Research Article

Effects of Endothelin-1 Antagonist BQ610 onHypoxia-Induced Injury and [Ca2+]i Changes in

Cultured Neonatal Rat CardiomyocytesLi Lin and Wen-Jun Yuann

Department of Physiology, Second Military Medical University, Shanghai, China

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ABSTRACT Hypoxia is a strong stimulus for endothelin-1 (ET-1) synthesis, which is involved inmyocardial ischemia. To shed light on the role and calcium mechanism of endogenous ET-1 in myocardialischemia, the effects of the ET-1 antagonist BQ610 on hypoxia-induced injury and [Ca2+]i changes wereobserved in cultured neonatal rat cardiomyocytes. Hypoxia-induced injury was assessed by determiningsupernatant activity of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in cardiomyocytesexposed to hypoxia produced by a 3% O2, 5% CO2 atmosphere. [Ca2+]i was measured with the Ca2+-sensitive dye Fluo-3 under a confocal microscope. The hypoxia model used for [Ca2+]i measurement wasprepared by perfusing cells with 95% N2, 5% CO2 saturated DMEM solution containing 1mM Na2S2O4.The results showed that: 1) Hypoxia significantly increased ET-1 content in culture supernatant. 2) Thesupernatant LDH was significantly increased after 12- or 24-h hypoxia. On the other hand, SOD activitywas decreased significantly. BQ610 0.2–5 mM dose-dependently reduced LDH release and enhanced SODactivity. 3) [Ca2+]i transient was observed in the normal cultured beating cardiomyocytes. Exposure tohypoxia caused termination of [Ca2+]i transient in 3–5min and a continuous rapid increase in [Ca2+]i afterabout 20min. In the presence of BQ610, the termination of [Ca2+]i transient was postponed to 15–20mininto hypoxia; the [Ca2+]i increase was rather slow until approximately 45min into hypoxia. These resultsindicate that endogenous ET-1 contributed to hypoxia-induced injury in the cultured neonatal ratcardiomyocytes and that the injury was associated with a [Ca2+]i mechanism partially mediated by ET-1.Drug Dev. Res. 58:74–78, 2003. �c 2003 Wiley-Liss, Inc.

Key words: endothelin; myocardial ischemia; calcium; rats

INTRODUCTION

Endothelin (ET) is a family of four isopeptides,ET-1, ET-2, ET-3, and ET-4 (vasoactive intestinalconstrictor). ET-1, the primary isopeptide of ET in thecardiovascular system, with potent vasoconstrictiveactivity, is synthesized mainly by endothelial cells, butalso by vascular smooth muscle cells and cardiomyo-cytes [Suzuki et al., 1993]. A dense distribution of ET-1receptors has been demonstrated in cardiomyocytes ofvarious species including neonatal rats, suggesting thatET-1 may act on the heart. ET-1 has been shown toregulate cardiac activities such as contractility andrhythm. During ischemia, production and secretion ofET-1 was elevated in cardiomyocytes and plasma

ET-1 was elevated as well [Tonnessen et al., 1995].Such elevation of ET-1 has been shown to contributeto ischemic cardiac injury and arrhythmia. The

DDR

Contract grant sponsor: the Major State Basic ResearchDevelopment Program of China; Contract grant number:G2000056905; Contract grant sponsor: the National NaturalScience Foundation of China; Contract grant numbers: 39770855,30070306.

nCorrespondence to: W.-J. Yuan, Department of Physio-logy, College of Basic Medical Sciences, Second Military MedicalUniversity, Shanghai 200433, P.R. China.E-mail: yuanwj@smmu.edu.cn

Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ddr.10134

DRUG DEVELOPMENT RESEARCH 58:74–78 (2003)

�c 2003 Wiley-Liss, Inc.

intracellular mechanisms underlying actions of ET-1involve mobilization of intracellular calcium ([Ca2+]i)and activation of protein kinase C (PKC). The [Ca2+]i

mechanism is so important that it directly correlateswith cardiomyocyte contraction, rhythm, and prolifera-tion. However, effects of ET-1 on [Ca2+]i have beenobserved mostly with exogenous ET-1 [Kohmoto et al.,1993]. To shed light on the actions of endogenous ET-1on cardiomyocyte [Ca2+]i under the condition ofischemia/hypoxia, we observed the effects of the ET-1antagonist BQ610 on hypoxia-induced [Ca2+]i changesin cultured neonatal rat cardiomyocytes. In addition,hypoxia-induced injury to cardiomyocytes was alsodetermined in the presence of BQ610 to illuminate therole of endogenous ET-1 in hypoxia.

MATERIALS AND METHODS

Preparation of Cultured Neonatal RatCardiomyocytes

Neonatal rat cardiomyocytes were cultured from1–3-day-old Sprague-Dawley rats with a modificationof the method described previously [Simpson andSavion, 1982]. In brief, the pups were decapitated andtheir ventricles were removed aseptically and mincedin chilled Ca2+- and Mg2+-free Hanks’ solution. Theventricles were cut into small pieces and digested with0.2% trypsin (Amresco, Solon, OH, USA) with constantagitation (100 cycles/min) at 371C. After digestion eachsupernatant except the first was collected every 10 minand centrifuged immediately at 1,000 rpm/min for 10min. The pellet was dispersed, filtered through a nylonmesh, and suspended in Dulbecco’s Modified EagleMedium (DMEM; GIBCO, Grand Island, NY, USA)containing 20% neonatal bovine serum. After a 30-minperiod of preplating allowing noncardiomyocytes toattach, the nonattached cells were seeded at aconcentration of 3� 105, and cultured in a 5% CO2,95% air humidified incubator at 371C. The mediumwas added to 0.1 mM 5-bromo-20-deoxyuridine (Sigma,St. Louis, USA) that presumably prevented noncardio-myocytes proliferation without cardiomyocyte toxicity[Simpson and Savion, 1982]. Studies were performedafter 4–5 days of culture in cells that had been serum-deprived for 24 h.

Supernatant ET-1 Determination

The supernatant was sampled prior to and 24 hafter hypoxia. Samples were collected into a prechilledpolypropylene tube, stored at �701C, and tested within2 weeks. ET-1 was extracted with C18 Sep-Pak columnand quantitatively assayed with a radioimmunoassay kit(Peninsula Laboratories, Belmont CA, USA).

Assessment of Hypoxia-Induced Injury

To assess hypoxia-induced injury to culturedcardiomyocytes, supernatant activity of lactate dehy-drogenase (LDH), and superoxide dismutase (SOD)were determined. LDH was determined with anautomatic biochemical analyzer (Hitachi 7150, Japan)and SOD was determined by the method described byMarklund and Marklund [1974]. In this protocol, cellswere restricted to hypoxia by incubating in a humidi-fied incubator with 3% O2, 5% CO2 atmosphere at371C.

Measurement of Intracellular Free Calcium

Intracellular free calcium was measured with theCa2+-sensitive fluorescent dye Fluo-3. To be loadedwith Fluo-3, a glass coverslip with cultured cells wasincubated with 5 mM Fluo-3/acetoxymethyl ester (Fluo-3/AM; Molecular Probes, Eugene, OR, USA) for30 min at 371C and then rinsed with fresh DMEM.Afterwards, the coverslip was mounted in a chamber onthe stage of a laser scanning confocal microscope (ZeissLSM510, Thornwood, NY, USA), and continuouslyperfused with DMEM solution. Fluo-3 in cells wasexcited with light at 488 nm and emitted fluorescencewas detected at Lp505 nM. Fluorescence images wereacquired at 200-ms intervals. Since Fluo-3 is notratiometric, absolute [Ca2+]i was not calculated.

Unlike that described above, the hypoxia modelused in [Ca2+]i measurement was produced by perfus-ing cells with 95% N2, 5% CO2 saturated DMEMcontaining 1 mM sodium dithionite (Na2S2O4).Na2S2O4 lowers oxygen pressure (pO2) instantly, while95% N2 bubbling maintained pO2 at a low level. pO2 ofthe solution in the chamber was assayed to be2.770.8 kPa. To observe the effects of the ET-1antagonist BQ610 on hypoxia-induced changes inintracellular calcium, BQ610 was perfused 20 minbefore hypoxia and lasted until the end of hypoxia.

Data and Statistics

All data are expressed as mean7SD. SupernatantET-1 content, LDH activity, and SOD activity beforeand after hypoxia were compared by Student’s pairedt-test, since samples were taken from the same culture.[Ca2+]i in the hypoxia control group and in BQ610groups were assessed by one-way ANOVA andsignificant differences examined by the Student-New-man-Keuls test. Po0.05 was considered statisticallysignificant.

ET-1 IN HYPOXIC CARDIOMYOCYTES 75

RESULTS

Effects of Hypoxia on Characterization of CulturedCardiomyocytes

The neonatal rat cardiomyocytes were observedto contract spontaneously after 1–2 days in culture andreached confluence after 4–5 days. The confluent cellscontracted synchronously and rhythmically at a fre-quency of 30–60 beats/min. During exposure tohypoxia produced by 3% O2, 5% CO2, the contractionbecame visibly weaker, arrhythmic, and nonsynchro-nous. After 24-h hypoxia almost no visible contractionwas observed. However, in 5 mM BQ610-incubatedcells the contraction was maintained, although slowerthan prehypoxia.

In the present preparation, the purity of cardio-myocytes was about 80%, which was determined bymorphological discrimination and by counting con-tracting cells.

Effects of Hypoxia on Supernatant ET-1 Content

After incubation in a 3% O2, 5% CO2 hypoxicatmosphere for 24 h, ET-1 in supernatant of culturedcardiomyocytes significantly increased from 6.5372.94pg/ml to 10.6274.28 pg/ml (Po0.01).

Effects of BQ610 on Hypoxia-Induced Changes inSupernatant LDH and SOD Activity

In normal cultures, supernatant LDH and SODactivity remained stable after 12- or 24-h incubation.When exposed to hypoxia, LDH activity significantlyincreased from 20.870.8 U/L to 31.871.0 (Po0.01)and to 37.071.0 U/L (Po0.01), respectively, whileSOD activity significantly decreased from 32.170.8U/ml to 15.071.2 (Po0.01) and to 12.171.4 U/ml(Po0.01), respectively. BQ610 dose-dependently re-duced LDH release and enhanced SOD activity. In thepresence of 5 mM BQ610, LDH was 22.271.6 and23.371.4 U/L, respectively, after 12-h and 24-h hypox-ia, both significantly lower than that in the hypoxia

group (Po0.01); SOD was 27.371.2 and 27.971.3U/ml, respectively, both significantly higher than thehypoxia (Po0.01) (Table 1).

Effects of Exogenous ET-1 on [Ca2+]i

Spontaneous [Ca2+]i transient synchronous to cellcontraction was observed in normal cultured cardio-myocytes, while no [Ca2+]i oscillation was observed innoncardiomyocytes (fibroblasts). As previously de-scribed [Kohmoto et al., 1993], [Ca2+]i are presentedas diastolic and systolic values. Systolic [Ca2+]i wasdetermined as the average of the highest point of eachtracing of [Ca2+]i transient over a 30-sec interval, whilediastolic [Ca2+]i was the average of the lowest point.Exposure of cells to 10 nM ET-1 resulted in asignificant increase in diastolic [Ca2+]i and a slightincrease in systolic [Ca2+]i (Fig. 1). In addition, thefrequency of [Ca2+]i transient increased by 40–70%.

Effects of BQ610 on Hypoxia-Induced [Ca2+]iChanges

Hypoxia produced by 95% N2, 5% CO2 bubbledDMEM solution added to Na2S2O4 caused arrhythmiafollowed by termination of [Ca2+]i transient in 3–5 min(Fig. 2) and a continuous rapid increase in diastolic[Ca2+]i after about 20 min (Fig. 3). In more than 300normal cardiac cells/cell clusters, calcium wave wasobserved in only one cell cluster. However, in the 46hypoxic cardiac cells/cell clusters, calcium wave wasobserved in three cells/cell clusters, showing that theincidence of calcium wave in cardiomyocytes wasincreased by hypoxia.

In BQ610-incubated hypoxic cardiomyocytes, thetermination of [Ca2+]i transient was postponed to15–20 min into hypoxia and the increase in [Ca2+]i

was rather slow until approximately 45 min intohypoxia. After 30 min of hypoxia, diastolic [Ca2+]i ofthe 5 mM BQ610 group was significantly lower thanthat of the hypoxia control (Fig. 3).

TABLE 1. Effects of BQ610 on Supernatant Activity of LDH and SOD in Hypoxic Cultured Neonatal Rat Cardiomyocytes

LDH (U/L) SOD (U/ml)

Group n Prehypoxia 12 h 24h Prehypoxia 12 h 24h

Normal control 16 20.471.1 22.071.5 22.371.4 31.670.7 33.871.3 35.471.5Hypoxia 25 20.870.8 31.871.0nn## 37.071.0nn## 32.170.8 15.071.2nn## 12.171.4nn##

BQ610 0.2 mM+hypoxia 8 19.871.5 29.771.3nn## 33.771.4nn## 30.271.6 16.371.4nn## 12.771.2nn##

BQ610 1 mM+hypoxia 14 20.271.3 26.570.9nn#– 30.371.2nn##– 31.271.2 21.670.9nn##– 21.071.5nn##–BQ610 5 mM+hypoxia 12 19.571.5 22.271.6– – 23.371.4– – 31.771.0 27.371.2#– – 27.971.3#– –

nnPo0.01 vs. pretreatment (compared by Student’s paired t-test).#Po0.05, ##Po0.01 vs. normal control group at the same time point (analyzed by ANOVA).–Po0.05, i.e.,– –Po0.01, –Po0.01 vs. hypoxia group at the same time point (analyzed by ANOVA).

76 LIN AND YUAN

DISCUSSION

In the present preparation of cultured neonatalrat cardiomytes, supernatant ET-1 content increasedduring hypoxia, suggesting that synthesis and release ofET-1 was enhanced by hypoxia, corresponding to thatdescribed in in vivo cardiomyocytes [Tonnessen et al.,1995]. Furthermore, the ET-1 antagonist BQ610alleviated hypoxia-induced release of LDH and de-

crease of SOD activity, supporting a promotive role ofincreased endogenous ET-1 in hypoxic injury.

When exposed to hypoxia, early termination of[Ca2+]i transient and rapid elevation of [Ca2+]i wereobserved in the present cultured cardiomyocytes,similar to previous studies [Miyata et al., 1992].Although endogenous ET-1 has been demonstrated toincrease during hypoxia and contribute to hypoxicinjury, how ET-1 affects [Ca2+]i during hypoxia remainsunknown. We first observed that BQ610 hindered thetermination of [Ca2+]i transient and the followingelevation of [Ca2+]i, suggesting a contributive role ofET-1 in [Ca2+]i changes. Possibly, preventing theeffects of ET-1 may help to inhibit hypoxia-induced[Ca2+]i overload, which is believed to lead to myocyteinjury, including death. As regards exogenous ET-1,both positive and negative effects on [Ca2+]i have beendemonstrated in cardiomyocytes [Kohmoto et al.,1993]. These variable responses are possibly species-and age-related. We observed significant elevation ofdiastolic [Ca2+]i in myocytes exposed to 10 nM ET-1.This supported the possibility of the contribution ofendogenous ET-1 to hypoxia-induced diastolic [Ca2+]i

elevation.Not only [Ca2+]i transient but also [Ca2+]i wave

have been described in cultured cardiomyocytes[Tanaka et al., 1996]. The former is a transientsimultaneous whole-cell increase in [Ca2+]i, dependenton [Ca2+]o influx and coupled to normal contractions.Conversely, the latter is a local [Ca2+]i increase

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Fig. 1. Effects of 10 nM ET-1 on systolic [Ca2+]i (Fmax) and diastolic[Ca2+]i (Fmin) in spontaneously beating cultured neonatal rat cardio-myocytes. nPo0.05, nnPo0.01 vs. �3min (prior to ET perfusion).

Hypoxia

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Fig. 2. A representative tracing of hypoxia-induced changes in [Ca2+]i transient in spontaneously beatingcultured neonatal rat cardiomyocyte (//=an interval of approximately 140 sec).

ET-1 IN HYPOXIC CARDIOMYOCYTES 77

propagating in the cytoplasm, triggered by Ca2+

released from sarcoplasmic reticulum (SR) [Tanakaet al., 1996]. [Ca2+]i wave was supposed to be aconsequence of membrane damage and/or SR Ca2+

overload [Williams, 1993]. In the present work, underthe condition of hypoxia, the greatly increased in-cidence of [Ca2+]i wave possibly reflected the disorderof cardiomyocytes.

In this model of cultured neonatal rat cardio-mytes, the preventive effects of BQ610 on hypoxia-induced [Ca2+]i changes indicated that the [Ca2+]i

mechanism, which is believed to be a commonmechanism for endogenous chemical mediators in-

volved in myocardial ischemia, was a mechanism of ET-1 in hypoxic/ischemic injury. On the other hand, itindicated that hypoxia-induced injury was associatedwith a [Ca2+]i mechanism partially mediated by ET-1.However, the mechanisms of transsarcolemmal Ca2+

movement and SR Ca2+ handling underlying theeffects of ET-1 on [Ca2+]i during hypoxia remain tobe clarified.

REFERENCES

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Marklund S, Marklund G. 1974. Involvement of the superoxideanion radical in the autooxidation of pyrogallol and a convenientassay for superoxide dismutase. Eur J Biochem 47:469–474.

Miyata H, Lakatta EG, Stern MD, Silverman HS. 1992. Relation ofmitochondrial and cytosolic free calcium to cardiac myocyterecovery after exposure to anoxia. Circ Res 71:605–613.

Simpson P, Savion S. 1982. Differentiation of rat myocytes in singlecell cultures with and without proliferating nonmyocardial cells.Circ Res 50:101–116.

Suzuki T, Kumazaki T, Mitsui Y. 1993. Endothelin-1 is produced andsecreted by neonatal rat cardiac myocytes in vitro. BiochemBiophys Res Commun 191:823–830.

Tanaka H, Kawanishi T, Matsuda T, Takahashi M, Shigenobu K.1996. Intracellular free Ca2+ movements in cultured cardiacmyocytes as shown by rapid scanning confocal microscopy.J Cardiovasc Pharmacol 27:761–769.

Tonnessen T, Giaid A, Saleh D, Naess PA, Yanagisawa M,Christensen G. 1995. Increased in vivo expression and productionof endothelin-1 by porcine cardiomyocytes subjected to ischemia.Circ Res 76:767–772.

Williams DA. 1993. Mechanisms of calcium release and propagationin cardiac cells. Do studies with confocal microscopy add to ourunderstanding? Cell Calcium 14:724–735.

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Fig. 3. Effects of BQ610 on hypoxia-induced changes in diastolic[Ca2+]i (Fmin) in spontaneously beating cultured neonatal rat cardio-myocytes (0: hypoxia) #Po0.05 1 mM BQ610 vs. hypoxia; nPo0.05,nnPo0.01 5mM BQ610 vs. hypoxia.

78 LIN AND YUAN