42 Experientia 41 (1985), Birkh/iuser Verlag, CH-4010 Basel/Switzerland Changes in the cardiac glutathione status after isehemia and reperfusion S. Curello ~, C. Ceconi, C. Bigoli, R. Ferrari 2, A. Albertini* and C. Guarnieri** Chair of Cardiology and *Institute of Chemistry, University of Brescia, Brescia (Italy) and **Institute of Biochemistry, University of Bologna, Bologna (Italy), 9 January 1984 Summary. In the isolated and perfused rabbit heart ischemia induced a rapid decline of contractility, associated with a reduction of the content of tissue GSH with no significant changes in GSSG. Reperfusion induced a small recovery of contractility, a substantial release of total glutathione and a further decrease in the content of tissue GSH with a significant increase of tissue GSSG. Glutathione reductase and glutathione peroxidase activities were not affected by ischemia and reperfusion. This study suggests a possible role for glutathione in the determination of functional damage induced by myocardial ischemia and reper- fusion. Key words. Glutathione heart; ischemia; reperfusion. The tripeptide glutathione is present in most living cells as re- duced glutathione (GSH), oxidized glutathione (GSSG) and as mixed glutathione disulfide (X-SSG). Glutathione metabolism has been widely studied, particularly in the liver where it plays an important role in defense mechanisms against the toxic ef- fects of reactive metabolites of oxygen and of chemical inter- mediates including various drugs 3. Relatively few studies have been undertaken to investigate the metabolism and the func- tion of gtutathione in heart muscle. Wendel et al? found in isolated and perfused hearts a GSH/ GSSG ratio close to 50, as a consequence of the glutathione reductase activity. In addition, Harfish and Mahmound 5 and Harrop et al. 6 demonstrated that X-SSG concentration in the myocardium is more elevated than in the liver. This finding is important because the turnover of cardiac glutathione is slow 7 and GSH content can be rapidly reduced by different situa- tions such as starvation 5 or chemotherapeutic treatment 8. We have previously shown 9' ~0 that hypoxia also causes a depletion of myocardial GSH content, associated with its release into the coronary effluent. The aim of the present study was to investigate the effect of myocardial ischemia, a condition different from hypoxia, and reperfusion on glutathione status, and to correlate these changes with myocardial function. Materials and methods. Adult male rabbits were used. The hearts were excised and subjected to Langendorff non-recircu- lating perfusion with Krebs-Henseleit buffer at pH 7.4, con- taining ll mM glucose and gassed with 95% O2 5% CO 2 as previously described u. Each heart was paced at 180 beats/min. After 30 rain of aero- bic perfusion (coronary flow 22 ml/min), the hearts were made ischemic by reducing the flow to 1 ml/min. During the reper- fusion the flow was restored to 22 ml/min. Left ventricular temperature was maintained at 37 ~ irrespective of coronary Effect of ischemia and reperfusion on rabbit heart tissue glutathione content and on glutathione reductase and glutathione peroxidase activi- ties Control Ischemia Ischemia + reperfusion GSH 11.76 4- 0.67 6.60 4- 0.55* 4.50 • 0.58* GSSG 0.26 4- 0.08 0.17 4- 0.03 (NS) 0.554- 0.02* (45.2) (38.8) (8.2) Glutathione reductase 45.6+2.8 48.14- 1.8 (NS) 41.0• (NS) Glutathione peroxidase 20.94- 1.2 22.5• 1.9 (NS) 23.04- 1.6 (NS) The hearts were perfused under ischemic conditions for 90 min, fol- lowed by 30 rain of reperfusion. Glutathione content was expressed as nmoles - mg prot-l. Numbers in parentheses indicate GSH/GSSG ratio. The enzymatic activities are expressed as nmole NADPH oxid.mg prot -1 9 min-I. Each result is expressed as mean of six experiments 4- SE. * Significantly different from control (p < 0.01); NS, not signifi- cantly. flow. Left ventricular pressures were monitored by means of a fluid-filled balloon inserted in the left ventricle 12. Total glutathione was assayed using the catalytic assay with 5,5'-dithiobis (nitrobenzoate) and glutathione reductase j3. GSSG was measured after preliminary reaction of GSH with 20 mM N-ethyl maleimide followed by complete removal of unreacted sulfhydryl reagent with diethylether. Glutathione re- ductase activity was measured following the oxidation of NADPH 14, and glutathione peroxidase activity as described by Grankvist et alY. Proteins were estimated by the method of Bradford 16. Coronary effluents were collected and assayed for CPK activity 12 and total glutathione is. Results. The effects of ischemia and reperfusion on mechanical function are shown in the figure A. Reduction of the coronary flow induced a rapid decline of systolic pressure and a slight increase of diastolic pressure. The following reperfusion pro- duced a further marked increase of diastolic pressure and a partial recovery of systolic function (32% of the preischemic value). The ischemic perfusion did not cause a significant increment of CPK (fig., B) or glutathione release (fig., C). On the contrary, reperfusion induced a progressive leakage of CPK and a sub- stantial release of glutathione, which reached a peak in about 3 min. The table shows that ischemia induced a significant dimi- nution of tissue GSH together with a slight reduction of GSSG Control Ischaemia . = Reperf_ 800[ ~ = 6000li 40 ~ .... ,~ ~+l A B_-r ._i E 1000~- *."'i o 750 l- } = ~ 5 --.-" ~ "--~ ~ 500~- # -~ ,=_ +1 * B ~ ~ 25Ol- o =,.E 01-~......... ~ ! = - .o .E -.~ ' ~ i ~ ~ o'~ .......... ~ * ~ ~ , = -30 0 30 60 90 120 min o..-- - .-..oCoronary flow 22 mt/min : : Coronary flow lml/min Effect of ischemia and reperfusion on mechanical function (A) and on the release of CPK (B) and glutathione (C) in isolated perfused rabbit hearts. The results are mean values + SE of six experiments. p relates to the significance of the difference between the results obtained during control and those obtained during ischemia and reperfusion.