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The failure of hydrogen peroxide to improve function in ischemically depressed myocardium
European Journal of Pharmacology, 60 (1979) 257--260 © Elsevier/North-Holland Biomedical Press
257
Short c o m m u n i c a t i o n T H E F A I L U R E OF H Y D R O G E N PEROXIDE TO IMPROVE FU N CT IO N IN ISCHEMICALLY DEPRESSED MYOCARDIUM * MICHAEL GOODLETT, KRYAN DOWLING and JAMES M. DOWNEY ** Department of Physiology, College of Medicine, University of South Alabama, Mobile, Alabama 36688, U.S.A.
Received 24 October 1979, accepted 25 October 1979
M. GOODLETT, K. DOWLING and J.M. DOWNEY, The failure of hydrogen peroxide to improve function in ischemically depressed myocardium, European J. Pharmacol. 60 (1979) 257--260. The hypothesis that hydrogen peroxide might lend inotropic support to ischemically depressed myocardium was critically tested in a canine model. A coronary branch was perfused at reduced flow to depress mechanical function. Since hydrogen peroxide infusion into the perfusate failed to alter the segment's contractile force, it was concluded that the previously described beneficial effects of hydrogen peroxide infusions are probably not related to a restoration of contractility in ischemic myocardium. Contractile force
Hydrogen peroxide
Ischemia
1. Introduction F o r some time h y d r o g e n peroxide has been proposed as a means of augmenting oxygen delivery to ischemic m y o c a r d i u m (Takahashi et al., 1969; Urschel, 1967). Theoretically tissue enzymes should liberate free oxygen from th e h y d r o g e n peroxide and make it available to th e tissues. Until recently, however, any clear beneficial effects have been difficult to demonstrate. Recent studies (O1son and Boerth, 1978; Olson et al., 1978), however, do d e m o n s t r a t e a protective effect o f h y d r o g e n peroxide infusion during the acute phase o f myocardial infarction. T hey concluded t h a t the increased survival time following total c o r o n a r y ligation provided by h y d r o g en peroxide could have been due to a direct action o f the agent on t he m y o c a r d i u m * Supported by a grant-in-aid from the American Heart Association and by grant HL 20648 from the USPHS. ** Send reprint requests to : James M. Downey, Ph.D., Department of Physiology, University of South Alabama, Mobile, Alabama 36688, U.S.A.
Oxygen
to maintain cardiac contractility (Olsen and Boerth, 1978). The present st udy was, therefore, undertaken t o directly test w h e t h e r h y d r o g e n peroxide has the ability to improve mechanical activity o f ischemically depressed myocardium. T hough Olson and Boerth (1978) perform ed their studies on rabbits, a bl ood perfused rabbit model of myocardial ischemia appropriate for these studies was n o t feasible, and we therefore used dogs. To achieve this goal a c o r o n a r y artery was cannulated and perfused at a reduced rate to render a segment ischemic while mechanical activity of the segment was m o n i t o r e d with a force gauge. A ny restoration in the inotropic state o f the ischemic segment caused by an i nt racoronary infusion o f h y d r o g e n peroxide should be reflected in t he response of the force gauge.
2. Materials and m e t h o d s Mongrel dogs o f either sex were anesthetized with sodium pentobarbital (30 mg/kg, i.v.),
258 supplemented as needed. The hearts were exposed by a left t h o r a c t o m y and the lungs were ventilated by a positive pressure respirator using room air. Coagulation of blood in the perfusion tubing was prevented by intravenous injection of heparin (10 000 units). Aortic blood pressure was measured from a vinyl catheter introduced into the thoracic aorta via right femoral artery. Another vinyl catheter was advanced through the left carotid artery into the left ventricle to measure ventricular pressure. Blood gases were not measured in these experiments. Coronary blood flow was measured by an extracorporeal electromagnetic flowmeter (Carolina Medical Electronics) in the perfusion circuit. Perfusion pressure was recorded from a branch in the circuit near the coronary cannula. The subclavian artery, exposed by a blunt dissection, was cannulated with one end of the perfusion circuit. The left anterior descending coronary artery (LAD) was then dissected free in the region between the first and the second diagonal branches and cannulated with the other end of the perfusion circuit. The perfusion tubing passed through the fingers of a Sigmamotor peristaltic pump so that the flow could be controlled when the pump was engaged. Contractile force of the LAD region was measured with a Walton-Brodie type force gauge (metal encased type with movable foot) modified by adding a perpendicular prong to each foot (Downey, 1976). The prongs ensured good transmission of force from the deep myocardial fibers. The gauge was oriented at right angels to the base-apex axis of the heart and extended to 130% of its initial attachment length (Downey, 1976). To ensure that the force gauge was responsive to ischemia in the test region, the perfusion line was clamped for 5-10 sec and released. If the force gauge did not demonstrate an abrupt fall in amplitude as a result of the transient ischemia, the gauge was readjusted, repositioned, or the preparation was discarded. The coronary artery was autoperfused by the animal's own aortic pressure until it
M. GOODLETT ET AL. stabilized (10-15 min). The pump was then engaged and the coronary flow was decreased to one half of the autoperfused rate to induce ischemia. After contractile force had fallen to a steady state, the solution under study was infused into the coronary perfusate for 3-5 min using a Harvard Apparatus syringe pump. The animal was allowed to recover with auto° perfusion between infusions. Isoproterenol, 2 pg/ml in 5% glucose, 0.17% hydrogen peroxide in 5% glucose, and 5% glucose were infused in randomized fashion. Six dogs were successfully prepared and subjected to the above protocol.
3. Results Resting coronary flow in the LAD averaged 33 ml/min and wash reduced to 18.3 ml/min with the pump. This represented an average reduction to 46% + 7 (S.D.) of the resting flow rate. This caused contractile force to fall to 47% +- 8 (S.D.) of its resting value. Fig. 1 shows the effect of infusions on contractile force of the depressed segment. None of the rates caused any significant changes in contractile force for either hydrogen peroxide or the 5% glucose carrier. Infusion rates above 3 ml/min of peroxide caused bubbles to form in the perfusate and therefore represented an upper limit to the dose which we could administer. Because the terminal LAD perfuses such a small percentage of the left ventricle, alterations in the perfused segment's contractility were not reflected in systemic hemodynamics. Aortic pressure averaged 101 + 3 (S.D.)/79 + 17 (S.D.) mm Hg for these experiments while diastolic ventricular pressure averaged 4 + 2.5 (S.D.) mm Hg. Likewise, none of the infusions, including isoproterenol, altered heart rate which averaged 153 + 20 S.D. beats/ min for the group. A slight trend toward increased force development was observed at 1.5 and 3 ml/ min for the hydrogen peroxide. This was not significant, however, and equal or greater
259
HYDROGEN PEROXIDE IN ISCHEMIA 250"
period, contractile force fell to essentially the same level for the next infusion. When the pump was opened and flow allowed to return to control values, contractile force promptly returned to normal as well. Hydrogen peroxide infusions when flow was normal had no effect on contractile force.
-r
• Isuprel
oH=OI &Glucose
200"
I
W
2$E
u
O
LL
150"
4. Discussion
LLJ
F0 c~ P Z 0
lOO-
Z L~
© z < I
50-
O"
-5O0
tI
t2
3
I N F U S I O N RATE ( M L / M I N )
Fig. 1. Percent change in contractile force realized from intracoronary infusions of 5% glucose (triangles), 0.17% hydrogen peroxide in 5% glucose (squares) or 2 pg/ml isoproterenol (circles). Changes are relative to a state of ischemia in which coronary blood flow and contractile force were reduced to approximately ½ of their resting values.
increments were achieved with the glucose carrier alone. In contrast to the hydrogen peroxide, isoproterenol (2 #g/ml) caused a massive increase in the contractile force of the ischemic segment indicating that the preparation was indeed sensitive and capable of increased force production. This preparation has the advantage that the relationship between the rate of coronary perfusion and contractile force development is very reproducable (Downey, 1976). As a result each time the pump was re-engaged after a recovery
In the canine model presented here hydrogen peroxide failed to restore the forcegenerating ability of ischemic myocardium. In the past, the dog has been considered to be a poor model for hydrogen peroxide studies since its blood catalase levels are much lower than that in the rabbit or the human (Urschel et al., 1966; White and Teasdale, 1966). In earlier studies it was assumed that oxygen would be liberated into the arterial blood by the hydrogen peroxide reacting with catalase resulting in a supersaturated solution (Urschel et al., 1966). Olson and Boerth (1978), however, found that even when their infusions did not completely saturate the arterial blood they still realized a protective effect. Furthermore, they were unable to duplicate hydrogen peroxide's action b y simply raising arterial PO: to an equal or greater level b y breathing with pure oxygen (Olson and Boerth, 1978) and their conclusion was that this was not an oxygen effect. That conclusion was reinforced when they subsequently found that the beneficial action of the drug could only be realized when it was administered in 5% glucose. In those studies, hydrogen peroxide in saline had no effect (Olson et al., 1978). All of the above evidence would indicate that blood catalase does not play an important role in mediating the protective action of hydrogen peroxide and therefore there is no reason to believe that the canine model is inappropriate for this study. At the same time, however, a species difference cannot be ruled out entirely. The depression o f the force gauge was reversible in these experiments b y either
260
restoring the blood flow or by the action of isoproterenol. The former action was to provide molecular oxygen and wash out metabolites thereby reversing the adverse effects of myocardial ischemia and should be considered a beneficial effect. The latter, though showing a short term improvement, must be considered an undesirable response since isoproterenol reportedly depresses the long term survival of myocardium in ischemia. Thus even if an improvement in force development were observed, it obviously would have been difficult to determine whether it were a wholly beneficial effect especially since the protective effects of hydrogen peroxide in ischemia do not appear to involve the evolution of molecular oxygen.
M. GOODLETT ET AL.
References Downey, J.M., 1976, Myocardial contractile force as a function of coronary blood flow, Amer. J. Physiol. 230, 1. Olson, R.D. and R.C. Boerth, 1978, Hydrogen peroxide: beneficial effects in rabbits following acute coronary occlusion, Amer. J. Physiol. 234, H28. Olson, R.D., E.J. Okediji and R.C. Boerth, 1978, Hydrogen peroxide versus glucose, insulin and potassium in coronary occlusion (abstract), Federation Proc. 37, 1358. Takahashi, M., Y. Horiguchi and K. Murakami, 1969, Effects of epicardial perfusion with hydrogen peroxide for ischemic myocardium, Jap. Heart J. 10, 53. Urschel, H.C., Jr., 1967, Cardiovascular effects of hydrogen peroxide;current status Dis. Chest 51,180. Urschel, H.C., Jr., A.R. Morales, J.W. Finney, G.A. Balla, G.J. Race and J.T. Mallams, 1966, Cardiac resuscitation with hydrogen peroxide, Ann. Thorac. Sur. 2,665. White, D. and P. Teasdale, 1966, The oxygenation of blood by hydrogen peroxide: in vitro studies, Brit. J. Anesthesia 38,339.
257
Short c o m m u n i c a t i o n T H E F A I L U R E OF H Y D R O G E N PEROXIDE TO IMPROVE FU N CT IO N IN ISCHEMICALLY DEPRESSED MYOCARDIUM * MICHAEL GOODLETT, KRYAN DOWLING and JAMES M. DOWNEY ** Department of Physiology, College of Medicine, University of South Alabama, Mobile, Alabama 36688, U.S.A.
Received 24 October 1979, accepted 25 October 1979
M. GOODLETT, K. DOWLING and J.M. DOWNEY, The failure of hydrogen peroxide to improve function in ischemically depressed myocardium, European J. Pharmacol. 60 (1979) 257--260. The hypothesis that hydrogen peroxide might lend inotropic support to ischemically depressed myocardium was critically tested in a canine model. A coronary branch was perfused at reduced flow to depress mechanical function. Since hydrogen peroxide infusion into the perfusate failed to alter the segment's contractile force, it was concluded that the previously described beneficial effects of hydrogen peroxide infusions are probably not related to a restoration of contractility in ischemic myocardium. Contractile force
Hydrogen peroxide
Ischemia
1. Introduction F o r some time h y d r o g e n peroxide has been proposed as a means of augmenting oxygen delivery to ischemic m y o c a r d i u m (Takahashi et al., 1969; Urschel, 1967). Theoretically tissue enzymes should liberate free oxygen from th e h y d r o g e n peroxide and make it available to th e tissues. Until recently, however, any clear beneficial effects have been difficult to demonstrate. Recent studies (O1son and Boerth, 1978; Olson et al., 1978), however, do d e m o n s t r a t e a protective effect o f h y d r o g e n peroxide infusion during the acute phase o f myocardial infarction. T hey concluded t h a t the increased survival time following total c o r o n a r y ligation provided by h y d r o g en peroxide could have been due to a direct action o f the agent on t he m y o c a r d i u m * Supported by a grant-in-aid from the American Heart Association and by grant HL 20648 from the USPHS. ** Send reprint requests to : James M. Downey, Ph.D., Department of Physiology, University of South Alabama, Mobile, Alabama 36688, U.S.A.
Oxygen
to maintain cardiac contractility (Olsen and Boerth, 1978). The present st udy was, therefore, undertaken t o directly test w h e t h e r h y d r o g e n peroxide has the ability to improve mechanical activity o f ischemically depressed myocardium. T hough Olson and Boerth (1978) perform ed their studies on rabbits, a bl ood perfused rabbit model of myocardial ischemia appropriate for these studies was n o t feasible, and we therefore used dogs. To achieve this goal a c o r o n a r y artery was cannulated and perfused at a reduced rate to render a segment ischemic while mechanical activity of the segment was m o n i t o r e d with a force gauge. A ny restoration in the inotropic state o f the ischemic segment caused by an i nt racoronary infusion o f h y d r o g e n peroxide should be reflected in t he response of the force gauge.
2. Materials and m e t h o d s Mongrel dogs o f either sex were anesthetized with sodium pentobarbital (30 mg/kg, i.v.),
258 supplemented as needed. The hearts were exposed by a left t h o r a c t o m y and the lungs were ventilated by a positive pressure respirator using room air. Coagulation of blood in the perfusion tubing was prevented by intravenous injection of heparin (10 000 units). Aortic blood pressure was measured from a vinyl catheter introduced into the thoracic aorta via right femoral artery. Another vinyl catheter was advanced through the left carotid artery into the left ventricle to measure ventricular pressure. Blood gases were not measured in these experiments. Coronary blood flow was measured by an extracorporeal electromagnetic flowmeter (Carolina Medical Electronics) in the perfusion circuit. Perfusion pressure was recorded from a branch in the circuit near the coronary cannula. The subclavian artery, exposed by a blunt dissection, was cannulated with one end of the perfusion circuit. The left anterior descending coronary artery (LAD) was then dissected free in the region between the first and the second diagonal branches and cannulated with the other end of the perfusion circuit. The perfusion tubing passed through the fingers of a Sigmamotor peristaltic pump so that the flow could be controlled when the pump was engaged. Contractile force of the LAD region was measured with a Walton-Brodie type force gauge (metal encased type with movable foot) modified by adding a perpendicular prong to each foot (Downey, 1976). The prongs ensured good transmission of force from the deep myocardial fibers. The gauge was oriented at right angels to the base-apex axis of the heart and extended to 130% of its initial attachment length (Downey, 1976). To ensure that the force gauge was responsive to ischemia in the test region, the perfusion line was clamped for 5-10 sec and released. If the force gauge did not demonstrate an abrupt fall in amplitude as a result of the transient ischemia, the gauge was readjusted, repositioned, or the preparation was discarded. The coronary artery was autoperfused by the animal's own aortic pressure until it
M. GOODLETT ET AL. stabilized (10-15 min). The pump was then engaged and the coronary flow was decreased to one half of the autoperfused rate to induce ischemia. After contractile force had fallen to a steady state, the solution under study was infused into the coronary perfusate for 3-5 min using a Harvard Apparatus syringe pump. The animal was allowed to recover with auto° perfusion between infusions. Isoproterenol, 2 pg/ml in 5% glucose, 0.17% hydrogen peroxide in 5% glucose, and 5% glucose were infused in randomized fashion. Six dogs were successfully prepared and subjected to the above protocol.
3. Results Resting coronary flow in the LAD averaged 33 ml/min and wash reduced to 18.3 ml/min with the pump. This represented an average reduction to 46% + 7 (S.D.) of the resting flow rate. This caused contractile force to fall to 47% +- 8 (S.D.) of its resting value. Fig. 1 shows the effect of infusions on contractile force of the depressed segment. None of the rates caused any significant changes in contractile force for either hydrogen peroxide or the 5% glucose carrier. Infusion rates above 3 ml/min of peroxide caused bubbles to form in the perfusate and therefore represented an upper limit to the dose which we could administer. Because the terminal LAD perfuses such a small percentage of the left ventricle, alterations in the perfused segment's contractility were not reflected in systemic hemodynamics. Aortic pressure averaged 101 + 3 (S.D.)/79 + 17 (S.D.) mm Hg for these experiments while diastolic ventricular pressure averaged 4 + 2.5 (S.D.) mm Hg. Likewise, none of the infusions, including isoproterenol, altered heart rate which averaged 153 + 20 S.D. beats/ min for the group. A slight trend toward increased force development was observed at 1.5 and 3 ml/ min for the hydrogen peroxide. This was not significant, however, and equal or greater
259
HYDROGEN PEROXIDE IN ISCHEMIA 250"
period, contractile force fell to essentially the same level for the next infusion. When the pump was opened and flow allowed to return to control values, contractile force promptly returned to normal as well. Hydrogen peroxide infusions when flow was normal had no effect on contractile force.
-r
• Isuprel
oH=OI &Glucose
200"
I
W
2$E
u
O
LL
150"
4. Discussion
LLJ
F0 c~ P Z 0
lOO-
Z L~
© z < I
50-
O"
-5O0
tI
t2
3
I N F U S I O N RATE ( M L / M I N )
Fig. 1. Percent change in contractile force realized from intracoronary infusions of 5% glucose (triangles), 0.17% hydrogen peroxide in 5% glucose (squares) or 2 pg/ml isoproterenol (circles). Changes are relative to a state of ischemia in which coronary blood flow and contractile force were reduced to approximately ½ of their resting values.
increments were achieved with the glucose carrier alone. In contrast to the hydrogen peroxide, isoproterenol (2 #g/ml) caused a massive increase in the contractile force of the ischemic segment indicating that the preparation was indeed sensitive and capable of increased force production. This preparation has the advantage that the relationship between the rate of coronary perfusion and contractile force development is very reproducable (Downey, 1976). As a result each time the pump was re-engaged after a recovery
In the canine model presented here hydrogen peroxide failed to restore the forcegenerating ability of ischemic myocardium. In the past, the dog has been considered to be a poor model for hydrogen peroxide studies since its blood catalase levels are much lower than that in the rabbit or the human (Urschel et al., 1966; White and Teasdale, 1966). In earlier studies it was assumed that oxygen would be liberated into the arterial blood by the hydrogen peroxide reacting with catalase resulting in a supersaturated solution (Urschel et al., 1966). Olson and Boerth (1978), however, found that even when their infusions did not completely saturate the arterial blood they still realized a protective effect. Furthermore, they were unable to duplicate hydrogen peroxide's action b y simply raising arterial PO: to an equal or greater level b y breathing with pure oxygen (Olson and Boerth, 1978) and their conclusion was that this was not an oxygen effect. That conclusion was reinforced when they subsequently found that the beneficial action of the drug could only be realized when it was administered in 5% glucose. In those studies, hydrogen peroxide in saline had no effect (Olson et al., 1978). All of the above evidence would indicate that blood catalase does not play an important role in mediating the protective action of hydrogen peroxide and therefore there is no reason to believe that the canine model is inappropriate for this study. At the same time, however, a species difference cannot be ruled out entirely. The depression o f the force gauge was reversible in these experiments b y either
260
restoring the blood flow or by the action of isoproterenol. The former action was to provide molecular oxygen and wash out metabolites thereby reversing the adverse effects of myocardial ischemia and should be considered a beneficial effect. The latter, though showing a short term improvement, must be considered an undesirable response since isoproterenol reportedly depresses the long term survival of myocardium in ischemia. Thus even if an improvement in force development were observed, it obviously would have been difficult to determine whether it were a wholly beneficial effect especially since the protective effects of hydrogen peroxide in ischemia do not appear to involve the evolution of molecular oxygen.
M. GOODLETT ET AL.
References Downey, J.M., 1976, Myocardial contractile force as a function of coronary blood flow, Amer. J. Physiol. 230, 1. Olson, R.D. and R.C. Boerth, 1978, Hydrogen peroxide: beneficial effects in rabbits following acute coronary occlusion, Amer. J. Physiol. 234, H28. Olson, R.D., E.J. Okediji and R.C. Boerth, 1978, Hydrogen peroxide versus glucose, insulin and potassium in coronary occlusion (abstract), Federation Proc. 37, 1358. Takahashi, M., Y. Horiguchi and K. Murakami, 1969, Effects of epicardial perfusion with hydrogen peroxide for ischemic myocardium, Jap. Heart J. 10, 53. Urschel, H.C., Jr., 1967, Cardiovascular effects of hydrogen peroxide;current status Dis. Chest 51,180. Urschel, H.C., Jr., A.R. Morales, J.W. Finney, G.A. Balla, G.J. Race and J.T. Mallams, 1966, Cardiac resuscitation with hydrogen peroxide, Ann. Thorac. Sur. 2,665. White, D. and P. Teasdale, 1966, The oxygenation of blood by hydrogen peroxide: in vitro studies, Brit. J. Anesthesia 38,339.