ / / .
~/////, "0
l
i i i i i I i i / / / i
o
0
~/////, C
C+SOD
P
P+80D
Fig. 1. Lactate dehydrogenaterelease 0U/kg BW) from control (C) and preconditioned(P) hearts in the absence and presence of MnSOD. Note that preconditionedhearts (n = 10) had a much lower enzyme release compared to controls (n = 14). M.n-SODtreated, nonpreconditionedhearts (C+ SOD, n = 6) had a significantlylower LDH release comparedto controls, but was significantlylower than preconditioned hearts. Mn-SOD treatted preconditionedhearts (P+ SOD. n = 5) had also a significantly lower LDH release, which was not differentfrom preconditioningalone.
SOD treated hearts had also a lower LDH release than controls (80 _+ 7 IU/kg BW, P < .01). Mn-SOD treated preconditioned hearts, however, had an LDH release of 31 +_ 9 IU/kg BW, which was not significantly different from nontreated preconditioned hearts.
Functional recovery data
Fig. 2 shows the percent recovery in developed tension for control and preconditioned hearts with and without Mn-SOD. Control hearts recovered by 16 1% of their preischemic developed tension. Preconditioned hearts had a significantly better recovery of 74 +- 3% (P < .001). Mn-SOD treated hearts also had a better recovery of developed tension compared to controis (37 -+ 4, P < .05). Mn-SOD treated preconditioned hearts, however, had a recovery of 72 -+ 4%, which was not significantly different from nontreated preconditioned hearts. The average preischemic developed tension for all hearts was 98 -+ 6 mmHg, and the average developed tension of preconditioned hearts after a 5-rain ischemic preconditioning episode was 97 + 8 mmHG (nonsignificant compared to preischemic). The average preischemic heart rate was 216 + 17 beats/rain, and the average postischemic heart rate was 179 -- 11 beats/ rain, with no significant difference among the various groups. The average flow rates for all hearts were 61 -- 9 ml/min, 40 __. 7 ml/min, and 34 -- 10 ml/min during preischemia, at reperfusion and at the end of 1 h of reperfusion, respectively. There was no significant difference among the various groups with regard to flow.
lschemic ~ e c o n d i f i o ~ not mediated by fzee radicals 100 ,
P
.
n.J. - - - = - I
i" ]~'2o
/ / / / / /
//////
C
C+SOD
P
//////
P+SOD
Fig. 2. Percent n~overy of developed tension (relative to preischemic) in control (C) and preconditioned (P) hemls with or without Mn-SOD. Preconditioned hearts (n = 10) had a much better functional recovezy compmcd to controls (n = 14). Mn-SOD treated, n o n ~ h e a ~ (C + SOD, n = 6) had a better recovery cempemd to coatmls, but was si~ificanfly lower than preconditioned hearts. Mn-SOD Ueated preconditioned hearts (P + SOD, n = 5) had alao a significant recovery of developed tension, which was not different from lXecondilioning alone.
DISCUSSION
The current study demonstrates that iscbemic preconditioning may be achieved in the isolated rabbit heart and that it results in improved developed tension and lower enzyme release. A beneficial effect of preconditioning on infarct size and arrhythmias has been reported in the literature. 1-4'6 In this study we have chosen a single 5-min episode of ischemia followed by 5 min of reperfusion as the preconditioning event. Preconditioning protocols vary among the different studies. While some investigators use several episodes of ischemia and reperfusion, 1's'9 others find a single episode to be as p r o t e c t i v e . 2'4'12 Although the minimum ischemic time for a preconditioning episode is unknown, a dual 2-min episode of ischemia and reperfusion was reported to be nonprotective, while a single or double 5-min ischemic episode was found to be protective in the same study. 4 The mechanism of ischemic preconditioning is unknown. Some investigators suggest that it could be mediated by sturming, 3'7 or by free radicals, which cause stunning, m Our results demonstrate that there was no significant decrease in developed tension in the hearts due to the 5 rain of ischemia since there was almost complete recovery of developed tension at the end of the 5 min of reperfusion. This is important because it rules out stunning as a prerequisite for preconditioning in this model. Myocardial stnnning has been shown to be mediated by free radicals, and to be ameliorated by radical scavengers in several systems. 13-16 Therefore, the absence of stunning in our preconditioning protocol could mean that no sufficient free radicals
519
were generated to cause stunning of the myocardium. To clarify this point further, we used Mn-SOD at 5 mg/L, a dose that we have previously shown to be optimal in this protocol. ~7 bin-SOD si£mificantly ameliorated stunning in nonpreconditioned hearts as revealed by the improved recovery of developed tension and lower LDH release. If free radicals were to play a role in the preconditioning event, one would expect that M_n-SOD should attenuate the recovery of developed tension in preconditioned hearts, at least to the level of SOD-treated, nonpreconditioned hearts. Nevertheless, as our results show, SOD did not cause any decrease in the recovery of developed tension or rise in enzyme release in preconditioned hearts. Except for the postischemic recovery of developed tension, the hemodynamic data in our study do not show any significant difference between control and preconditioned hearts and, therefore, cannot account for the beneficial effects of preconditioning observed. A recent study in the literature showed improved biochemical and ultrastructural end-points of ischemic preconditioning in the canine heart in vivo during ischemia alone, s In the current study we addressed the effect of ischemic preconditioning on the ability of the myocardium to recover from 1 h of ischemia by measuring developed tension and enzyme release at the end of 1 h of reperfusion. The effect of ischemic preconditioning on the postischemic stunned myocardinm has not been examined previously in the literature, while its effect on infarct size and tissue necrosis has been clearly documented. 1-4 The amelioration of stunning by preconditioning in our study could be due to attenuation of free radical generation at repeffnsion. Therefore, ischemic preconditioning may actually be modulating reperfusion injury as well as influencing the ischemic insult. However, the exact mechanism by which it may decrease either form of injury is not known yet. The fact that free radical scavenging alone cannot attenuate ischemia/reperfusion injury to the same extent as preconditioning implies that other components of injury, such as calcium entry, could be important. The marked attenuation of enzyme release by preconditioning implies that preconditioning caused less cell death and tissue necrosis, in addition to decreasing stunning, in our study. In conclusion, we have demonstrated a protective effect of ischemic preconditioning in the isolated rabbit heart, which precludes the involvenmnt of neutrophils or other blood elements, against enzyme release and functional loss. We have shown that the preconditioning episode does not cause stunning and that Mn-SOD does not attenuate the protective effect of preconditioning, therefore excluding free radicals as causative agents in preconditioning. Finally, the fact that preconditioning attenuates postischemic stunning implies that its
520
B. A. OM~ et al.
action is partially mediated by amelioration of free radical generation. Acknowledgement -- This work was supported by a Cardiovascular
Discovery Grant from Glaxo.
REFERENCES
1. Murry, C.E.; Jennings, R.B.; l~im~, K.A. Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 74:1124-1136; 1986. 2. Li, G.C.; Vasqnez, J.A.; Gallagher, K.P.; Lucchesi, B.R. Preconditioning with single or multiple brief coronary artery occlusions limit infarct size. 3. Mol. Cell. Cardiol. 21(~qspi ID:161 abstr. (1989) 3. Schott, R.J.; Rohmann, S.; Braun, E.R.; Schaper, W. Ischemic preconditioning reduces infarct size in swine myocardium. Circ. Res. 66:1133-1142; 1990. 4. Downey, J.M.; Van Winkle, D.M.; Pollard, J.D. Preconditioning in rabbit hearts: Effect of varying number and duration of transient ischemic events. FASEB Y. 4(3):A852 abstr. (1990). 5. Meerson, F.Z.; Malyshev, I.Y. Adaptation to stress increases the heart resistance to ischemic and reperfusion arrbyflunias. J. Mol. Cell. Cardiol. 21:299-303; 1989. 6. Shiki, K.; Hearse, D.J. Preconditioning of ischemic myocardium: reperfesion-induced arrbythmias. Am. J. Physiol. 253(6 pt 2):HI470-HI476; 1987. 7. Schott, R.J.; Schaper, W. Effects of transient coronary occlusion: Experience with myocardial stunning and preconditioning. lsr. J. Med. Sci. 25:479-482; 1989. 8. Murry, C.E.; Richard, V.J.; Reimer, K.A.; Jennings, R.B. Ischemic preconditioning slows energy metabolism and delays ul-
tras~ damage during a sustmned ischemic episode.Circ. Res. 66(4):913-931; 1990.
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