Effects of oxygen radicals on substrate oxidation by cardiac myocytes

Biochimica et Biophysica Acta 926 (1987) 127-131 Elsevier

127

BBA 22829

E f f e c t s of o x y g e n radicals on substrate o x i d a t i o n by cardiac m y o c y t e s

K a t h l e e n H . M c D o n o u g h , J a n e J. H e n r y a n d J o h n J. Spitzer Department of Physiology, Louisiana State University Medical Center, New Orleans, LA (U.S.A.)

(Received 29 January 1987) (Revised manuscript received 29 June 1987)

Key words: Superoxide dismutase; Catalase; High energy phosphate; Xanthine; Xanthine oxidase; Myocardial metabolism; (Rat)

Freshly isolated adult rat heart cells were used to study the effects of oxygen-free radicals on the myocardial oxidation of different substrates. The calcium-tolerant quiescent cells were incubated with xanthine plus xanthine oxidase as the source of free radicals. The oxidation of exogenous glucose, lactate and octanoate was severely inhibited (approx. 70%) by products of xanthine oxidase activity. Superoxide dismutase plus catalase effectively prevented the inhibition of oxidation. Cellular high energy phosphate levels were decreased in the presence of the oxygen free radical generating system although cell viability determined by Trypan blue exclusion and light microscopic assessment of normal morphology was not affected. These data suggest that oxygen free radicals decrease myocardial substrate oxidation which may contribute to the functional and ultrastructural changes in the myocardium under conditions such as reoxygenation after hypoxia and reperfusion after ischemia.

Introduction

Superoxides, generated either physiologically, e.g., by cells of the immune system, or in artificial enzyme systems, have been shown to have a multitude of effects upon biological systems [1]. Oxygen radicals have been implicated in cellular injury resulting from myocardial ischemia and reperfusion [2-5] and from hypoxia and reoxygenation [6]. Several groups of investigators have shown that reoxygenation-induced cardiac injury is in large part prevented if agents which scavenge oxygen radicals are present during the reoxygenation [6]. Oxygen radical scavenging agents have Abbreviation: Mops, 4-morpholinepropanesulfonic acid. Correspondence: K.H. McDonough, Louisiana State University Medical Center, Department of Physiology, 1901 Perdido Street, New Orleans, LA 70112, U.S.A.

also been implicated in protection of the myocardium after short-term [2,5] and longer-term occlusion followed by reperfusion [3] and in hypothermic cardioplegic reperfusion [4]. Peroxidation of unsaturated lipids, which may then alter membrane functions, may represent the initial phase of injury. Kramer et al. [7] have recently shown that sarcolemmal and microsomal enzymes are depressed by free radical generating systems. Coincidental with the depression of membrane enzyme activities is the accumulation of malondialdehyde as a marker of membrane lipid peroxidation [7]. In this study, the sarcolemma appeared to be more susceptible to free radical-induced damage than were the microsomes. Since the superoxide anion and hydrogen peroxide can cause peroxidation of membrane lipids, we hypothesized that myocardial metabolism would also be altered by oxygen-derived free radi-

0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

128 cals. Thus the aim of these studies was to investigate the effects of oxygen-derived free radicals on myocardial substrate oxidation. From our previous studies, in which hypoxia followed by reoxygenation induced alterations in glucose oxidation after short periods of oxygen deprivation [8], we predicted that glucose oxidation might be particularly sensitive to sarcolemmal membrane changes induced by oxygen radicals. Since Guarnieri et al. [9] have demonstrated alterations in pyruvate oxidation in mitochondria isolated from rats treated with a superoxide dismutase inhibitor, we also determined whether mitochondrial oxidative metabolism was depressed by the extracellular generation of oxygen free radicals by incubating cells with lactate or octanoate. We used myocytes in this study in order to determine direct effects of oxygen-derived free radicals on metabolism. Burton et al. [10] and Ytrehus et al. [11] have recently shown that oxygen radicals can cause changes in myocardial morphometry in the isolated perfused septal preparation and in contractile performance in the Langendorff perfused rat heart. Since myocardial metabolism is normally finely matched to myocardial performance, changes in contractile function can induce alterations in metabolism. Thus, the quiescent, freshly isolated ventricular myocytes are advantageous for these investigations, since the direct effects of superoxides and hydrogen peroxides on substrate utilization can be readily assessed independently of contractile activity. Materials and Methods Myocytes were prepared by the method of Montini et al. [12] as we have previously described [8]. In brief, hearts were excised from pentobarbital-anesthetized, male, Sprague-Dawley rats (250-350 g body weight). Cells were obtained by perfusing the hearts with Jokliks phosphate buffer (minimum essential medium) supplemented with 8 mM glutamate, 1.25 m M MgSO4, 0.1% bovine serum albumin, and 0.1% collagenase (type II, Cooper Biomedical) followed by mincing of the ventricles and mechanical dispersion. Cells were purified in Jokliks-magnesium-glutamate buffer containing 1% bovine serum albumin (essentially fatty acid-free) and finally taken up in Jokliks-

magnesium buffer supplemented with 1% bovine serum albumin (fatty acid-flee), 1.5 mM CaC12 and the substrate to be studied (glucose, lactate or octanoate). Substrate oxidation. Aliquots of cells (approx. 2 - 3 mg protein) were incubated with either 5 mM [U-14C]glucose, 1 mM [U-14C]lactate or 0.5 mM [1-aaC]octanoate in 1% bovine serum albumin (fatty acid-free), Jokliks, containing 1.5 mM Ca e+, for 40 rain in a 37 ° C water bath shaking at 60 cycles per min. In flasks in which superoxide effects were to be assessed, 0.2 mM xanthine and 27 or 13 mU xanthine oxidase (Boehringer Mannheim) were included in the incubation medium (final volume 3 ml). Superoxide dismutase and catalase (at either 20 # g / m l or 40 /~g/ml) were added to some flasks in order to reverse or prevent any oxygen radical-induced damage. An equal aliquot of cells was rinsed twice with bovine serum albumin-free Jokliks and assayed for protein by the method of Lowry et al. [13]. Viability was assessed on cells treated identically to those used in the oxidation studies except that no radiolabeled substrate was added. Viability was determined by light microscopy using both Trypan blue exclusion and elongated, clearly striated, non-granular appearance as the criteria for normal morphology. For the oxidation studies, cells were incubated in 25 ml flasks fitted with rubber stoppers containing centerwells. The centerwells contained hyamine hydroxide to collect CO 2 produced by oxidation. After the 40 rain incubation of the cells with the radiolabeled substrate, oxidation was terminated by the addition of perchloric acid and CO 2 was collected into the hyamine-filled centerwells for another 60 min. Centerwells were then removed, blotted and the radioactivity quantitated by liquid scintillation spectrometry. Radioactivity of the radiolabeled substrate was also assessed and used to calculate specific activity of the substrate. Oxidation rates were expressed per mg protein. In a separate series of experiments, cells (3-4 mg) were incubated with xanthine plus xanthine oxidase at similar concentrations as in the oxidation studies but in a final volume of 1 ml. After 40 min cells were precipitated by the addition of ice-cold perchloric acid. The perchloric acid supernatant was neutralized with K O H and 10 mM

129

Mops and the neutralized extract assayed for adenosine triphosphate (ATP) and creatine phosphate by fluorometric assays as described by Lowry and Passonneau [14]. Results

The incubation of myocytes with xanthine and xanthine oxidase resulted in a marked depression in oxidation of all three substrates studied (Fig. 1). The oxidation rate of glucose was decreased by 77%, that of octanoate by 80%, and that of lactate by 60%. However, the viability measured after 40 min of incubation with xanthine plus xanthine oxidase was not significantly altered (Table I). When cells were incubated with xanthine plus xanthine oxidase for 40 min at 37°C, ATP and creatine phosphate levels were significantly decreased (Table II). The lowered ATP and creatine phosphate levels were also present in the cells incubated with the free radical generating system plus superoxide dismutase and catalase. The changes in substrate oxidation induced by oxygen-derived free radicals were prevented if superoxide dismutase and catalase were present when t,m

E

+

& E

10

+

E

TABLE I VIABILITY OF CONTROL CELLS A N D CELLS EXPOSED TO X A N T H I N E PLUS X A N T H I N E OXIDASE FOR 40 M I N Viability is expressed as the percentage of elongated cells that excluded Trypan blue and exhibited a striated appearance with no obvious granulation under the light microscope. Values are means 5- S.E.; number in parentheses is the number of experiments. Substrate

Control

Xanthine + xanthine oxidase

Glucose Octanoate Lactate

86 + 2 (13) 845:2 (7) 82 5- 5 (7)

84 5- 2 (13) 8 0 + 5 (6) 80 5- 5 (6)

the xanthine and xanthine oxidase were incubated with the cells (Fig. 2). For all three substrates, oxidation rate in the presence of superoxide dismutase and catalase was equal to or greater than the rate in control cells. When glucose, lactate and octanoate oxidation was studied in control cells in the presence of 0.2 mM xanthine, an unexpected stimulation of oxidation was observed. In the presence of xanthine, glucose oxidation increased from 2.53 _ 0.43 to 3.50 ___0.35 n m o l / m g per min, octanoate increased from 3.78 + 0.74 to 6.62 _ 1.37 n m o l / m g per min, and lactate from 7.89 + 0.97 to 9.49 + 0.91 n m o l / m g per rain. Since data were obtained from paired samples, all of these changes were significant ( p < 0.05).

c

+

_o

6

,1

2'

a

TABLE II ATP A N D C R E A T I N E PHOSPHATE C O N T E N T OF CELLS EXPOSED TO V A R Y I N G C O N D I T I O N S OF O X Y G E N F R E E RADICAL G E N E R A T I O N

m

x

o

Values are means+S.E.; n = 6; ATP and creatine phosphate values are in n m o l / m g . * P < 0.05, different from control.

uJ rr IU~

(n

[]

5 mM

0.5 mM

GLUCOSE

OCTANOATE

LACTATE

(11)

(17)

(20)

CONTROL

[]

XANTHINE

1 mM

+

X A N T H I N E OXIDASE

Fig. 1. Rate of oxidation of glucose, lactate and octanoate under control conditions and in the presence of 0.2 mM xanthine plus 13 or 27 mU of xanthine oxidase in a final volume of 3 ml. Numbers in parentheses indicate the number of experiments with each substrate.

Condition

ATP

Creatine phosphate

Control

36.9 + 3.3

40.4_+ 8.6

+ Xanthine

37.7 _+3.3

42.1 5-10.4

+ Xanthine and xanthine oxidase

25.9 + 4.9 *

28.6 + 6.4 *

+ Xanthine, xanthine oxidase, superoxide dismutase and catalase

25.8 + 3.9 *

24.1 + 6.4 *

130 []

CONTROL

[]

XANTHINE + XO

[]

XANTHINE

•

XANTHINE + XO + SOD + CAT

l'st

150

1oo[ 125 l

Oae

7st 50

25 0 GLUCOSE (n=5)

LACTATE (n=9)

OCTANOATE (n=7)

Fig. 2. Oxidation rates expressed as percent of control cells. Three conditions were studied: cells plus 0.2 mM xanthine; cells in the presence of 0.2 mm xanthine plus xanthine oxidase (XO), plus superoxide dismutase (SOD) and catalase (CAT). Substrate concentrations were the same as in Fig. 1. Oxidation rates in control cells were 2.53+0.43 nmol/mg per min for glucose, 9.92_+0.91 nmol/mg per min for lactate and 3.78_+0.78 nmol/mg per rain for octanoate. All values with xanthine and xanthine plus xanthine oxidase were significantly different from control ( P < 0.05).

Discussion Our data demonstrate that the extracellular generation of superoxides a n d / o r hydrogen peroxides has a powerful inhibitory effect on myocardial utilization of the exogenously supplied substrates glucose, lactate and octanoate. Since all three substrates were affected to approximately the same extent, there does not appear to be a differential effect on cytosolic metabolic processes and mitochondrial oxidative processes. In previous studies, we had noted such a differential effect occurring after short periods of oxygen deprivation followed by reoxygenation [8]. In that study, glucose hydrolysis was impaired whereas octanoate and pyruvate oxidation were unaltered by 5 rain of 0 2 deprivation followed by reoxygenation. Kramer and co-workers [7] have observed a

greater sensitivity to oxygen free radical-induced lipid peroxidation in sarcolemmal membranes as compared to microsomal membranes. Along with the faster rate of lipid peroxidation of sarcolemmal membranes there was a greater magnitude of depression of the sarcolemmal enzyme ( N a + + K+)-ATPase than there was of the microsomal enzyme rotenone-insensitive N A D H - c y t o c h r o m e c reductase. These investigators also showed an enhanced lipid peroxidation of sarcolemmal vesicles preincubated with physiologically occurring amphiphiles [15]. In our study no comparable differences were observed, either because mitochondria are as sensitive to oxygen free radical-induced alterations as are the cytosolic enzyme systems, or because the damage was severe enough to obliterate such differences. Our results are consistent with those of Guarnieri et al. [9] in which injection of diethyldithiocarbonate into rats inhibited mitochondrial and cytosolic superoxide dismutase and resulted in elevated malondialdehyde levels in the isolated mitochondria as well as decreased oxidative phosphorylation. Interestingly, viability, as determined by light microscopic assessment of morphology and Trypan blue exclusion, showed no significant change after a 40 min incubation with the oxygen free radical generating system. More specific methods of measuring membrane damage [16,17] may be required to detect subtle morphologic and plasma membrane changes in the myocytes. Noronha-Dutra and Steen [16] have used scanning and transmission electron microscopy and shown plasma membrane and mitochondrial ultrastructural changes in myocytes incubated with agents which induce lipid peroxidation. Scott et al. [17] have used uptake of fluorescent antimyosin as an indication of sarcolemmal damage in cultured myocytes incubated with a free radical generating system. These methods are more sensitive to changes in myocyte sarcolemma than were our methods. Myocyte high energy phosphate levels were reduced by a 40 min incubation with xanthine plus xanthine oxidase even when superoxide dismutase and catalase were present. This difference between high energy phosphates and oxidation may represent differential sensitivities or different time courses for response. The decreased ATP levels

131 m a y lead ultimately to changes in viability not seen at the time point used in the present study. Ytrehus et al. [11] have shown changes in m y o c a r dial high energy phosphate levels in L a n g e n d o r f f hearts perfused with h y p o x a n t h i n e and xanthine oxidase by as early as 10 min after perfusion. After reperfusion creatine phosphate levels returned to normal but A T P levels were still decreased. Adenosine triphosphate levels were also not returned to those of the control in a study of Przyklenk and Kloner [5] in which superoxide dismutase plus catalase protected the function of the m y o c a r d i u m f r o m a short-term ischernic insult followed by reperfusion but did not improve the A T P response. A l t h o u g h the oxygen free radicals in our experiments were exogenously supplied, xanthine oxidase can be physiologically derived from xanthine dehydrogenase, which is located on the capillary endothelium, under ischemic conditions [1,18]. Recent evidence would suggest that oxygen free radicals m a y be involved in d a m a g e to the reprefused, ischemic myocardium. Jolly et al. [3] have shown that early reperfusion with superoxide dismutase plus catalase can limit the size of infarction in dogs in which the circumflex c o r o n a r y artery was occluded for 90 min. More recently, neutrophils have been implicated as an important source of oxygen free radicals, since reperfusion studies with b l o o d depleted of leukocytes has led to m u c h smaller infarctions of the heart [3]. Activated leukocytes have also been shown by Rowe et al. [19] to inhibit Ca 2+ uptake by sarcoplasmic reticulum vesicles. O u r data would suggest that some of the reperfusion d a m a g e induced by oxygen free radicals m a y result f r o m depression of m y o c a r d i a l metabolism in the area of the m y o c a r d i u m at risk. Since leukocyte infiltration correlates with histological evidence of infarction [20], leukocytic production of superoxides, if not sufficiently counteracted by endogenous free radical scavenging systems, m a y depress m y o c y t e oxidative metabolism, high energy phosphate production, and thus cell viability. Administration of superoxide dismutase plus catalase can prevent this sequence of events.

Thus, in addition to the other deleterious effects of oxygen free radicals, they also appear to depress the oxidative utilization of substrates b y the myocardium.

Acknowledgement This work was supported by N I H G r a n t H L 32749.

References 1 McCord, J.M. and Roy, R.S. (1982) Can. J. Physiol. Pharmacol. 60, 1346-1352 2 Gross, G.J., Farber, N.E., Hardman, H.F. and Warltier, D.C. (1986) Am. J. Physiol. 250, H372-H377 3 Jolly, S.R., Kane, W.J., Baile, M.B., Abrams, G.D. and Lucchesi, B.R. (1984) Circ. Res. 54, 277-285 4 Otani, H., Vmemoto, M., Kagawa, K., Nakamura, Y., Omoto, K., Tanaka, K., Sato, T., Nonoyama, A. and Kagawa, T. (1986) J. Surg. Res. 41, 126-133 5 Przyklenk, K. and Kloner, R.A. (1986) Circ. Res. 73, 148-156 6 Gaudel, Y. and Duvelleroy, M.A. (1984) J. Mol. Cell. Cardiol. 16, 459-470 7 Kramer, J.H., Mak, I.T. and Weglicki, W.B. (1984) Circ. Res. 55, 120-124 8 McDonough, K.H. and Spitzer, J.J. (1983) Proc. Soc. Exp. Biol. Med. 173, 519-528 9 Guamieri, C., Flamigni, F., Ventura, C. and RossoniCaldarera, C. (1981) Biochem. Pharmacol. 30, 2174-2176 10 Burton, K.P., McCord, J.M. and Ghai, G. (1984) Am. J. Physiol. 246, H776-H783 11 Ytrehus, K., Mykelbust, R. and Mjos, O.D. (1986) Cardiovasc. Res. 20, 597-603 12 Montini, J., Bagby, G.J., Bums, A.H. and Spitzer, J.J. (1981) Am. J. Physiol. 240, H659-H663 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 266-275 14 Lowry, O.H. and Passonneau, J.V. (1972) A Flexible System of Enzymatic Analysis, pp. 151-153, Academic Press, New York 15 Mak, I.T., Kramer, J.N. and Weglicki, W.B. (1986) J. Biol. Chem. 261, 1153-1157 16 Noronha-Dutra, A.A. and Steen, E.M. (1982) Lab. Invest. 47, 346-353 17 Scott, J.A., Khaw, B.A., Locke, E., Haber, E. and Homcy, C. (1985) Circ. Res. 56, 72-77 18 Hearse, D.J., Manning, A.S., Downey, J.M. and Yellon, D.M. (1986) Acta Physiol. Scand. (Suppl.) 548, 65-78 19 Rowe, G.T., Manson, N.H., Caplan, M. and Hess, M.L. (1983) Circ. Res. 53, 584-591 20 Lucchesi, B.R. and Mullane, K.M. (1984) Annu. Rev. Pharmacol. Toxicol. 26, 201-224