Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): Lethal peroxidative membrane injury

JOURNAL OF CELLULAR PHYSIOLOGY 149:347-364 (1991)

Hydrogen Peroxide-Induced Oxidative Stress to the Mammalian Heart-Muscle Cell (Cardiomyocyte): Lethal Peroxidative

Membrane Injury DAVID R. JANERO,* DAVID HRENIUK, AND HAAMID M. SHARIF

Research Department, Pharmaceuticals Division, CIBA-CHCY Corporation, Summit, New jersey 0790 1

Oxidative stress induced by hydrogen peroxide (H,O,) may contribute to the pathogenesis of ischemic-reperfusion injury in the heart. For the purpose of investigating directly the injury potential of H,O, on heart muscle, a cellular model of H,O,-induced myocardial oxidative stress was developed. This model employed primary monolayer cultures of intact, beating neonatal-rat cardiomyocytes and discrete concentrations of reagent H,O, in defined, supplement-free culture medium. Cardiomyocytes challenged with H,O, readily metabolized it such that the culture content of H,O, diminished over time, but was not depleted. The consequent H,O,-induced oxidative stress caused lethal sarcolemmal disruption (as measured by lactate dehydrogenase release), and cardiomyocyte integrity could be preserved by catalase. During oxidative stress, a spectrum of cellular derangements developed, including membrane phospholipid peroxidation, thiol oxidation, consumption of the major chain-breaking membrane antiperoxidant (a-tocopherol), and ATP loss. No net change in the protein or phospholipid contents of cardiomyocyte membranes accompanied H,O,-induced oxidative stress, but an increased turnover of these membrane constituents occurred in response to H,O,. Development of lethal cardiomyocyte injury during H,O,-induced oxidative stress did not require the presence of H,O, itself; a brief ”pulse” exposure of the cardiomyocytes to H,O, was sufficient to incite the pathogenic mechanism leading to cell disruption. Cardiomyocyte disruption was dependent upon an intracellular source of redox-active iron and the iron- dependent transformation of internalized H,02 into products (e.g., the hydroxyl radical) capable of initiating lipid peroxidation, since iron chelators and hydroxyl- radical scavengers were cytoprotective. The accelerated turnover of cardiomyocyte-membrane protein and phospholipid was inhibited by antiperoxidants, suggesting that the turnover reflected molecular repair of oxidized membrane constituents. Likewise, the consumption of a-tocopherol and the oxidation of cellular thiols appeared to be epiphenomena of peroxidation. Antiperoxidant interventions coordinately abolished both H,O,-induced lipid peroxidation and sarcolemmal disruption, demonstrating that an intimate pathogenic relationship exists between sarcolemmal peroxidation and lethal compromise of cardiomyocyte integrity in response to H,O,-induced oxidative stress. Although sarcolem- ma1 peroxidation was causally related to cardiomyocyte disruption during H,O,-induced oxidative stress, a nonperoxidative route of H,O, cytotoxicity was also identified, which was expressed in the complete absence of cardiomyocyte- membrane peroxidation. The latter mode of H,O,-induced cardiomyocyte injury involved ATP loss such that membrane peroxidation and cardiomyocyte disruption on the one hand and cellular de-energization on the other could be completely dissociated. The cellular pathophysiology of H,O, as a vectorial signal for cardiomyocyte necrosis that “triggers” irreversible peroxidative disruption of the sarcolemma has implications regarding potential mechanisms of oxidative injury in the postischemic heart.

Myocardial ischemia is a disease process established when the coronary blood supply to heart muscle be- comes insufficient for continued oxidative metabolism by the basic unit of pump function, the heart-muscle cell (cardiomyocyte) (Janero, 1991). Although timely

0 1991 WILEY-LISS, INC.

reestablishment of nutritive coronary flow is the fundamental clinical strategy for limiting ischemic cardi-

Received April 5, 1991; accepted June 28, 1991. T o whom reprint requestskorrespondence should be addressed.

348 JANERO ET AL.

omyocyte necrosis, this therapeutic approach carries with it a component of myocardial damage termed “reperfusion injury” (Flaherty and Weisfeldt, 1988). The factors responsible for reperfusion injury to the cardiomyocyte are ill-defined and may include phos- pholipase activation (Chien et al., 19841, fatty-amphiphile overload (Janero et al., 1988), calcium overload (Opie, 1989a) mitochondria1 dysfunction (Shin et al., 1989), formation/action of lipid mediators (Jan- ero and Burghardt, 1990), and sarcolemmal disruption (Jennings et al., 1990). Since the prognosis for the ischemic heart-disease patient is inversely related to the extent of myocardial necrosis (Bigger et al., 19841, increased mechanistic understanding of the pathogenesis of myocardial ischemic-reperfusion injury represents a fundamental concern of both experimental pathology and clinical cardiology.

Species of partially-reduced oxygen are increasingly being implicated as causing tissue damage during myocardial ischemia-reperfusion (Kloner et al., 1989; Janero, 1990a). These nontetravalent reduction products of molecular oxygen are of two general types: “free radicals” containing an unpaired electron, such as the superoxide anion radical (O,?) and the hydroxyl radical (‘OH), and a nonradical inorganic hydroperoxide, hydrogen peroxide (HzOz) (Pryor, 1986). Tissue injury associated with partially-reduced oxygen reflects a state of “oxidative stress,” under which bioactive oxidants present in excess of tissue detoxifying capabilities reach local levels sufficient to incite and promote tissue damage (Janero, 1990a). The myocardial content of partially-reduced oxygen increases dramatically with postischemic reperfusion of mammalian (Bolli et al., 1989) [including human (Davies e t al., 199011 hearts. Multiple sources of partially-reduced oxygen have been identified in the reperfused myocardium (Schmid-Schonbein and Engler, 1987). Furthermore, antioxidant therapy reduces the extent of postischemic myocardial dysfunctioninecrosis in a t least some organ (Konz et al., 1989; Massey and Burton, 1989) and animal (Buchwald et al., 1989) models and may afford cardioprotection in humans (Janero, 1991).

Despite experimental and clinical evidence that oxidative stress has a causative role in cardiac ischemic-

~

BME DPPD DTNB EDTA GSH HEPES HPLC

LDH MDA NAD OH 0 2 - PBS SDS SOD TBA TBARS TCA Tris

H,O,

Abbreviations

Eagle’s basal medium NJV’-diphenyl-l,4-phenylenediamine 5,5’-dithiobis-(2-nitrobenzoic acid) ethylene diaminetetraacetic acid reduced glutathione N-2-hydroxyethylpiperazine-N‘-2‘-ethanesulfonic acid high-pressure liquid chromatography hydrogen peroxide lactate dehydrogenase malondialdehyde nicotinamide adenine dinucleotide hydroxyl radical superoxide anion radical phosphate-buffered saline sodium dodecylsulfate superoxide dismutase thiobarbituric acid thiobarbituric acid-reactive substances trichloroacetic acid tris(hydroxymethy1)aminomethane

reperfusion injury, the pathogenic relationships between individual species of partially reduced oxygen and cardiomyocyte necrosis remain obscure (Kloner et al., 1989; Janero, 1990a). Recent studies with heart- muscle tissue and isolated mammalian hearts ex vivo have provided compelling, albeit inferential, evidence that H,Oz is the primary form of partially reduced oxygen responsible for postischemic myocardial derangements (Meyers et al., 1985; Brown et al., 1989a,b; Konz et al., 1989; Kraemer et al., 1990; Shlafer et al., 1990). Yet the mechanism(s) by which H,O, establishes the cardiomyocyte injury at the basis of postischemic myocardial dysfunctionlinfarction and the identities of the H20,-sensitive cardiomyocyte targets are not known.

The lack of information concerning the cellular pathology of H,02 in the cardiomyocyte and the availability of reliable methods for cardiomyocyte isolation prompted the present work, which was designed to examine directly the mechanistic pathology associated with H,02-induced oxidative stress on the ventricular heart-muscle cell. For this purpose, we have formulated a model of H,O,-induced cardiomycyte injury employ- ing intact neonatal-rat cardiomyocytes in primary monolayer culture under defined, supplement-free conditions. This report places particular emphasis on the degenerative changes in cardiomyocyte membranes with H,O,-induced oxidative stress and their pathogenic importance to irreversible cell injury. In these regards, alterations in membrane biochemistry and function have been observed under conditions of both tissue oxidative stress and cardiac ischemia-reperfusion, yet the role of such membrane derangements in the establishment of cardiomyocyte necrosis is a matter of considerable debate (Kloner et al., 1989; Janero, 1990a). Our results define a peroxidative mechanism for the pathogenesis of H,O,-.induced lethal cardiomyocyte injury involving the rapid transformation of H20, via iron-mediated reactions within the cardiomyocyte itself to products that initiate membrane lipid peroxidation. The peroxidation elicits defects in sarcolemmal permeability and the irreversible loss of cardiomyocyte integrity in an autocatalytic fashion independent of the continued presence of H,Oz. This peroxidative mode of cardiomyocyte necrosis is not, however, the sole injury mechanism operative during H,O,-induced oxidative stress, for a H,02-dependent, nonperoxidative route of cardiomyocyte de-energization appears to exist as well.

MATERIALS AND METHODS Materials

Tissue-culture media and media components were from Gibco (Grand Island, NY). Culture-ware was from Becton-Dickinson (Oxnard, CA). H20z was obtained as a 30% (wiv) aqueous solution at highest chemical purity from Fluka Chemie AG (Buchs, Switzerland). Metabo- lite standards for chromatography, sodium dodecylsulfate (SDS), trichloroacetic acid (TCA), ethylene diaminetetraacetic acid (EDTA), tris(hydroxymethy1) aminomethane (Tris), N-2-hydroxyethylpiperazine-N’- 2’-ethanesulfonic acid (HEPES), cysteine, a-tocopherol, p-carotene, 2-thiobarbituric acid (TBA), N-acetylcysteine, mannitol, 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), 0-phenanthrolene, and reduced glutathione

349 H,O,-INDUCED CAKUIOMYOCYTE INJURY

(GSH) were obtained a t tissue-culture (or highest avail- able) grade from Sigma (St. Louis, MO). Trolox, NJV- diphenyl-l,4-phenylenediamine (DPPD), and Freon were from Aldrich (Milwaukee, WI). Deferoxamine was from Ciba-Geigy, and ferioxamine was synthesized by iron-loading deferoxamine, as described (Hoe et al., 1982). Superoxide dismutase (02-:02- oxidoreductase, EC 1.15.1.1.; analytical preparation from bovine erythrocytes, 5,000 Uimg) (SOD) and catalase (H,02:H,0, oxidoreductase, EC 1.11.1.6; analytical preparation from beef liver, 65,000 Uimg) were purchased from Boehringer-Mannheim (Indianapolis, IN). Catalase was thermally inactivated according to VerDonck et al. (1988). [3,4,5-3HlLeucine (155 Ciimmol, sp. act.), [5,6,8,9,11,12,14, 15-3Hlarachidonic acid (240 Ciimmol, sp. act.), and Solvable’” solubilizer were from New England Nuclear (Boston, MA). Ready-flow I11 scintillation fluid was from Beckman (Fullerton, CA). Sol- vents were high-pressure liquid chromatography (HPLC) grade (Burdick and Jackson, Muskegon, MI), and water was purified to low conductivity with a Milli-Q-plus apparatus (Millipore, Bedford, MA). HPLC analyses were carried out on a Beckman system equipped with a spectrophotometric detector. All radioactive samples were analyzed in a Beckman LS 9800 liquid scintillation spectrometer; the efficiency of tri- tium counting was >50%, and the efficiency of 14C counting was >80%.

Buffers, media, and media supplements Phosphate-buffered saline (PBS) consisted of 137.0

mM NaC1,2.7 mM KC1,8.0 mM Na,HPO,, and 1.0 mM KH,PO,, pH 7.4. Complete Eagle’s basal medium (BME), pH 7.4, consisted of Earle’s salts, BME amino acids (without glutamine), BME vitamins, 0.22% (wiv) Na-bicarbonate, 5% (viv) horse serum, 5% (viv) newborn calf serum, glucose (5.5 mM), penicillin (10,000 unitsiml), and streptomycin (10 mgiml). “Supplement- free” BME contained these same constituents except for horse serum, calf serum, and vitamins and was buffered by 10 mM HEPES instead of bicarbonate. Media did not contain phenol red. All physiological solutions were 300-310 mOsmol, as determined by osmometry (Preci- sion Instruments, Sudbury, MA). Solutions were ster- ilized by passage through 0.22-pm cellulose-acetate filters (Nalgene, Rochester, NY). Media additions were generally from concentrated, sterile stock solutions made with the corresponding medium. In certain cases (DPPD, 0-phenanthrolene, a-tocopherol), concentrated stock solutions were prepared in ethanol and diluted with the appropriate medium such that the final ethanol concentration in the culture dish was well below that at which a vehicle effect was noted.

Cardiomyocyte culture Ventricular cardiomyocytes were isolated under

aseptic conditions from 3-day-old Sprague-Dawley rats, as previously detailed (Janero et al., 1988; Dunnmon et al., 1990). The isolated myocytes in complete BME were plated in 60-mm Falcon “Primaria” tissue-culture dishes a t 1.5 x lo6 viable cells/dish and incubated a t 37°C under humidified 95% air:5% CO,. As estimated by dye exclusion, over 90% of the isolated cardiomyocytes were viable a t plating. The seeded cardiomyo-

cytes reached confluency by 72 hr postplating, a t which time they were used experimentally. The primary cultures have been well characterized (Janero et al., 1988; Janero and Burghardt, 1989a) and consist of a monolayer network of cardiomyocytes beating synchro- nously a t -140 beatsimin and containing 330 * 16 pg cell proteini60-mm culture dish (mean * SD; n = 10). Such cultures were comprised almost exclusively (>95%) of ventricular cardiomyocytes, as determined with myosin light-chain kinase antisera (Dunnmon et al., 1990) (data not shown).

Establishment of H,O,-induced oxidative stress Confluent cardiomyocyte monolayers were washed

free of complete BME with 15.0 ml supplement-free BME. The washed monolayers were then incubated under culture conditions (37°C; 95% air:5% CO,) with 2.0 ml of prewarmed supplement-free BME containing reagent H,Oz a t a desired final concentration. Substi- tution of HEPES for bicarbonate in the supplement- free BME obviated the need for atmospheric preequil- ibration of the medium and thereby aided reliable, acute assessment of cell status. HEPES buffer did not affect cardiomyocyte viability as assessed by dye exclusion and maintenance of cellular energy charge (data not shown). Preliminary experiments with H,Oz in bicarbonate-buffered, supplement-free BME demonstrated that the observed cardiomyocyte responses to H,O, were not influenced by HEPES (data not shown). The H,O,-induced oxidative stress was terminated by adding 5,000 U catalase to each culture dish; this quantity of catalase dismutates 5.0 mM H20z within seconds (data not shown). The culture dishes were then placed on ice and immediately processed for analysis, depending upon the experimental parameter being evaluated.

Assessment of cardiomyocyte sarcolemmal permeability defects and cellular integrity

Lactate dehydrogenase [L-lactate: nicotinamide adenine dinucleotide (NAD) oxidoreductase, EC 1.1.1.271 (LDH) release into the culture medium was used to assess sarcolemmal disruption and quantify cardiomyocyte necrosis (Kehrer and Starnes, 1989). LDH was assayed spectrophotometrically by determining the reduction of pyruvic acid to lactic acid and measuring colorimetrically the lactate as its phenylhydrazone derivative (Cabaud and Wroblewski, 1958). LDH activity was expressed in international units. The extent of lethal cardiomyocyte injury during H,O,-induced oxidative stress was taken as the net LDH release from H,O,-exposed cells above the negligible basal release observed from contemporaneous control cells that had been incubated with supplement-free BME only. Total cardiomyocyte LDH activity was determined in 1.0% (wiv) SDS cell extracts. As noted elsewhere (Rao et al., 1990) and confirmed by us (data not shown), the levels of H202 employed in this study did not affect LDH activity.

Evaluation of cardiomyocyte-membrane protein and phospholipid degradation

Degradation of cardiomyocyte-membrane protein was assessed in cells that had been incubated in

350 JANERO ET AL.

complete BME containing 1.0 FCi [3Hlleucineiml for 24 hr prior to establishment of H,O,-induced oxidative stress. Cardiomyocyte membranes were isolated by hypotonic lysis and centrifugation (Thandroyen et al., 1989) in the presence of protease inhibitors (Janero and Lane, 1983; Olson and Lane, 1987). Membrane protein was precipitated with 1.0 ml ice-cold 5% (wiv) TCA-5.0 mM EDTA. The precipitates were collected by centrifugation in a Beckman microfuge B, solubilized in 0.5 ml Solvable and analyzed for incorporated u3H1leu- cine by liquid scintillation spectrometry in 10.0 ml Ready-flow 111 and for protein content with a dye- binding microassay (Fanger, 1987).

Since cardiomyocyte-membrane acyl lipids are largely phospholipids (Rogers, 19741, degradation of cardiomyocyte-membrane phospholipid was assessed by the net loss of fatty-acyl esters from total membrane lipid, as detailed (Janero et al., 1988; Janero and Burghardt, 1989a). In brief, equilibrium labeling of cardiomyocyte-membrane phosphoglycerides was achieved by incubating confluent monolayers for 24 h r with 3.0 ml complete BMEidish containing 1.5 FCi ['Hlarachi- donic acid. The labeled cardiomyocytes were then rap- idly washed free of unassimilated radioactivity with 20.0 ml supplement-free BME and subjected to H,Oz- induced oxidative stress. Cardiomyocyte membranes were isolated by hypotonic lysis and centrifugation (Thandroyen et al., 1989), and the membrane lipids were quantitatively extracted (Janero and Barrnett, 1981) and analyzed for incorporated [3H]arachidonic acid by liquid scintillation spectrometry. The fatty-acyl ester content of the membrane lipid was determined microchemically after derivatizing the esters to their hydroxamic acids (Skidmore and Entenman, 1962).

Assessment of oxidative damage to cardiomyocyte-membrane lipid and protein

Conjugated diene formation was used as an index of cardiomyocyte lipid peroxidation. Cardiomyocyte lipids were quantitatively extracted (Janero and Barrnett, 1981) and dissolved in 1.0 ml spectro-grade cyclohexane. An absorption spectrum of the lipids was taken against cyclohexane from 190 nm to 400 nrn, and a difference spectrum was obtained between the lipid spectrum of cardiomyocytes exposed to H,Oz and the lipid spectrum of cocultured cells not exposed to H,Oz. The net formation of lipid conjugated dienes in response to H,O,-induced oxidative stress was calculated from the difference spectrum using the molar absorptivities given (Watson et al., 1984).

Formation of TBA-reactive substances (TBARS) was used as an empirical indicator of cardiomyocyte lipid damage due to H,02-induced oxidative stress (Janero, 1990b). The TBA test was carried out under acidic (pH 2.4) conditions with a colorimetric microassay (Janero and Burghardt, 1989b), and the level of TBARS was expressed relative to the response of the assay to malondraldehyde (MDA), which was synthesized as detailed (Janero and Burghardt, 1989b). Since H,02 may interfere with color development in this assay (Kostka and Kwan, 19891, all samples were treated with excess (5,000 Uiml) catalase prior to acidification and reaction in the TBA test.

Thiols were quantified with the following modifica-

tion of the method of Albano et al. (1985). Cardiomyo- cyte monolayers were rinsed with 15.0 ml ice-cold 0.1 mM Tris HC1, pH 7.4, containing 5.0 mM EDTA prior to isolation of cardiomyocyte membranes by hypotonic lysis-centrifugation (Thandroyen et al., 1989). Mem- brane proteins were precipitated with 1.0 ml ice-cold 5.0% (wiv) TCA-5.0 mM EDTA, and each protein precipitate was washed with 5.0 ml of the same TCA- EDTA solution. The membrane protein pellets were each dissolved in 1.0 ml 0.1 M Tris-HC1, pH 7.4, containing 5.0 mM EDTA and 0.5% (wh) SDS. To each dissolved sample was added 250 ~ 1 0 . 7 5 M Tris-HC1, pH 8.6, containing 0.75 M EDTA. The resulting alkaline mixture was reacted with 0.1 mM (final concentration) DTNB in methanol in a volume of 4.0 ml. The absorption of the nitromercaptobenzoic acid anion produced from DTNB reduction by sample thiol groups was read spectrophotometrically at 412 nm and was subtracted from the background value obtained by treating a parallel sample with 5.0 mM (final concentration) N-ethylmaleimide before reaction with DTNB. The results were expressed relative to the response of the assay to known amounts of cysteine.

a-Tocopherol determination a-Tocopherol was assayed by reverse-phase HPLC

(Nierenberg and Lester, 1985). a-Tocopherol was quantitatively extracted from cardiomyocytes (Janero and Burghardt, 1989c) and dissolved in acetonitrile for injection onto the HPLC column. Quantification was by computerized regression analysis from a detector response curve generated with pure a-tocopherol.

GSH quantification Cardiomyocyte GSH was quantified in HC10, ex-

tracts by a technique modified from Timerman et al. (1990) with the anion-exchange HPLC procedure of Reed et al. (1980). Briefly, cardiomyocyte monolayers were rinsed with 15.0 ml PBS, scraped into 0.7 ml ice-cold PBS, and centrifuged for 1.0 min a t 4°C in a Beckman microfuge B through 0.35 ml dibutylphtha- late and into 0.25 ml 10.0% (wiv) HC10, containing 25.0 pM a-glutamyl glutamic acid internal standard and 1.0 mM EDTA. After removal of the PBS and the organic layer below it, the cell metabolites extracted into the HClO, were recovered and subjected to a two-step derivatization in which free thiols were first S-car- boxymethylated and, subsequently, free amino groups were dinitrophenylated (Fariss and Reed, 1983). The resulting solution was analyzed by radial-compression anion-exchange HPLC, as described (Reed et al., 1980). Quantification was based upon the recovery of internal standard and the spectrophotometric detector response to known amounts of GSH.

Nucleotide analyses Cardiomyocyte ATP content was determined in 6.0%

(wiv) HClO, cell extracts with the ion-pair HPLC method of Juengling and Karnmermeir (1980). The acidic metabolite extracts were neutralized with Freon- trioctylamine (Khym, 1975). Identities of the eluted substances were established by their spectral properties, coelution of internal standards, and the enzymatic peak-shift techniques of Brown (1970). Quantification

H,O,-INDUCED CARDIOMYOCYTE INJURY 351

of ATP was by computer-assisted regression analysis with respect to the response of the HPLC spectropho- tometer to known standard.

Intervention studies Substances were evaluated for their potential to

inhibit H20,-induced cardiomyocyte injury usually after either a 24-hr or a 15-min pretreatment period and at the concentrations specified in the text, Tables 4 and 5, and Fig. 8. For a 24-hr pretreatment, cultures a t confluency were rinsed with 15.0 ml BME and incubated under culture conditions for 24 hr in 2.0 ml complete BME containing test substance. For a 15-min pretreatment, cultures at confluency were rinsed with 15.0 ml supplement-free BME and incubated under culture conditions for 15 min in 2.0 ml supplement-free BME containing test substance. At the end of either preincubation, the monolayers were rinsed with 15.0 ml supplement-free BME containing test substance and incubated under culture conditions in 2.0 ml supplement-free BME containing test substance and 500 pM H,0, for 60 min. In some cases, the test substance was introduced into the cultures at a specific point during the progression of H,O,-induced oxidative stress. To terminate the experiment, any remaining H,O, was eliminated with 5,000 U catalase, and the dishes were placed on ice and immediately processed for analysis, depending upon the parameter to be assessed. Evalua- tion of each experimental parameter in parallel control cultures pretreated with test substance but not exposed to H,O, provided the basis for determining whether the intervention had exerted protection against H,O,-induced oxidative stress. Preliminary experiments verified that the substances tested did not interfere with the end-point assays used, nor did they induce cardiomyocyte injury themselves (data not shown).

Reversibility studies The reversibility of H,O,-induced cardiomyocyte in-

jury was taken as the degree to which LDH loss from injured cells could be attenuated by alleviating the oxidative stress. Three parallel groups of cardiomyocyte cultures were defined for each reversibility experiment: (1) cells incubated in 2.0 ml supplement-free BME containing 5,000 U catalase for 120 min; (2) cells incubated in 2.0 ml supplement-free BME containing 500 pM Hz02 for 120 min; and (3) cells incubated in 2.0 ml supplement-free BME containing 500 p.M H20, for intervals ranging from 2 min to 30 min, a t which time excess (5,000 U) catalase was added to eliminate any H,O, remaining, the medium was removed and replaced with 2.0 ml fresh supplement-free BME containing 5,000 U catalase, and the cells were allowed a further incubation up to a total incubation time of 120 min, including the interval of oxidative stress. The net LDH loss a t 120 min in groups 2 and 3 above any basal release in group 1 was calculated and taken as the extent of cardiomyocyte injury. The degree to which the net LDH loss in group 3 was less than that of group 2 a t 120 min defined the extent to which the progression of lethal cardiomyocyte injury was reversed by allevi- ation of the H,O,-induced oxidative stress.

H,O, quantification H,O, was quantified with a chemical procedure

(Hildebrandt et al., 1978) that depends on the stoichi- ometric oxidation of ferrous ammonium sulfate by H,O, in the presence of potassium thiocyanate to yield a yellow product (trithiocyanoferrate) measured spectrophotometrically a t 480 nm. In all samples matrices, catalase (5,000 U) prevented product formation, veri- fying that the assay had indeed measured H,O,.

Statistical analyses The significance of the difference between group

means was evaluated statistically by using analysis of variance (Bishop, 1966). The confidence interval was set a t 95%. Unless otherwise indicated, the effects of H,O,-induced oxidative stress on cardiomyocyte biochemistry and physiology reached statistical significance when compared to contemporaneous control cultures not exposed to H,Oz.

RESULTS Cardiomyocyte H,O, utilization

In order to ensure that our cell-culture system reflects H,O,-induced cardiomyocyte injury, we first investigated the potential interactions among H,O,, culture medium, and the cardiomyocytes. Reagent H,O, was chemically stable in supplement-free BME (Fig. 1). No change in the HzOz content of such solutions over a wide H,Oz concentration range (1.0 pM- 5.0 mM) was observed even during a 24-hr incubation in a humidified incubator a t 37°C. Supplement-free BME solutions containing H,O, did not elicit either the SOD-inhibitable reduction of ferricytochrome C (Cohen and Chovaniec, 1978) or the mannitol-inhibitable oxidation of deoxyribose (Halliwell and Gutteridge, 1981) (data not shown), demonstrating a lack of spontaneous 0,- or 'OH formation from H,O, in supplement-free BME.

Since it is membrane-permeable (Ramasarma, 1982 j, extracellular H,O, could traverse the cardiomyocyte sarcolemma, inviting potential reaction with, for example, cardiomyocyte catalase, GSH peroxidase (GSH: H,O, oxidoreductase, EC 1.11.1.9), and cellular stores of redox-active iron (Thayer, 1986; Chevron, 1988). Consequently, we studied whether reagent H,Oz exogenously supplied to cardiomyocyte monolayers as a bolus in supplement-free BME was consumed by the cells. A maximal, initial rate of H,02 loss of 16.9 * 1.4 nmol H,Oz/min (mean ? SD; n = 4) was observed during the first 10-15 min of cardiomyocyte incubation with H,O,. This rate was invariant over a wide (50- 1,000 pM) H,O, concentration range (Fig. 1 and data not shown). At these initial H,O, concentrations, however, the cardiomyocytes were not able to metabolize completely the supplied H,Oz, as demonstrated by the H,O, remaining in cardiomyocyte cultures 60 min after bolus introduction of 250 pM or 500 pM H,Oz (Fig. 1): had the initial maximal rate of H,O, utilization been sustained, the cultures would have been totally depleted of H,O, well before the 60-min sampling time. The inability of the cardiomyocytes to maintain their initial rate of H,O, utilization may reflect such phe-

352 JANERO ET AL.

I

f

I I I I I I I I I 0 2 4 6 8 10 12 14 16 ’

TIME (min)

Fig. 1. Kinetics of cardiomyocyte H,O, utilization. Confluent cardiomyocyte monolayers were incubated at 37°C in 2.0 ml supplement- free BME containing either 250 p,M (*) or 500 pM ( & ) H,O, (initial concentrations) for up to 60 min. Supplement-free BME containing

nomena as catalase inactivation by oxidants, including H,O, itself (Altomare et al., 1974). From these data, it is evident that any indicated H,O, bolus introduced exogenously into the cardiomyocyte cultures should be regarded as an initial, maximal concentration.

Influence of H202 on sarcolemmal permeability and cardiomyocyte integrity

Leakage of the soluble cytoplasmic enzyme LDH is experimentally and clinically a quantitative index of compromised cardiomyocyte integrity and lethal cell damage (Kehrer and Starnes, 1989). We have employed this principle to determine whether H,O,-induced oxidative stress elicits cardiomyocyte sarcolemmal disruption and supports cell necrosis. To this intent, culture medium was assayed for LDH during incubation of the cardiomyocytes with H,O,. In response to oxidative stress established with H,02, cardiomyocytes leaked LDH into the culture medium, and the rate and magnitude of cellular LDH loss depended upon the initial H,O, concentration and the duration of the oxidative stress (Fig. 2A). An initial H,O, bolus of at least 50 pM was required to elicit the injury process, and the time-course of sarcolemmal disruption in response to bolus H,O, above 1.0 mM did not differ from that observed a t 1.0 mM (data not shown). A 60-min incubation of cardiomyocytes with an initial H,O, concentration of, minimally, 500 p.M was necessary for quantitative LDH release (i.e., an apparent total loss of culture viability). Consequently, the 60-min data in F ig 2A were reexpressed as a “survival curve” (Fig. ZB), from which the H,Oz bolus required to kill 50% of the cardiomyocyte monolayer within 60 min (i.e., the LD50 for H,OJ was calculated to be 210 pM.

either 250 p,M (0) or 500 pM ( A ) H,O, was incubated in parallel at 37°C without cells. During this time, media samples were taken, and their H,O, content was determined by direct chemical microassay. The H,O, content is expressed here as mean values 2 SD (n = 4).

H20,-induced degenerative changes to cardiomyocyte-membrane protein and lipid

Cardiomyocyte sarcolemmal disruption in response to H,O, suggested that H,O,-induced oxidative stress might elicit the degradation of cardiomyocyte-membrane structural components-i.e., proteins and phospholipids. To investigate this possibility, we first assessed whether a net loss of cardiomyocyte-membrane protein or phospholipid took place over the course of development of lethal cardiomyocyte injury in response to 500 pM H,O,. Direct quantification of potential membrane changes necessitated subfractionating the cardiomyocytes to obtain the cellular membrane complement. In preliminary experiments similar to those detailed elsewhere (Janero and Burghardt, 1989c), it was determined that over 90% of the phospholipid present in the starting cardiomyocyte homogenate was recovered in the membranes isolated therefrom by hypotonic lysis-centrifugation (Thandroyen et al., 1989). Since myocardial phospholipid is localized almost exclusively in heart-muscle membrane (Wheel- don et al., 19651, the high phospholipid recovery in our membrane fraction indicates that the isolated membrane is representative of that in situ.

As evaluated by appropriate chemical microassays, a concentration of H,Oz that induced cardiomyocyte disruption by 60 min did not elicit any significant change in cardiomyocyte-membrane protein or phospholipid contents (Table 1). Consequently, net degradation of cardiomyocyte-membrane protein or phospholipid did not accompany H,O,-induced oxidative stress sufficient to compromise cardiomyocyte integrity.

Static mass measurements cannot reveal whether changes in membrane turnover accompanied H,O,-

H,O,-INDUCED CARDIOMYOCYTE INJURY

0 10 20 30 40 50 MI ” 90 120 TIME ( m i d

100 100

m a - L 2

20 ’ 20

353

F----

0 0

o 0.2 0 4 0 6 o a 1 0 IHz021. mM

Fig. 2. Release of LDH and development of lethal cardiomyocyte injury during H,O,-induced oxidative stress. Confluent cardiomyocyte monolayers were incubated with 2.0 ml supplement-free BME alone (“control” cells) or supplement-free BME containing H,O, at an initial concentration ofeither 50 ( A 1,100 (m), 250 (01,500 (A), or 1,000 (0) pM for up to 120 min. During this time, cultures were removed from the incubator, and their media were analyzed for LDH activity,

which is expressed in A as the net release of LDH from cardiomyocytes exposed to H,O, relative to the low basal release from contemporaneous control cells. B represents a re-expression of data from A as a “survival curve” for cardiomyocytes exposed to various initial H,O, concentrations for 60 min; from this curve, an LD50 of 210 pM H,O, was calculated. Data are expressed as mean values 2 SD (n = 4).

TABLE 1. Membrane protein and phospholipid-ester contents of cardiomyocytes during HzOz-induced oxidative stress’

Incubation conditions

0 zM HzOz 0 min

120 min 500 pM HzOz

5 min 15 rnin 30 rnin 60 rnin

120 min

Membrane protein cpm/dish cpm/pg protein

pg/dish ( x 10-4) (XlOW)

98 t 3 2.0 f 0.1 2.0 f 0.1 99 i 4* 1.7 f 0.1 1.7 f 0.1

93 i 5* 1.9 f 0.1* 2.0 k 0.1* 98 i 3* 1.8 i 0.2* 1.8 f 0.1* 95 f 3* 1.5 i 0.1 1.6 f 0.1

101 f 6* 1.1 f 0.1 1.1 f 0.1 99 f 4* 1.2 i 0.1 1.2 f 0.1

Membrane phospholipid ester cpm/pEquivalent

pEquivalents/ cpm/dish phospholipid ester dish (xlo-5) (xlo--z)

1,327 i 102 5.3 + 0.3 4.0 + 0.2 1,395 k 119* 4.6 f 0.2 3.3 f 0.2

1,382 + 106* 5.2 k 0.3* 3.8 f 0.3* 1,364 * 89* 4.5 i 0.2 3.3 i 0.2 1,379 + 120* 3.6 i 0.2 2.6 i 0.2 1,343 * 83% 3.0 f 0.1 2.2 f 0.1 1,373 * 65* 3.0 f 0.2 2.2 f 0.1

’Confluent cardiomyocyte monolayers were incubatedfor 24 hr in complete BME containing either 1.0 pCi [”HI leucine/mlor0.5 pCi [jH] arachidonic acidlml. After 24 hr, unassimilated radioactivity was washed from the cultures,and the membrane complement wasimmediately isolatedfrom some ofthe cardiomyocytes (0 pM Hz02,O minj by hypotonic lysis-centrifugation. The remaining labeledcardiomyocyte monolayers were incubated either for 120 minin supplement-free BME, 0 p M HzOa, or for 5-120 min in supplement-free BME containing 500 pM H202 (initial concentration). At the end of each incubation, the cultures were harvested, and their membranes were isolated. The isolated membranes from rAH] leucine-labeled cells were analyzed microchemically for protein content (given as pg/dishj and radiochemically to quantify leucine-labeled TCA-precipitahle protein (given as cpm/dish). Membranes from [3H] arachidonic acid-labeled cells were analyzed microchemically for phospholipid ester content (given as pEquivalents/dish) and radiochemically to quantify arachidonic acid-labeled, lipid-extractable membrane phospholipid (given as cpmldishj. These data were used to calculate the specific radioactivity of membrane protein (given a s cpm/pg protein) and membrane phospholipid (given as cpm/pEquivalent phospholipid ester). Data are means + SD (n 2 5j. *Value not significantly (P 2 0.05, analysis of variance) different from respective mean value a t 0 pM H202, 0 min.

induced cardiomyocyte injury. In order to address this question, cardiomyocytes were metabolically prela- beled with either [3H]leucine or [3H]arachidonic acid prior to establishment of oxidative stress. After a 24-hr incubation with radioactive precursor, -60% of the total cellular protein labeled from [3Hlleucine was recovered as membrane protein, whereas >85% of the cellular label from [3HJarachidonic acid became assim- ilated into membrane phospholipid. Since membrane

protein and phospholipid contents remained constant throughout the oxidative stress (Table l), any H,O,- induced decreases in the specific radioactivities of [3HJleucine-labeled membrane protein or [“Iarachi- donic acid-labeled membrane phospholipid relative t o the changes observed in membranes from cells not exposed to H,O, would be indicative of accellerated protein or phospholipid turnover, respectively. The results of these labeling experiments demonstrate that

354 JANERO ET AL.

I 1 I I

OL 0 10 20 30 40 50 60 ‘‘ 90 120

TIME (mid

1 150

Fig. 3. Peroxidation of cardiomyocyte-membrane phospholipid during H,O,-induced oxidative stress. Confluent cardiomyocyte monolayers were incubated with 2.0 ml supplement-free BME containing H,O, at an initial concentration of either 0 (O), 50 (A ), 250 (01, or 500 ( A) pM for up to 120 min. At various times during the experiment, cultures were removed from the incubator, and the cardiomyocyte lipids were extracted. The purified lipids were analyzed spectrophotometrically for their content of conjugated-diene intermediates of peroxidation, given here as mean values -t SD (n = 4).

H,O,-induced oxidative stress elicited a marked increase in cardiomyocyte-membrane protein and phospholipid turnover (Table 1). At 120 min of oxidative stress established by a concentration (500 pM) of H,O, which caused an apparently complete disruption of cardiomyocyte integrity, the specific activities of both cardiomyocyte-membrane protein and phospholipid were some 30% below contemporaneous control cultures incubated with supplement-free BME only.

Development of peroxidative damage to cardiomyocyte-membrane phospholipid during

H,O,-induced oxidative stress The possibility was investigated that degenerative

transformations of cardiomyocyte-membrane phospholipids more subtle than rank degradation had occurred during H,02-induced oxidative stress. In biological systems, oxidative stress carries with it the potential for polyunsaturated fatty acid peroxidation (Janero, 1990a). In heart-muscle tissue, as in the cardiomyocyte, polyunsaturated fatty acids are largely arachidonic- acid esters of membrane phospholipid (Rogers, 1974; Janero and Burghardt, 1989~) . Consequently, we studied whether peroxidative membrane damage is an injury component of H,02-induced oxidative stress to the cardiomyocyte by evaluating whether conjugated- diene intermediates of lipid peroxidation were produced with Hz02 exposure. As summarized in Figure 3, lipid conjugated dienes were generated in cardiomyocytes during Hz02-induced oxidative stress. Net conjugated diene production was correlated with the severity of the initial oxidative insult. By 30-45 min of oxidative stress, the cardiomyocyte content of lipid conjugated dienes began to decrease and approach, but not reach, the low initial level. The decline in lipid conju-

I I 1 1 I I 2- 1

Fig. 4. Formation of TBARS in cardiomyocytes during H,O,-induced oxidative stress. Confluent cardiomyocyte monolayers were incubated with 2.0 ml supplement-free BME containing H,O, at an initial concentration of either 0 ( O ) , 50 ( A), 250 (O), or 500 ( A ) KM for up to 120 min. At various times during the experiment, cultures were removed from the incubator and analyzed by a colorimetric microassay for their content of TBARS, given here as mean nmol MDA equivalentsiculture dish t SD (n = 4).

gated dienes with progression of oxidative stress suggested that the rate of formation of diene intermediates had become less than the rate of conversion of the dienes to hydroperoxides and secondary decomposition products (Janero and Burghardt, 1989b).

Empirical evidence for the formation and decomposition of fatty peroxides during H,O,-induced oxidative stress was obtained by assaying cardiomyocyte TBARS (Janero, 1990b). A progressive increase in cardiomyocyte TBA-reactivity was associated with the first 45-60 min of H,O, exposure, and the rate and extent of TBARS formation were potentiated with oxidative insults of increased severity (Fig. 4). The general decline in TBARS formation after 60 min of oxidative stress suggests that TBARS degradation or reactivity with cellular constituents exceeded the rate of TBARS formation at the later stages of H,O,-induced cardiomyocyte injury (Janero and Burghardt, 198913).

a-Tocopherol has recently been identified as the principal membrane-associated antiperoxidant in the myocardium (Janero and Burghardt, 1989~) . Consump- tion of a-tocopherol is generally regarded to reflect its reactivity with peroxyl radicals generated during the propagation phase of lipid peroxidation (McCay, 1985). Because of the association between H,O,-induced oxidative stress and cardiomyocyte-membrane phospholipid peroxidation (Figs. 3 and 4), we investigated whether cardiomyocyte a-tocopherol status changed in response to H,Oz. Our results demonstrate that cardiomyocyte-membrane a-tocopherol is depleted by H20,- induced oxidative stress (Fig. 5). A maximal, initial rate of a-tocopherol consumption was established very acutely upon H,O, exposure and was largely independent of the initial H,O, concentration. However, the extent of a-tocopherol loss reflected both the severity of the H20, insult and the degree of membrane phospho-

355 H,O,-INDUCED CARDIOMYOCYTE INJURY

u

U P 4o EE

- 8 $ u >

I I I I I I

0 10 20 30 40 50 60 " 90 120 TIME fmini

U P 4o EE

- 8 $ u >

I I I I I I I >, I I

TIME fmini 0 10 20 30 40 50 60 " 90 120

Consumption of cardiomyocyte a-tocopherol during H,O,- induced oxidative stress. Confluent cardiomyocyte monolayers were incubated with 2.0 ml supplement-free BME containing H,O, a t an initial concentration of either 0 ( O ) , 50 (A), 250 (O), or 500 (A) pM for up to 120 min. At various times during this period, cultures were removed from the incubator, and the cardiomyocyte a-tocopherol was extracted and quantified by HPLC. Data are mean values 2 SD (n 2 3).

lipid peroxidation (cf. Figs. 3 and 4). The partial a-tocopherol loss observed during H202-induced oxidative stress with 50 pM H20z suggests that membrane lipid peroxidation was quenched by endogenous cellular antiperoxidants; i.e., the oxidative insult was not sufficient to peroxidize all of the susceptible targets.

Alterations in cardiomyocyte-membrane thiols during H,O,-induced oxidative stress

In addition to being potential direct targets of oxidative stress, thiol moieties in cellular biomolecules serve as antioxidant defenses against protein oxidation and lipid peroxidation (DiMascio et al., 1991). Conse- quently, we defined the cardiomyocyte thiol profile and monitored the status of cellular and, particularly, membrane thiols during H,O,-induced oxidative stress. As in heart muscle (Lesnefsky et al., 1991), protein and nonprotein sulfhydryl-containing biomolecules consti- tuted the two major cardiomyocyte thiol pools, whereas all membrane thiols were protein-bound (Table 2). GSH accounted for some 85% of the nonprotein cellular thiols; the remaining nonprotein thiols likely included a variety of low-molecular-weight, water-soluble metabolites such as cysteine (DiMascio et al., 1991).

Under H20,-induced oxidative stress, cardiomyocyte thiols, including the thiols in membrane proteins, became oxidized (Fig. 6A). Within an initial H,O, concentration range of 50-1,000 pM, the loss of membrane-protein thiols was particularly dramatic, with their virtual depletion by 30 min after exposure to 500 pM H,O, (Fig. 6B). The extent of membrane thiol loss was dependent upon the severity of the oxidative stress, although, as with a-tocopherol depletion (cf. Fig. 51, the initial rate of membrane thiol loss was largely independent of the magnitude of the H,02 insult.

TABLE 2. Cardiomyocyte thiol pools'

Thiol content Cell fraction pmol/dish nmol/mg protein

Cardiomyocytes 138.8 i- 6.3 404.9 k 20.3 Cell protein 93.2 ? 3.6 271.9 k 12.3 Cell nonprotein 45.6 k 2.9 133.0 k 6.32 Membranes 38.5 + 1.6 189.7 + 7.3 Membrane protein 38.1 + 1.3 187.7 k 8.2

'Confluent cardiomyocyte monolayers were harvested and either assayed by a microchemical procedure for total-cell thiol content, subjected to TCA-precipitation to isolate cell protein, or subjected to hypotonic lysis-centrifugation to isolate the cellular membrane complement. In turn, some of the membranes were subjected to TCA-precipitation, and the membrane-protein precipitate was recovered. Each cardiomyocyte subfraction was analyzed microchemically for thiol content, tabulated here a s mean values + SD (n 2 3). The cellular nonprotein thiol content represents the calculated difference between the cardiomyocyte and cell-protein values. 'GSH constitutes 112 * 8.7 nmol/mg protein (mean * SD, n = 4) of the cellular nonprotein thiol pool, as quantified directly by HPLC.

Cardiomyocyte ATP depletion during H,O,-induced oxidative stress

The cardiomyocyte-membrane peroxidation observed during H202-induced oxidative stress (Figs. 2 and 3 ) , along with evidence that peroxidation may elicit mito- chondrial dysfunction (Bindoli, 19881, invited study of the potential for H,O, to compromise the cardiomyocyte ATP pool. A progressive depletion of cardiomyocyte ATP was characteristic of H,O,-induced oxidative stress, and the rate and extent of ATP loss were potentiated with increased oxidative stress established by a bolus of H,O, 3 50 pM (Fig. 7). With any given H,O, insult, the maximal rate of ATP depletion was preceded by a lag period that could be diminished, but not abolished, by increasing the initial H,O, bolus. A complete loss of cardiomyocyte ATP was observed over a 60-90 min period following exposure of the cells to 1 .O mM H20,: Oxidative insults above 1.0 mM H,O, did not significantly potentiate the maximal rate of ATP depletion (data not shown).

Reversibility of lethal cardiomyocyte injury during H,O,-induced oxidative stress

Cardiomyocyte necrosis (i .e., sarcolemmal disruption quantified as LDH release) in response to H,O,-induced oxidative stress was positively correlated with the severity of the initial oxidative insult from 50 pM to 1.0 mM H,O, (Fig. 2). Yet cardiomyocyte injury developed over a period within which the H,02 content of the cultures declined (Fig. 1). These data suggested that the development of lethal cardiomyocyte injury during H,O,-induced oxidative stress may not have depended upon the continued presence of H,O, itself. In order to investigate this possibility, cardiomyocytes were exposed to either 0 p.M, 250 pM, or 500 pM H,02 in supplement-free BME for intervals up to 30 min, after which time any remaining H,O, in the culture dish was eliminated by the addition of catalase. The cardiomyocytes were then incubated in supplement- free BME containing catalase such that the total incubation time (i.e., H202 exposure plus subsequent H,O,-free incubation) was 120 min. At 120 min, the cultures were harvested and analyzed for net LDH leakage with respect to "control" cultures that had been incubated for 120 min with supplement-free BME only.

356 JANERO ET AL

I I I I I I 1 2 -

0 I 10 I 20 I 30 I 40 I 50 I 60 I TIME (min)

Fig. 6. Consumption of cardiomyocyte and cardiomyocyte-membrane thiols during H,O,-induced oxidative stress. A: Cardiomyocytes were incubated with supplement-free BME containing 500 pM H,O, (initial concentration) for up to 120 min. At various points during the 120 min, replicate cultures were removed from the incubators and processed to determine the thiol content of the whole cell, the TCA- precipitable cell protein, the TCA-precipitable nonmembrane protein (m), or the TCA-precipitable membrane protein (0) . The difference between the whole-cell thiol content and the cell-protein thiol content was taken as an estimate of the cellular nonprotein thiol pool (A ), which is mainly GSH (cf. Table 2). The status of each cardiomyocyte thiol pool in cells exposed to H,O, was expressed as a percentage relative to that thiol pool in contemporaneous “control” cells not

The extent of lethal injury in cardiomyocyte cultures “pulsed” with H,O, was compared to contemporaneous cultures that had been exposed to supplement-free BME containing H,02 for the entire 120 min (cf. Fig. 2).

The results of these experiments (Table 3) demonstrated that amelioration of the H,O,-induced oxidative stress as early as 2.0 min after cardiomyocyte Hz02 exposure did not reduce the extent of sarcolemmal disruption (i.e., net LDH leakage) as compared to cultures incubated with the same initial H,O, concentration for the entire 120-min interval. This result was comparable to the failure of complete BME to “rescue” cardiomyocytes after an acute H,O, insult and halt lethal cardiomyocyte injury (data not shown). Conse- quently, irreversible cardiomyocyte injury developed independently of the maintenance of H,02-induced oxidative stress.

Inhibition of lethal cardiomyocyte oxidative injury induced by H202

In order to help define the pathogenic importance of H,O, metabolites and the various degenerative processes identified above to the establishment of lethal H,O,-induced cardiomyocyte injury, the effects of a variety of interdictions were evaluated. Evaluations were made in cardiomyocytes exposed to an initial H,O, concentration of 500 pM for 60 min. Under these conditions, cardiomyocytes consistently evidenced a

0 10 20 30 40 50 60 TIME (minl

exposed to oxidative stress. Data are mean values ? SD (n = 4). The absolute thiol content of each pool in control cells is given in Table 2. B Time-course of cardiomyocyte-membrane thiol depletion and its dependence upon the degree of H,O,-induced oxidative stress. Cardi- omyocytes were incubated in supplement-free BME containing 50 ( A), 250 (U), or 500 (0) p,M H,O, (initial concentrations) for up to 120 min. During this time, cultures were periodically removed from the incubator, and cardiomyocyte membranes were isolated and analyzed for their protein thiol content. The results are given as the protein thiol level relative to the thiol content of membranes from contemporaneous “control” cells not exposed to H,O, (means ? SD; n = 4). The absolute protein thiol content of membranes from cardiomyocytes not exposed to oxidative stress was 187.7 -t 8.2 nmolimg protein (Table 2).

0 10 20 30 40 50 60 TIME (mid

Fig. 7. Kinetics of cardiomyocyte ATP loss during H,O,-induced oxidative stress. Confluent cardiomyocyte monolayers were incubated for up to 120 min with supplement-free BME containing either 0 (o), 250 (o), 500 ( A ) , or 1,000 (0) p,M H,O, (initial concentrations). During this time, cultures were removed from the incubator, and the cells were extracted with ice-cold 6% (wiv) HC10,. The neutralized HC10, exlracts were analyzed by reverse-phase HPLC to quantify the cardiomyocyte ATP content, expressed here as mean values k SD (n 2 4).


TABLE 3. Effect of H202 removal on the progression of lethal cardiomyocyte injury during HzOz-induced oxidative stress’

Incubation conditions Supplement-free BME Supplement-free BME

500 pM HzOz + +

2500 U catalase/ml

0 min 120 min 120 min 0 min

2 min 0 min 2 min, then: 118 min

10 min 0 min 10 min, then: 110 min 15 rnin 0 min 15 min, then: 105 min 30 rnin 0 min 30 min, then: 90 min

Net LDH release I.U./culture dish ?6 Total-cell LDH

0 0

<1.0 <1.0 26.3 i 2.0

24.8 k 2.1 2.3 i 0.1

26.6 k 2.3 4.1 f 0.3

27.4 + 2.1 10.3 f 0.9 26.9 i 1.9

96.5 i 5.6

94 3 f 4.8 9.2 f 0.4

96.4 i 6.3 17.2 5 1.1

101.3 i 6.7 42.1 f 3.2 97.3 f 4.9

‘Confluent cardiomyocyte monolayers were incubated for the indicated time intervals in either supplement-free BME containing 500 pM H202, supplement-free BME containing 2,500 U catalasehnl, or Hn02-containing, supplement-free BME followed by catalase-containing, supplement-free BME. All media were collected after each incubation, and the cellular LDH released into the media was determined. The net LDH release relative to control cultures incubatedin supplement-free BME alone for 120 min was calculated for each sample and isexpressed here as absoluteinternational units and a s a percentageof the total LDH content of the cardiomyocytes. Data are means * SD (n = 4).

TABLE 4. Inhibition of cardiomyocyte injury under conditions of HzOz-induced oxidative stress’

Inhibition of iniurv (%)

Test agent

Catalase SOD Deferoxamine 0-Phenanthrolene p-Carotene Mannitol Sodium benzoate N-Acetylcysteine a-Tocopherol Trolox DPPD

Treatment protocol Pretreatment

Concentration interval

50 U/ml 15 rnin 50 U/ml 15 rnin 10 mM 15 min

250 pM 15 rnin 200 pM 24 hr 100 mM 15 rnin 100 mM 15 rnin

1.0 mM 15 rnin 200 pM 24 hr 500 pM 24 hr

1.0 NM 15 rnin

Cardiomyocyte Sarcolemmal ATP GSH

disruption depletion depletion

9 9 i 3 9 8 t 4 9 9 5 5 10 rt 1 <3 <2 96 f 4 0 12 f 1 9 6 + 3 2 0 f 3 1 4 f 1

<4 0 0 39 t 2 0 0 56 i 4 0 0 98 f 3 0 26 f 3

100 f 3 <2 <5 100 f 4 <6 <3 98 5 4 <5 <8

Cardiomyocyte-membrane Phospholipid Phospholipid Protein peroxidation turnover turnover

100 i 2 101 i 2 9 8 f 4 (2 0 0

95 k 7 91 + 4 8 2 f 4 98 i 5 9 7 + 5 9 o i 3

<3 <5 <5 49 i 4 4 3 i 4 3 6 i 3 46 k 3 4 8 k 4 4 3 i 3

100 * 4 9 7 i 3 8 6 i 4 100 t 2 9 6 t 5 8 3 i 4 97 rt 4 9 5 f 3 8 4 i 5

102 * 5 9 4 i 3 8 0 f 5

Thiol depletion

96 i 3 <5

32 f 1 39 rt 3

<3 12 i 1 1 o i 1 38 + 3 58 f 4 52 + 4 66 + 5

‘Confluent cardiomyocytemonolayers were incubatedeither for 24 hr with agiven concentration of a particular test agent in complete BME or for 15 min with test agent in supplement-free BME, as indicated. After thistreatment period, themedia were removed and replaced with 1.9 ml supplement-free BME containing that test agent. Avolume (100 pl) of H202 solution in supplement-free BME was added to each culture dish to result in the final concentration of each test agent indicated and a final H202 concentration of 500 pM in a total volume of 2.0 ml supplement-free BME. After 60 min, any remaining H202 was eliminated by adding 5,000 U catalase/dish, and the cultures were processed to evaluate each experimental end point indicated, either at the cellular level or in isolated cardiomyocyte membranes, as detailed in Materials and Methods. The development of cardiomyocyte and membrane injury in the presence of each intervention was compared to the maximal injury displayed by cultures which had been incubated for 60 min withouttest agent in supplement-freeBME containing 500 pM H202, and any attenuation of the particular injury parameter was expressed as a relative (percent) inhibition of the damage by that intervention. Data are means ? SD (11 = 4).

>90% loss of cellular integrity (Fig. 2) and an -75% loss of ATP (Fig. 7). Preincubation of test agents with cardiomyocytes for either 15 rnin or, in some cases, 24 hr was routinely carried out to optimize the potential for inhibition. Seven end points of cardiomyocyte injury were evaluated: cell integrity [i.e., LDH leakage (Fig. 2)], ATP depletion (Fig. 7), GSH depletion (Fig. 61, membrane phospholipid peroxidation [i.e., TBARS formation (Fig. 4)], membrane phospholipid and protein turnover (Table l), and membrane-protein thiol status (Fig. 6). Preliminary experiments verified that, under the culture and incubation conditions utilized, the test agents themselves did not induce cardiomyocyte injury and did not interfere with the analytical procedures employed to quantify cell damage (data not shown).

The results of these investigations are summarized in Table 4 for either a concentration of test substance

that totally inhibited a t least one aspect of H202- induced cardiomyocyte injury or for the highest concentration of test substance attainable in supplement- free BME. The inhibitory effects of all interventions were concentration-dependent (Fig. 8 and data not shown). Of the interventions tested, only catalase prevented all aspects of H,O,-induced cardiomyocyte injury. Heat-inactivated catalase was without effect (data not shown). Since catalase specifically dismutates H,O, to water (Calabrese and Canada, 1989), the complete cytoprotection by catalase demonstrates con- clusively that the development of cardiomyocyte pathology in our culture system was reflective of H,O,- induced oxidative stress.

The total inhibition of cardiomyocyte damage in our injury model by catalase (Table 4) and the chemical stability of H,O, in supplement-free BME (Fig. 1) do

358 JANERO ET AL.

TABLE 5. Coordinate antiperoxidant inhibition of progressive cardiomyocyte lipid peroxidation and sarcolemmal disruption during HzOs-induced oxidative stress'

Treatment protocol Inhibition (%)

Treatment Sarcolemmal Lipid peroxidation disruption Test agent Concentration interval

Catalase 50 U/ml 55 min 0 0 a- To cop her o 1 200 pM 55 min 5.1 +_ 1.0 10.3 f 1.3 Trolox 500 p M 55 min 54.8 2 5.3 48.2 f 3.6 DPPD 1.0 pM 55 min 62.4 f 5.0 57.4 2 4.1

'Confluent cardiomyocytemonolayers were incubated for 6 min in supplement-free BME containing500 pM H20~. At 5 min, some of the dishes were removed from the incubator, and the HZOl-containing medium was replaced with supplement-free BME containing one of the test agents listed at the specified final concentration. The cultures were then allowed 55 min of further incubation, after which time they were processed to determine the extent of lipid peroxidation (as net TBARS formation) and sarcolemmalrupture (as net LDH release). The TBARS formation and LDH release in cultures with test agent were compared to the levelsdisplayed by cells which had been incubated with supplement-free BME alone after the 5-min H202 "pulse," and any relative reduction in net TBARS formation and net LDH release due to the test agent was expressed as a percent inhibition. Data are mean values * SD (n = 4).

not allow conclusion that Hz02 itself elicited the observed cytopathology. Metabolites of H,Oz may have been generated intracellularly, for transition metals (mainly iron in biological systems) readily catalyze the transformation of H,O, into other partial-reduction species of molecular oxygen, especially 'OH (Miller et al., 1990). Consequently, two cell-permeant iron chelators (deferoxamine, O-phenanthrolene) (Halli- well, 1989), an enzymatic scavenger of 0,' (SOD) (McCord and Fridovich, 1969), a small-molecule scavenger of singlet oxygen (@-carotene) (DiMascio et al., 1991), two 'OH scavengers (mannitol, sodium benzoate) (Anbar and Neta, 1967; Goldstein and Czapski, 1984), and a readily oxidizable sulfhydryl compound (N-acetylcysteine) (Sochman et al., 1990) were evaluated as potential inhibitors of H,02-induced cardiomyocyte oxidative injury. Deferoxamine and O-phenanthrolene simultaneously preserved cell integrity, prevented lipid peroxidation, and inhibited the H,O,- induced increases in membrane protein and phospholipid turnover (Table 4). Iron-loaded deferoxamine was without effect (data not shown). Iron-chelation, however, offered only limited protection against ATP depletion and (membrane) thiol oxidation. SOD and @-carotene exerted negligible protective effects, whereas mannitol and sodium benzoate partially (by -3040%) inhibited LDH leakage, TBARS formation, and the enhanced membrane turnover without much effect on ATP depletion or thiol status. N-Acetylcysteine blocked sarcolemmal disruption and membrane peroxidation and inhibited H,02-induced membrane turnover and thiol depletion, but exerted no effect against ATP loss.

The acute occurrence of cardiomyocyte lipid peroxidation during HzOz-induced oxidative stress (Figs. 3 and 4) prompted study of several antiperoxidative agents (a-tocopherol, Trolox, DPPD) (Farber et al., 1990; Janero et al., 1990). All antiperoxidants tested coordinately preserved cardiomyocyte integrity and abolished cardiomyocyte lipid peroxidation; they also significantly inhibited membrane thiol depletion and the accelerated turnover of membrane molecular constituents, but had negligible effects on H,02-induced GSH depletion and ATP loss (Table 4).

In order to examine further whether a pathogenic link exists between cardiomyocyte-membrane peroxi-

dation and lethal cardiomyocyte sarcolemmal disruption as suggested by the data in Table 4, additional intervention studies were performed. One line of in- quiry was predicated on the idea that, if peroxidation were the causative factor in establishing cardiomyocyte necrosis via sarcolemmaI disruption during H,O,-induced oxidative stress, then introduction of an antiperoxidant into cardiomyocyte cultures exposed to H,O, should block ongoing membrane lipid peroxidation and limit sarcolemmal disruption (i.e., LDH leakage). The results of such experiments (Table 5) demonstrate that antiperoxidant intervention a t 5 min of oxidative stress established with 500 pM H,O, was effective in bringing to a halt both lipid peroxidation and the progressive loss of cardiomyocyte integrity. The lack of cardiomyocyte preincubation with each antiperoxidant intervention in these studies likely accounts for the seemingly weaker potency of the antiperoxidants with respect to the data in Table 4. Examination of the concentration- dependency of both processes to inhibition by a-tocopherol (Fig. 8) and Trolox (data not shown) revealed a close correlation between the susceptibilities of cardiomyocyte-membrane peroxidation and sarcolemmal disruption to inhibition by antiperoxidants (Fig. 8). It is noteworthy that the concentration-response curve for antiperoxidant inhibition of cardiomyocyte LDH release (Fig. 8) evidenced a steep slope characteristic of processes mediated by lipid peroxidation (Janero et al., 1989).

DISCUSSION Despite extensive data from animal models of myo-

cardial ischemia, the operative interrelationships between oxidative stress and postischemic cardiomyocyte injury remain elusive. Attempts to define the cellular pathobiology of oxidative myocardial injury from in vivo data have been complicated, if not confounded, by such factors as the corruption of many in vivo studies by analytical methods not predictive of myocardial necrosis (Ooiwa et al., 1991) and the predominance of nonmuscle cells in the mammalian heart (Schaper et al., 1985). Consequently, although recent ex vivo and in vivo studies provide indirect evidence that H,02 is the central species of partially-reduced oxygen responsible for postischemic myocardial dysfunctionhecrosis (Myers et al., 1985; Brown et al., 1989a,b; Konz et al.,


4- 100

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80 i - 0

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- 4 0 r !? n 5 i

- 2 0 p

In -

60 g

>

1 I I I I I &+<-I

0 1 2 3 4 5 10 200 [a-TOCOPHEROLI, pM

Fig. 8. Coordinate inhibition by a-tocopherol of cardiomyocyte- membrane lipid peroxidation and sarcolemmal disruption during H,O,-induced oxidative stress. Confluent cardiomyocyte monolayers were preincubated for 24 hr with the indicated concentrations of a-tocopherol in 2.0 ml complete BME. The cells were then washed with 15.0 ml supplement-free BME and incubated for 60 min with supplement-free BME containing the original concentration of a-tocopherol and 500 pM H,O,. After 60 min, any H,O, remaining in the cultures was eliminated by adding 5,000 U catalase/dish, and the cells were analyzed for the extents of H,O,-induced membrane lipid peroxidation (as net TBARS formation) (0) and sarcolemmal disruption (as net LDH release) (0 ) relative to the low background TBARS content and LDH release in contemporaneous control cultures treated with a-tocopherol but not exposed to H,O,. Inhibition of peroxidation and sarcolemmal disruption is expressed relative to the 0% inhibition of these processes in contemporaneous cultures not treated with a-tocopherol but exposed to 500 p.M H,O, in supplement-free BME for 60 min. Data are mean values -+ SD (n 3 3).

1989; Kraemer et al., 1990; Shlafer et al., 1990), direct mechanistic information on the cytopathology of H,O, in the cardiomyocyte is conspicuously lacking. In the present report, we approach this point experimentally with the objectives of identifying a t least some of the H,O,-sensitive cardiomyocyte targets, characterizing the nature of the degenerative changes in these targets that accompany lethal cardiomyocyte injury in response to H,O,, and assessing the pathogenic importance of specific cellular changes to H,O,-induced cardiomyocyte necrosis.

To address these objectives, a cellular model of H,O,-induced cardiomyocyte oxidative stress has been defined. Neonatal-rat cardiomyocytes in primary monolayer culture were selected as the study object, for these cultures constitute a homogeneous population of viable, beating cells representative of the basic unit of myocardial structure and function, allowing changes in cardiomyocyte status to be quantified directly and reliably (Janero et al., 1988; Timerman et al., 1990). Reagent H,Oz was employed to enable precise defini- tion and titration of the cardiomyocyte oxidative insult and to avoid the ambiguities inherent with the use of radical “scavengers” to help identify the H20, component of a chemically heterogenous oxidative insult

(VerDonck et al., 1988). Establishment of H,O,-induced oxidative stress in defined, supplement-free medium was considered necessary to eliminate indirect cellular responses to, for instance, oxidatively-modified, cytotoxic serum lipoproteins (Hessler e t al., 1979) or the antioxidant properties of certain micronutrients (Machlin and Bendich, 1987). Bolus administration of reagent H,Oz to the cardiomyocytes could be considered analogous to the H20, “burst” observed in vivo during early postischemic myocardial reperfusion (Brown et al., 198913). The analogy is strengthened by evidence that oxidative injury to postischemic cardiac muscle in vivo reflects an exogenous oxidant burden and not cardiomyocyte oxidant production (Shlafer et al., 1990).

The stability of H,02 in our oxidative-injury model (Fig. 1) makes i t unlikely that the cardiomyocyte pathology observed reflects the extracellular transformation of H,Oz into other species of partially reduced oxygen (Pryor, 1986). The cellular injury most probably reflects the activity of H202 within the cardiomyocyte and as such would be modulated by cardiomyocyte properties such as endogenous antioxidant defenses (Rikans and Moore, 19881, intracellular H,02 metabolism (Thayer, 1986), and the presence of oxidant- sensitive cellular targets. In this regard, our cell- culture model is a simplification of the in vivo situation, where processes such as H20, metabolism/ scavenging by erythrocytes (Brown et al., 1989a) and infiltration of activated leukocytes (Lucchesi et al., 1989) may modulate the establishmenticonsequences of H,02-induced oxidative stress. Nevertheless, in culture as well as in vivo, cardiomyocyte injury in response to H,02 can occur only in the presence of net H,Oz levels which exceed cellular H,Oz detoxifying capabilities. These considerations suggest that our cardiomyocyte model has the potential to reflect the types of cellular derangements which might accompany H,O,-induced myocardial oxidative injury in vivo.

The data presented demonstrate that acute H20, exposure incites lethal cardiomyocyte injury as assessed by a well-accepted and clinically recognized end-point, LDH leakage (i.e., loss of sarcolemmal integrity) (Kehrer and Starnes, 1989). The development and progression of cardiomyocyte necrosis were positively correlated with the magnitude of the oxidant insult (i.e., the initial H20, bolus) within a concentration range of 50 pM to 1.0 mM H,O,. The threshold concentration of 50 FM H,02 suggests that endogenous cardiomyocyte H,O, catabolism and antioxidant status are sufficient to prevent cardiomyocyte necrosis with exposure to H,O, a t a concentration tenfold that of normal human plasma (Yamamoto et al., 1987). On the other hand, 1.0 mM HzOz apparently overwhelmed cardiomyocyte antioxidant defenses, since oxidative stress above 1.0 mM H,02 did not potentiate LDH leakage. Levels of H,02 in excess of 1.0 mM can readily be achieved by activated neutrophils in vitro (Test and Weiss, 1984; Nathan, 1987) and at sites of tissue inflammation (Spector and Garner, 1981). Thus, bolus delivery of micromolar H,O, to cardiomyocyte monolayers reflects in magnitude an oxidative insult that could potentially be established in reperfused myocardium, although the precise sources of partially reduced

360 JANERO ET AL.

oxygen in the postischemic heart are undefined (Kloner et al., 1989).

Cardiomyocyte injury during H,O?-induced oxidative stress was characterized by specific degenerative cellular changes. These changes included loss of cardiomyocyte antioxidants (a-tocopherol, GSH/thiols), membrane phospholipid peroxidation, increased membrane lipid and protein turnover, ATP depletion, and plasmalemmal disruption. None of these derangements is a unique cardiomyocyte response to H,O,-induced oxidative stress. Hepatocytes (Farber et al., 19901, endothelial cells (Varani et al., 1990), and P388D1 cells (Schraufstatter et al., 1985) share with the cardiomyocyte a t least some of the degenerative responses to H,O, we have documented. Species of partially reduced oxygen other than H,O, and nonoxidative injury stim- uli to the cardiomyocyte elicit some of the same cellular derangements that developed during H,O,-induced oxidative stress (Janero et al., 1988; Massey and Burton, 1990). Consequently, identification of H,O,-sensitive cardiomyocyte targets cannot per se offer mechanistic information on the pathogenesis of the accompanying cellular necrosis or serve as diagnostic evidence for the occurrence of H,O,-induced oxidative stress. Although the qualitative similarities in the degenerative responses of divergent cell types to H,O, imply that the cytotoxicity reflects the disturbance of a common, oxidant-sensitive target, it has not been possible to identify a unifying mechanism that defines the initia- tion and progression of cell injury in response to H202 (Farber et al., 1990).

The rapid H,O, utilization by cardiomyocytes (Fig. 1) and the sustained development of lethal injury after a brief, transient H,O, exposure (Table 3) suggest that a50 FM bolus H,O, acts in the cardiomyocyte as a vectorial signal for cell necrosis by “triggering” one or more processes that, in turn, are responsible for sarcolemmal disruption. This line of reasoning gains at- tractiveness from the limited reactivity of H,O, with biomolecules (Pryor, 1986) and further implies that mediators other than H,02 itself act intracellularly to establish and extend cell injury subsequent to a lethal H20, signal. Since catalase fully prevented cardiomyocyte damage during H,O,-induced oxidative stress (Table 41, the expression of any intracellular processes that may mediate cardiomyocyte necrosis in our injury model was nonetheless dependent upon at least the transient availability of H,O,.

The differential responses of specific, H,O?-induced cardiomyocyte degenerative changes to inhibitors (Table 4) allow distinction between those cellular derangements that help establish cardiomyocyte necrosis in response to H,O, and those that are incidental to oxidative stress. Coincident prevention of both LDH leakage and TBARS formation by antiperoxidants (Table 4) strongly indicates that membrane (i.e., sarcolemmal) lipid peroxidation is a prime determinant of cardiomyocyte necrosis during H20,1induced oxidative stress. The nature of lipid peroxidation as an autocatalytic “chain reaction” (Janero, 1990a) could explain why cardiomyocyte disruption in response to H,O, required only a brief H,O, “pulse” and progressed independently of the continued presence of H,O, (Table 3). A causal role for peroxidation in lethal

sarcolemmal injury would also account for the similar sensitivities of both H,O,-induced TBARS formation and LDH leakage to antiperoxidants (Fig. 8) and the coordinate interruption of these two processes by antiperoxidants even after H,O,-induced oxidative stress had been established (Table 5).

The role of lipid peroxidation in oxidative cell damage (Tribble et al., 1987) and myocardial ischemia- reperfusion (Kloner et al., 1989; van der Kraaij et al., 1989) has been subject to continued debate, mainly because antiperoxidants are not invariably protective in these injury settings (Ooiwa et al., 1991). Evidence exists, however, linking lipid peroxidation to hepato- cyte necrosis elicited by hydroperoxides (Farber et al., 1990). Demonstration has also been made that free- radical reactions that can incite lipid peroxidation occur in the reperfused heart (Bolli et al., 1989) and contribute to postischemic myocardial dysfunction (Bolli et al., 1990). In the cardiomyocyte, the sarcolemma is particularly susceptible to peroxidative damage, both in vitro (Kramer et al., 1984) and in vivo (Romaschin et al., 19901, and sarcolemmal rupture has been regarded by some as the decisive event establishing irreversible cardiomyocyte injury and infarction in vivo after acute ischemia (Jennings et al., 1990). With these considerations, the evidence presented herein for an intimate causal association between sarcolemmal lipid peroxidation and loss of cardiomyocyte integrity suggests that the H,O, burden of the post ischemic heart (Brown et al., 198913) could incite the lipid peroxidation observed with reperfusion (Romaschin et al., 1987) and help precipitate myocardial necrosis/ infarction. Our data also provide a mechanistic ratio- nale for observations that catalase, as well as small- molecule antiperoxidants, are cardioprotective in a t least some animal models of myocardial ischemia (Opie, 198913; Holzgrefe et al., 1990; Jeroudi et al., 1990).

Iron chelators preserved cardiomyocyte integrity and simultaneously prevented peroxidation during H,O,- induced oxidative stress (Table 4). These data provide additional support for an intimate pathogenic link between membrane lipid peroxidation and cardiomyocyte necrosis in response to HzOz, since the reaction of H,O, with transition metals via Fenton-type chemistry is required to produce species of partially reduced oxygen capable of initiating lipid peroxidation (Y a- mazaki and Piette, 1990). Metal chelators inhibit the cardiac damage elicited by Hz02 perfusion of isolated rat hearts (Appelbaum et al., 1990) and protect against postischemic myocardial dysfunction in vivo (Liu et al., 1990). Furthermore, products of Fenton chemistry have been spin-trapped in postischemic myocardium (Bolli et al., 1989). Although the most important initiator of lipid peroxidation in biological systems has been pre- sumed to be ‘OH (Janero, 1990a1, multiple oxidizing species with similar reactivities to ‘OH (such as the ferry1 ion, FeO,+) are also produced by Fenton chemistry (Yamazalii and Piette, 1990). Consequently, the significant, but incomplete, inhibition of cardiomyocyte lipid peroxidation and necrosis during H,O,-induced oxidative stress by .OH scavengers (Table 4) implies that cardiomyocyte H,02 consumption (Fig. 1) results in the iron-dependent intracellular generation


of multiple initiators of lipid peroxidation including, but not exclusively, 'OH. The very limited cytoprotection and antiperoxidative effects of SOD in our model do not per se exclude the participation of 0,- in the oxidative reactions underlying cardiomyocyte-membrane peroxidation. The relative ineffectiveness of SOD could reflect a subsidiary role for 0,- as a redox intermediate during the iron-dependent conversion of H202 into OH (Pryor, 1986) or the enzyme's limited intracellular accessibility (Nakae et al., 1990). In contrast, the lack of effect of chronic p-carotene loading against H,O,-induced oxidative damage to the cardiomyocyte indicates that singlet oxygen is not a likely injury mediator (DiMascio et al., 1991).

The net consumption of cardiomyocyte antioxidants (a-tocopherol, thiols) during H,02-induced oxidative stress is further evidence of a prominent role of membrane peroxidation in the resultant cardiomyocyte necrosis. a-Tocopherol is the critical lipophilic chain- breaking antioxidant responsible for protecting myocardial-membrane phospholipid against peroxidation (Janero and Burghardt, 1989~) . Thiols are important antioxidants that protect membrane lipids by prevent- ing peroxidation and helping regenerate a-tocopherol from its one-electron oxidation product (DiMascio et al., 1991). As we have demonstrated in cardiomyocytes during H,O,-induced oxidative stress, and as has been observed by Kyle et al. (1990) in liver cells during hepatotoxin-induced oxidative stress, (protein) thiol depletion and membrane peroxidation share an intimate temporal relationship with one another. This relationship suggests that loss of endogenous target- cell antioxidants represents a secondary consequence of oxidant exposure, not a primary determinant of lethal cardiomyocyte disruption. Support for this conclusion rests with the finding that hepatocytes (Kyle et al., 1989, 1990) and cardiomyocytes (Table 4) may be depleted of at least 60% of their protein thiols without loss of integrity. Lewko (1987) has further demonstrated that total depletion of the soluble thiol pool in cardiomyocytes is not per se cytotoxic. Consequently, the appreciable preservation of cardiomyocyte-membrane protein thiols by chain-breaking antiperoxidants or scavengers of the 'OH initiator of lipid peroxidation (Table 4) indicates that a significant fraction of the cardiomyocyte-membrane thiol depletion associated with H,O,-induced oxidative stress represents an epiphenomenon of membrane lipid peroxidation. These data, however, do not exclude possible secondary effects of H,O,-induced cardiomyocyte thiol oxidation on, for example, enzyme function (Guarnieri et al., 1987) or ion-channellpump activity (Vile and Winterbourn, 1990), which may be important to contractility. Such effects, however, would not appear to be intimately coupled to lethal cardiomyocyte disruption during H,O,-induced oxidative stress (Table 4) (Lewko, 1987).

The increased turnover of membrane protein and phospholipid observed in cardiomyocytes during H,Oz- induced oxidative stress (Table 1) could be significantly diminished by antiperoxidants and iron chelators (Table 4). As with membrane thiol oxidation, the attenuation of the H,02-induced turnover of cardiomyocyte-membrane molecular constituents by antiperoxidants suggests that the accelerated turnover is largely

an epiphenomenon of peroxidation per se. Oxidative damage to rat-liver slices induced by bromotrichloromethane has been reported to stimulate the enzymatic degradation of hepatic (including, presumably, membrane) protein, and the proteolysis was inhibitable to a large degree by antiperoxidants (Zamora et al., 1990). Iron-mediated cardiac-membrane peroxidation is associated with membrane phospholipid breakdown in the ischemic-reperfused rat heart (Liu et al., 1990), and a defective phospholipid deacylation-reacylation cycle has been identified in the ischemic-reperfused pig heart (Das et al., 1986). In our oxidative-injury model, though, an accellerated loss of membrane-lipid acyl groups was apparently met by an increased level of reacylation or acyl-group transfer, leading to a net increase in membrane phospholipid turnover. This finding is reminiscent of the recent autoradiographic data of Miyazaki et al. (1990), which indicate that iodoacetate poisoning of neonatal myocytes accellerated sarcolemmal phospholipid turnover. Although potential cause-and-effect relationships between degra- dative changes in membrane molecular constituents and lethal cell injury were not addressed in these cited studies, they support the conclusion made in the present work that membrane lipid peroxidation stim- ulates the turnover of membrane molecular constituents, perhaps as a reflection of cellular repair mechanisms.

It is particularly noteworthy that abolition of cardiomyocyte peroxidation and preservation of cell integrity could be achieved in the absence of any effect on H,O,-induced ATP depletion (Table 4). These data demonstrate a clear mechanistic uncoupling between the metabolic injury supporting cardiomyocyte de-energization and the membrane peroxidation supporting cardiomyocyte disruption. The pathogenic relationship between ATP loss and cell necrosis has long been a matter of controversy (Andreoli, 1989). Our results correlate well with the finding of Siegmund et al. (1990) that ATP-depleted anoxic cardiomyocytes did not necessarily leak LDH. In contrast, other investiga- tors have concluded that maintenance of cardiomyocyte ATP content is a prerequisite for sarcolemmal integrity (Higgins and Bailey, 1983; Murphy et al., 1987). This conclusion, however, has been based largely on the temporal coincidence between the degree of ATP loss in chemically poisoned cardiomyocytes and the extent of cytoplasmic enzyme leakage, a coincidence we also have observed between the two process during H,O,- induced oxidative stress (Figs. 2 and 7). The finding that de-energized cardiomyocytes did not invariably leak LDH during H20,-induced oxidative stress (Table 4) although ATP depletion and sarcolemmal rupture occurred simultaneously clearly demonstrates that the coincident relationship was not a causal one. Subsequent reports will address the nonperoxidative mechanism responsible for cardiomyocyte de-energization during H202-induced oxidative stress.

The fact that sequellae of cardiomyocyte H20, exposure, including lipid peroxidation (Davies e t al., 19901, antioxidant depletion (Weisel et al., 1989), nucleotide depletion (Abd-Elfattah et al., 19901, and cardiomyocyte necrosis (Ooiwa et al., 1991), have been observed in ischemic-reperfused hearts in vivo does not allow con-

362 JANERO ET AL

clusion that postischemic cardiac injury solely, or even primarily, reflects H202-induced oxidative stress. The multifactorial nature of ischemic heart disease (Janero, 1990a) makes it prudent to consider neither oxidative stress nor H202 as an inevitable determinant of postischemic cardiomyocyte necrosis and myocardial infarction. Although catalase offers cardioprotection in at least some animal models of acute myocardial ischemia (e.g., Jeroudi et al., 19901, it is anticipated that increased mechanistic understanding of the biochemical pathology of H,O, in the cardiomyocyte would aid the design of more specific interventions with potential clinical utility to the ischemic heart-disease patient.

ACKNOWLEDGMENTS We thank our technical staff, N. Howie, R. Lappe,

and M. Worcel for support of this work, A. Lopata, M. Smith, S. Mikulicka, and C. Shulman for secretarial assistance, the Netter Scientific Library and Scientific Information Center for literature assistance, M. Meredith (Oregon Health Sciences University) for ad- vise regarding thiol quantification, and D. Feldman, J. Thakkar, and C. Yarwood for comments.

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Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): Lethal peroxidative membrane injury