J Mol
Cell
Cardiol
Interstitial
23, 149- 159 (1991)
Equilibration its Protective
of Superoxide Dismutase meet in the Isolated Rabbit
Bassam Webb- Waring Lung Institute,
Correlates Heart
with
and Joe M. McCord
A. Oznar
University of Colorado, Denver, Colorado 80262, USA
(Received 2 October 1989, accepted in revised form 22 August 1990) B. A. OMAR AND J. M. MCCORD. Interstitial Equilibration of Superoxide Dismutase Correlates with its Protective Effect in the Isolated Rabbit Heart. 30umal of Molecular and Cellular Cardiology (1991) 23, 149-159. We examined the effect of preischemic equilibration of the rabbit heart with superoxide dismutase (SOD) on the extent of recovery of contractile function following an episode of ischemia. First, hearts were perfused with Krebs-Henseleit buffer. The pulmonary artery was cannulated and its flow diverted as the vascular effluent, and all other orifices were tied off. The fluid seeping from the epicardial surface represented the interstitial outflow. SOD was added to the perfiusate and the interstitial and vascular entuents were assayed for SOD at regular intervals. Second, hearts were perfused in the LangendorfTmode. SOD was included in the perfusate at all times at 20 000 U/l. After either 15 or 50 min of equilibration the hearts were subjected to 1 h of ischemia followed by 1 h ofreperfusion. The developed tension was measured via a balloon in the left ventricle. Control hearts showed a recovery of developed tension of 63 f 12%. H uman recombinant (h) Cu,Zn-SOD, which equilibrated with the interstitial fluid by 20 * 10% and 92 f 7% after 15 and 60 min ofperfusion respectively, caused a recovery of 68 & 29% (non-significant) and 92 f 18% (P < 0.01) with 15 and 50 min of equilibration respectively. The positively charged hrMn-SOD and sheep Cu,Zn-SOD, however, equilibrated much faster reaching 84 + 139a and 95 f 11% at 15 min respectively, which correlated with a recovery of99 f 11% and 96 k 10% (P < 0.01) respetively. HrCu,Zn-SOD conjugated to polyethylene glycol equilibrated much slower reaching 38 5 10% after 1 h, which correlated with lack of protection even after 50 min of equilibration. Therefore, the protection afforded by SOD to the isolated rabbit heart correlates with the concentration of SOD in the interstitial fluid. The rate of equilibration depends on the charge as well as the size of the enzyme. KEY
WORDS:
Interstitial
equilibration;
Superoxide
dismutase;
Myocardial
ischemia;
Reperfusion
injuT;
Free
radicals.
Introduction The protection of the myocardium by superoxide dismutase (SOD) against reperfusion or reoxygenation injury has been disputed. While some investigators have established such a protecctive effect (McCord et al., 1989; Casale et al., 1983), others failed to document any protection (Myers et al., 1985; Menasche et al., 1986). In a recent study (Omar et al., 1990) we found that cardioprotection with SOD depends on the dose of SOD, the model of ischemia/reperfusion and the animal species used. Unexpectedly, the dose response curves in three different models all showed relatively sharp maxima, with SOD losing its ability to protect at higher concentrations. In this study we have addressed the problem of pretreatment with SOD prior to ischemia, since it is a relatively large molecule and, 0022-2828/91/020149+
I1 $03.00/O
therefore, may need a long time to equilibrate with the interstitial space. We believe that it is important for SOD to be present in the inter&Gum as early as possible, since free radical production is a long term process that starts with ischemia itself, but may continue for hours into reperfusion (Chi et al., 1989). We examined the ability of different SODS at equivalent doses to equilibrate between the vascular and interstitial spaces. This equilibration was correlated with the physicochemical characteristics of the SODS used, as a lower molecular weight enzyme with a positive net charge would be expected to better adhere to the endothelial surface and, perhaps, cross to the interstitial space. This vascular to interstitial equilibration correlated well with the length of preischemic treatment period required to elicit a protective effect by SOD. 0
1991 Academic
Press Limited
B. A. Omar
150
and J. M. McCord
Methods (I)
Enzyme analysis
Sheep Cu,Zn-SOD was isolated from sheep liver essentially as described by McCord and Fridovich ( 1969). Bovine Cu,Zn-SOD was kindly provided by DDI, Mountain View, California. HrCu,Zn-SOD modified by covalent attachment of polyethylene glycol (an average of 16 molecules of MW 3000 per SOD molecule) was a gift from Pharmacia, Uppsala, Sweden. Unmodified hrCu,Zn-SOD and hrMn-SOD were provided by Biotechnology General, New York. All SOD preparations were assayed and found to possess 4,000 standard units/mg.
(2) Determination of the apparent molecular weight of polyethylene glycol-mod$ed hrCu,
Cell
Cardiol
Interstitial
23, 149- 159 (1991)
Equilibration its Protective
of Superoxide Dismutase meet in the Isolated Rabbit
Bassam Webb- Waring Lung Institute,
Correlates Heart
with
and Joe M. McCord
A. Oznar
University of Colorado, Denver, Colorado 80262, USA
(Received 2 October 1989, accepted in revised form 22 August 1990) B. A. OMAR AND J. M. MCCORD. Interstitial Equilibration of Superoxide Dismutase Correlates with its Protective Effect in the Isolated Rabbit Heart. 30umal of Molecular and Cellular Cardiology (1991) 23, 149-159. We examined the effect of preischemic equilibration of the rabbit heart with superoxide dismutase (SOD) on the extent of recovery of contractile function following an episode of ischemia. First, hearts were perfused with Krebs-Henseleit buffer. The pulmonary artery was cannulated and its flow diverted as the vascular effluent, and all other orifices were tied off. The fluid seeping from the epicardial surface represented the interstitial outflow. SOD was added to the perfiusate and the interstitial and vascular entuents were assayed for SOD at regular intervals. Second, hearts were perfused in the LangendorfTmode. SOD was included in the perfusate at all times at 20 000 U/l. After either 15 or 50 min of equilibration the hearts were subjected to 1 h of ischemia followed by 1 h ofreperfusion. The developed tension was measured via a balloon in the left ventricle. Control hearts showed a recovery of developed tension of 63 f 12%. H uman recombinant (h) Cu,Zn-SOD, which equilibrated with the interstitial fluid by 20 * 10% and 92 f 7% after 15 and 60 min ofperfusion respectively, caused a recovery of 68 & 29% (non-significant) and 92 f 18% (P < 0.01) with 15 and 50 min of equilibration respectively. The positively charged hrMn-SOD and sheep Cu,Zn-SOD, however, equilibrated much faster reaching 84 + 139a and 95 f 11% at 15 min respectively, which correlated with a recovery of99 f 11% and 96 k 10% (P < 0.01) respetively. HrCu,Zn-SOD conjugated to polyethylene glycol equilibrated much slower reaching 38 5 10% after 1 h, which correlated with lack of protection even after 50 min of equilibration. Therefore, the protection afforded by SOD to the isolated rabbit heart correlates with the concentration of SOD in the interstitial fluid. The rate of equilibration depends on the charge as well as the size of the enzyme. KEY
WORDS:
Interstitial
equilibration;
Superoxide
dismutase;
Myocardial
ischemia;
Reperfusion
injuT;
Free
radicals.
Introduction The protection of the myocardium by superoxide dismutase (SOD) against reperfusion or reoxygenation injury has been disputed. While some investigators have established such a protecctive effect (McCord et al., 1989; Casale et al., 1983), others failed to document any protection (Myers et al., 1985; Menasche et al., 1986). In a recent study (Omar et al., 1990) we found that cardioprotection with SOD depends on the dose of SOD, the model of ischemia/reperfusion and the animal species used. Unexpectedly, the dose response curves in three different models all showed relatively sharp maxima, with SOD losing its ability to protect at higher concentrations. In this study we have addressed the problem of pretreatment with SOD prior to ischemia, since it is a relatively large molecule and, 0022-2828/91/020149+
I1 $03.00/O
therefore, may need a long time to equilibrate with the interstitial space. We believe that it is important for SOD to be present in the inter&Gum as early as possible, since free radical production is a long term process that starts with ischemia itself, but may continue for hours into reperfusion (Chi et al., 1989). We examined the ability of different SODS at equivalent doses to equilibrate between the vascular and interstitial spaces. This equilibration was correlated with the physicochemical characteristics of the SODS used, as a lower molecular weight enzyme with a positive net charge would be expected to better adhere to the endothelial surface and, perhaps, cross to the interstitial space. This vascular to interstitial equilibration correlated well with the length of preischemic treatment period required to elicit a protective effect by SOD. 0
1991 Academic
Press Limited
B. A. Omar
150
and J. M. McCord
Methods (I)
Enzyme analysis
Sheep Cu,Zn-SOD was isolated from sheep liver essentially as described by McCord and Fridovich ( 1969). Bovine Cu,Zn-SOD was kindly provided by DDI, Mountain View, California. HrCu,Zn-SOD modified by covalent attachment of polyethylene glycol (an average of 16 molecules of MW 3000 per SOD molecule) was a gift from Pharmacia, Uppsala, Sweden. Unmodified hrCu,Zn-SOD and hrMn-SOD were provided by Biotechnology General, New York. All SOD preparations were assayed and found to possess 4,000 standard units/mg.
(2) Determination of the apparent molecular weight of polyethylene glycol-mod$ed hrCu,
