Camp. Biochem. Physiol. Vol. 104C, No. I, pp. 171-173, 1993 Printed in Great Britain

0306~4492/93 $6.00 + 0.00 0 1993 Pergamon Press Ltd

INTERACTION OF H,O, AND DITHIOTHREITOL AND THE GENESIS OF CELLULAR DAMAGE IN THE

PERFUSED RAT HEART

STEPHANIE DANIELS and CHRISTOPHER J. DUNCAN

Department of Environmental and Evolutionary Biology, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. (Tel. 051 794-2000; Fax 051 708-6502)

(Received 17 July 1992; accepted for publication 28 August 1992)

Abstract-l. H,O, (0.3 mM) causes release of creatine kinase (CK) from the perfused rat heart, but only when activated by removal of extracellular Ca2+.

2. Dithiothreitol (DTT), 2 mM, provides partial protection against this damage induced by artificially- introduced 0, radicals.

3. Paradoxically, D’IT triggers CK release by H,O, in the presence of extracellular Ca2+. The interaction of these agents in stimulating and protecting against this damage to the sarcolemma is discussed.

INTRODUCYION

The production of OX free radicals has been linked to a variety of pathophysiological states (Halliwell and Gutteridge, 1984) including the damage process of both the Ca*+- and 02-paradoxes of the mammalian heart (Hess and Manson, 1984). This hypothesis is supported by the detection of active 0, metabolites in the perfusate from the damaged heart (Bolli et al.. 1988; Zweier, 1988; Zweier et al., 1989) and by the partial protection provided by a variety of O2 radical scavengers (Duncan, 1990). However, the generation and detection of 0, radicals during cellular damage does not necessarily mean that they are the causative agent; they may be simply the by-products of a network of events triggered by Ca’+. We report here on the effects of artificially generating O2 radicals by perfusing rat hearts with H,O,, and show that it causes CK release, but only when the system is already activated by the removal of extracellular Ca*+. However, dithiothreitol (DTT), a protective agent for -SH groups, gave apparently anomalous results.

METHODS

Wistar rats (25o-400 g) were used throughout the experiments; they were lightly anaesthetized in ether prior to intraperitoneal injection of heparin (1000 U/kg) and were killed by cervical dislocation 20 min later. The heart was dissected out, transferred to ice-cold perfusion medium and perfused at 37°C using a modified, non-recirculating Langendorff per- fusion apparatus with three limbs to allow rapid change of medium. Perfusion was at a constant pressure of 750mm water, and mechanical activity was recorded throughout the perfusion with a Bio- sciences Dl isometric transducer and a Washington

MD1 oscillograph. Coronary flow was measured throughout the perfusion.

Hearts were perfused with Krebs-Henseleit medium which contained (mM) NaCl 117, KC1 5, NaHCO, 25, MgC12 1, CaCl, 2.5, NaH,PO, 1, glu- cose 11 made up in distilled water that had been passed through a deionizing column and which was then gassed with 95% O2 + 5% CO2 or 95% N, + 5% CO,; pH = 7.4 + 0.05. Ca*+-free solutions had CaCl, omitted without ionic substitution.

Immediately after mounting, the hearts were per- fused with standard medium for a 20 min wash-out and equilibration period to establish baseline levels of CK release, mechanical activity and perfusion rates. Samples of coronary effluent were taken at regular intervals for measurements of CK activity, using the linked assay method of Jones et al. (1983), the production of NADPH being followed at 340 nm. Blank assays were also run with the various test agents included.

All inorganic salts were AnalaR grade; biochemi- cals and dithiothreitol (DTT) were obtained from Sigma Chemical Co., St Louis, U.S.A. and hexoki- nase glucosed-phosphate dehydrogenase preparation was obtained from Boehringer Mannheim U.K., Lewes, E. Sussex, U.K.

RESULTS

Hearts were perfused for 40 min with 0.3 mM H202 in a Ca’+-containing medium when there was a rapid decline in developed tension but only small amounts of CK released (18.8 f 6 IU/40 min perfusion per g dry wt).

Hearts were perfused for 40 min with 0.3 mM H202 in a Ca*+-free medium. After a delay of lOmin, a massive, linear release of CK was observed resulting in a total of 536 f 25 IU/g dry wt (Fig. 1). The

171

172 S. DANIELS and C. J. DUNCAN

0 10 20 30 40

T/ME [MINI

Fig. 1. 0.3 mM H,O, plus 2mM DTT in a Caz+-free medium (N = 5; solid circles). CK release was significantly reduced (P < 0.001) compared with perfusion with H,O, alone in a Ca*+-free medium (N = 4; open circles, dashed

line).

regression lines were compared directly by an analysis of covariance programme on an IBM PC AT, and this release of CK was significantly greater than that occurring during the perfusion of hearts with H,O, in the presence of Ca*+ (P < 0.001).

Hearts were perfused for 40 min with 0.3 mM H202 in a Ca*+-free medium in the presence of 2 mM DTT. H, O,-induced CK release was significantly reduced by approximately 50% by DTT (P < 0.001); the total CK release during the 40min perfusion period was 294 f 63 III/g dry wt (Fig. 1).

600 r

I

+ “2O2 /’

200 - -I #‘O

1” /-’ A

,?

,+

+DTT

~8~ - DTT

0_,~~__‘_~___~___~---~---‘1-” 0---O

, * ’ * ’ * * ’ ’ 0 IO 20 30 40

TIME IMINJ

Fig. 2. Perfusion with 0.3 mM H,O, in the presence of 2 mM DlT in a Ca’+containing medium (N = 5; solid circles). CK release was significantly increased (P < 0.001) com- pared with the results of perfusion with H,O, in the absence of DTT and in the presence of [Ca*+ 1, (N = 4; open squares, dashed line) but was significantly less (P < 0.001) than that found with perfusion of H,O, in Caz+-free medium (N = 4;

open circles, dashed line).

The introduction of 0.3 mM H202 plus 2 mM DTT in the presence of Ca2+ (when minimal CK release would be expected) caused a decline in developed tension (indicating damage to the contractile machin- ery) and a significant increase (P < 0.001) in CK release (Fig. 2) compared with the very small values observed in control hearts (i.e. hearts perfused with H,O, in a Ca*+-containing medium).

DISCUSSION

H,O, has been used in the present experiments as a way of generating O2 radicals; it is an active Or species that can either directly peroxidise susceptible cell components or be converted to the more reactive hydroxyl radical, ‘OH. The probable cellular targets are -SH groups; if these are on key proteins, severe damage may ensue from this oxidative attack. Perfusion with 0.3 mM H202 in a Ca2+-containing medium caused only a very low CK release. Previous work has revealed a small (non significant) increase in LDH release in rat hearts perfused with 0.13 mM H202 (Konz et al., 1989), and 2 mM H202 has been shown to produce leaky membranes (Deuticke et al., 1986). It is therefore concluded that the oxygen radicals generated by perfusion with H,O, may be able to act directly on the sarcolemma and produce micro-lesions which cause this very low leakage of CK, although this effect does not play a major role in the H,O,-induced damage recorded in this communication.

In contrast, a massive release of CK occurred in a Ca’+-free medium containing 0.3 mM H202 and it is concluded that the action of H,O, is similar to that of caffeine; both agents act to raise intracellular Ca*+ concentration by release from intracellular Ca*+ storage sites thereby causing the initial activation of the damage system, but massive CK release begins only when the molecular complex at the sarcolemma controlling damage is synergistically activated by removal of extracellular Ca*+ (Daniels and Duncan, 1990). Caffeine causes the release of Ca*+ from the sarcoplasmic reticulum; H,O, is known to depress the heart sarcolemma Ca*+-pump activity via the rapid oxidation of -SH groups in the sarcolemma causing an inhibition of the Ca2+-pump and the subsequent disruption of Ca*+ homeostasis (Kaneko et al., 1989). H202 also inhibits the Ca*+-ATPase of the heart sarcoplasmic reticulum, accompanied by a decrease in its -SH groups (Scherer and Deamer, 1986; Kukreja et al., 1988).

DTT maintains -SH groups in the reduced state (Cleland, 1964) and prevents both the inhibition of the sarcolemma Ca*+-pump (Kaneko et al., 1989) and the inhibition of the Ca*+-ATPase of the sar- coplasmic reticulum by H202 (Scherer and Deamer, 1986; Kukreja et al., 1988). DTT also decreased the H,O,-induced CK release (in the absence of Cat’) in the present experiments by 50%. Since H,O, has no major effect on CK release in the presence of

H,O, and dithiothreitol 173

extracellular Ca2+ but causes massive release on Caa+-depletion, it is evident that damage by the oxygen radicals does not promote an increased influx of Ca2+ across a damaged sarcolemma. It is suggested therefore that H202 inhibits the sarcoplasmic reticu- lum Ca2+-ATPase in perfused hearts via oxidation of sulphydryl groups, so raising [Ca2+li. However, as with exposure of the heart to caffeine, although a rise in [Ca2+li is an essential prerequisite for cellular damage, it is not normally sufficient alone, and for CK release to occur, a synergistic activation of the damage system at the sarcolemma by Cai+-depletion

is necessary. An unexpected and marked release of CK occurred

in hearts perfused with H202 plus DTT in a Ca2+- containing medium; under these conditions no release would be expected. Clearly DTT now has a stimu- latory and not a protective action. One explanation might be that H,O, plus DTT act together to aug- ment the production of 0, radicals (Reeves et al.,

1986), perhaps leading to a steeper and greater rise in [Ca2+li than that produced by H202 alone that is sufficient to activate the CK release mechanism with- out the additional, synergistic activation by removal of extracellular Ca2+. Alternatively, H202 acts to raise [Ca2+ Ii, as before, and DTT may, in some way, replace Ca2+-depletion as the synergistic activator of the damage process.

The results illustrated in Figs 1 and 2 therefore represent the resultant interaction of several conflict- ing processes: the active oxygen species oxidise -SH groups on the sarcoplasmic reticulum, thereby releas- ing Ca2+. DTT provides partial protection of these -SH groups and so, on the one hand, reduces CK in the synergistically activated system; on the other hand it interacts with H20z, thereby exacerbating the oxidative attack, raising [Ca2+], more dramatically and markedly augmenting CK release. Thus, the true protective effect of DTT on the -SH groups may be much greater than is suggested in Fig. 1. The major effect of DTT plus H,O, (probably via an additional and substantial rise in [Ca’+],) is shown in Fig. 2, where, in spite of DTT protection of -SH groups and in the absence of the synergistic activation of Ca2+-depletion, significant CK release is seen.

Acknowledgements-This work was supported by Smith Kline Beecham and Action Research. We thank Miss S. Scott for assistance in the preparation of the manuscript. S.D. was in receipt of an SERC studentship.

REFERENCES

Bolli R., Pate1 B. S., Jeroudi M. O., Lai E. K. and McCay P. B. (1988) Demonstration of free radical generation in “stunned” myocardium of intact dogs with the use of the spin trap phenyl N-tert-butyl nitrone. J. Clin. Invest. 82, 476-485.

Cleland W. W. (1964) Dithiothreitol, a new protective reagent for SH groups. Biochemisfry 3, 480482.

Daniels S. and Duncan C. J. (1990) Priming the damage system that causes the release of cytosolic proteins in the uerfused rat heart. Biochem. Sot. Trans. 18. 608-606.

Deuticke D., Heller K. B. and Haest C. W. M. (1986) Leak formation in human erythrocytes by the radical-forming oxidant t-butylhydroperoxide. Biochim. Biophys. Acta 854, 169-183.

Duncan C. J. (1990) Biochemical events associated with rapid cellular damage during the oxygen- and calcium- paradoxes of the mammalian heart. Experientia 48, 4148.

Halliwell B. and Gutteridge J. M. C. (1984) Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219, I-14.

Hess M. L. and Manson N. H. (1984) Molecular oxygen: friend and foe. The role of the oxygen free radical system in the calcium paradox, the oxygen paradox and ischemia/reperfusion injury. J. Mol. Cell. Cardiol. 16, 969-985.

Jones D. A., Jackson M. J. and Edwards R. H. T. (1983) The release of intracellular enzymes from an isolated mammalian skeletal muscle preparation. Clin. Sci. 65, 193-201.

Kaneko M., Elimban V. and Dhalla N. S. (1989) Mechan- ism for depression of heart sarcolemmal Ca2+ pump by oxygen free radicals. Am. J. Physiol. 257, H804H811.

Konz K.-H., Haap M., Hill K. E., Burk R. F. and Walsh R. A. (1989) Diastolic dysfunction of perfused rat hearts induced by hydrogen peroxide. Protective effect of sel- enium. J. Mol. Cell. Cardiol. 21, 789-795.

Kukreja R. C., Okabe E., Schrier G. M. and Hess M. L. (1988) Oxygen radical-mediated lipid peroxidation and inhibition of Ca2+ ATPase activity of cardiac sarcoplas- mic reticulum. Archs Biochem. Biophys. 261, 447457.

Reeves J. P., Bailey C. A. and Hale C. C. (1986) Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles. J. biol. Chem. 261, 49484955.

Scherer N. M. and Deamer D. W. (1986) Oxidative stress impairs the function of sarcoplasmic reticulum by oxi- dation of sulfbydryl groups in the Ca2+-ATPase. Archs Biochem. Biophys. 246, 589601.

Zweier J. L. (1988) Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanisms of reperfusion injury. J. biol. Chem. 263, 1353-1355.

Zweier J. L., Kuppusamy P., Williams R., Rayburn B. K., Smith D., Weisfeldt M. L. and Flaherty J. T. (1989) Measurement and characterization of postischemic free radical generation in the isolated perfused heart, J. biol. Chem. 264, 18890-18895.