Endogenous antioxidants in isolated hypertrophied cardiac myocytes and hypoxia-reoxygenation injury

J Mol Celt Cardiol 27, 263 272(1995)

Endogenous Antioxidants in Isolated Hypertrophied Cardiac Myocytes and

Itypoxia-Reoxygenation Injury Lorrie A. Kirshenbaum, Michael Hill and Pawan K. Sin gal Division of Car&ovascula" Sczences, St. Bonifl~ce General Host)ital Research Centre and Department (~" Phgsiology, FaculUd of Medicine, Universit9 qfi Ma.nitoba, Wimzit)e~l, Canada (Received 10 March 1994, accepted in revisedJorm 2 ]une 1994) I,. A. KIIlSH[~;N/~AUM,M, 1111.1, AND P. K, SlNGA/,, Endogem3us Antioxidants in Isolated Hypertrophied Cardiac Myocytes and Hypoxia-Reoxygenation Injury, ]eTernal of Molecular and (3ellllhu"Cardiolo(19 (1995) 27, 263-272, Effects of hypoxia-reoxygenation (H-R) on myocytes isolated f'rom 10 week hypertrophied and sham control rat hearts were studied. Myocyte hypertrophy was indicated by an increase in cell size. Superoxide dismutase (SOl)) and glutathione peroxidase (GSHPx) enzyme activities were signilicautly higher and lipid peroxidaiion (TBARS) was lower in hypertrophied myocytes prior to any t]--R. Hypertrophied myocyte population showed signilican/ly less damage to cell morphology due to H-R. In shmn as well as hypertrophied myocytes, Na ~ arid Ca2 L contents were increased by H-R. but Ca z~ accumulation was significantly less in the hypertrophied myocytes. Bolh SOD and GSltPx activities were depressed by the oxidative stress in the sham myocytes whereas these activities were not significantly changed iu the hypertrophied myocytcs. Catalase activil N in the prehypoxic sham and hypertrophied myocytes was comparable and lhis activity did not change during H-R. There was a signil]cant increase in lipid peroxidation due to H-R but this change was less in hypertrophied myocytes. This study shows less vulnerability of hypertrophied myocytes to oxidative stress and an increase in endogenous antioxidan/reserve may have an important role in medialing this protec/ion. Key Worms: Superoxide dismutase; Glntathkme peroxidase; Myocardial protection.

Introduction

this concept. Increased oxygen radical activity has been detected within lhe first few minutes of reperfusion (Bolli et aL, 1988; Zweier et aL, 1987). Furthermore, it has been s h o w n that increased oxyge[1 radical activity as well as contractile dysfunction could be attenuated with the use of exogenous antioxidants (Bolli et al., 1989; Kirshenbaum and Singal, 199 ~). Agents k n o w n to either inhibit or scavenge oxygen radicals have been shown to reduce the incidence of reperfusioninduced arrhythmias and improve contractile function in a dose dependent m a n n e r (Bernier el: al., 1989; Gaudel and Duvelleroy, 1984; Kirshenbamn and Singal, 1993; Werns et al., 1986). Cellular antioxidant enzyme activities have been reported to c h a n g e in response to both physiological

Several hypotheses have been proposed to explain ischemia-reperthsion injury. These include: the occurrence of a low energy state (]ennings et d., 1981); intracellular Na v and Ca ~ ~ overload (Tani and N eely, 1989); loss of cell structure (Ziegelhoffbr et al.. 1979); and increased lipid peroxidation (Meerson et al., 1982; Rao et al., 198 ~). There is n o w a general consensus that these different mechanisms m a y not be mutually exclusive and oxygen radicals generated during ischemia/reperfusion a n d / o r hypoxia/reoxygenation contribute to this injury (Bolli et al., 1988; Dhaliwal et al., 1991; Gaudel and Duvelleroy, 1984; Guarnieri et al., 1980). There are both direct and indirect evidences to support

Please address all correspondertce to: Dr P. K. Singal, St. Boniface General Hospital Research Centre. 351 Tache Ave., Winnipeg. Canada R2H 2A6. 0022-2828/95/010263 + 10 $08.00/0

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and pathophysiological conditions (Gerli et al., 1980; Gupta and Singal, ] 989; Kanter etal., 1985; Nohl and Hegner, 1979). We have previously reported that hypertrophied rat hearts with an increased endogenous antioxidant reserve were less vulnerable to oxidative stress injury (Gupta and Singal, 1989: Kirshenbaum and Singal, 1992a; Kirshenbaum and Singal. 1993). In these studies, the hypertrophied hearts displayed a lower incidence of reperfusion-induced arrhythmias, better recovery of contractile function and lower lipid peroxide content than corresponding sham hearts. Similarly, adriamycin, an agent believed to mediate its cardiotoxic effects through free radical mechanisms (Singal et al., 1987), was found to be less toxic in exercised-induced hypertrophied hearts (Kanter et al., 1985). Tolerance to oxidative stress injury has also been reported by others (Anderson et al., 1990; Gaasch et al., 1990). It is not clear, however, whether these observed differences between the sham control and hypertrophied hearts in response to oxidative injury are a consequence of differences in endogenous antioxidants occurring at the level of the cardiac myocyte. Alternatively, these differences can be a reflection of varied responses within the vascular component or secondary to vascular intluences of the oxidative stress imposed. In order to assess the probable involvement of cardiac myocytes as well as to fllrther defne the antioxidant changes reported in whole heart studies, we examined the effects of hypoxia-reoxygenation in myocytes isolated from sham and hypertrophied hearts.

Methods Animals and hypertrophyprocedure Male Spragueq)awley rats weighing ] 50_+.25 g were kept in individual (:ages and were provided with tbod and water ad libitum. Pressure overload was produced by narrowing of the abdominal aorta as described before (Gupta and Singal, 1989). Aninrta/s were anaesthetized (Nembutal, ~ %mg/kg i.p.) and access to the aorta was gained through a midline abdominal incision. A segment (about O. 5 cm) of the aorta in the subdiaphragmatic region was cleared from the adhering tissue. A blunt steel wire ( 1 . ] 5 m m diameter) was placed along the aorta, and a 3 - 0 silk ligature was tied snugly around the wire and aorta. The wire was then withdrawn from the ligature and the abdominal incision was closed. Sham-operated rats (sham controls) were handled in an identical m a n n e r except

for narrowing of the aorta. After surgery, hypertrophic and sham-operated rats were allowed to recover prior to returning them to cages.

Hemodynamic measurements Ten weeks after the surgery, animals were anaesthetized with sodium pentobarbital (Nembutal, 35 mg/kg i.p.). A catheter with a miniature pressure transducer (model PR249, Millar Instruments, Houston, Texas) was introduced into the right carotid artery and advanced in to the left ventricle (Gupta and Singal, 1989) to record heart rate, left ventricular peak systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), on a precalibrated multichannel dynograph (Beckman Instruments, Fullerton, Ca/itbrnia). Measurements were taken 15 min after catheterization of the left ventricle when steady state had been reached.

Myocyte isolation Calcium tolerant rat myocytes were obtained by a method previously described (Bihler et al., 11984; Kirshenbaum and Singal, 1992b). In briel; rats ] 0 weeks post-surgery were injected with sodium heparin (1000 U/Kg) and killed l h thereafter. The thoracic cavity was opened to expose the heart and the ascending aorta was cannulated. Coronary perfltsion was started in situ prior to remowfl of the heart from the animal. Hearts were excised and mounted on a modified Langendorff perlusion apparatus which allowed for switching between single-pass and recirculating perfusions at 37°C. The perfnsate consisted of a modified ]oklik miniinure essential medium (calcium free) supplemented with (raM) taurine, 60; glutmnate, 8; carnitine, 2; magnesium, 3.4; glucose, 15 and 0.1% bovine serum albumin with a low fatty acid content (ptI 7.4). Following a brief wash out period, the perfnsion system was switched to the recirculating mode with Joklik bulti~r containing: 25 #m calcium; 0.1% co/lagenase; and 0.1.% hyaluronidase for 20 rain. The extraneous tissue was removed and hearts were incubated i n the same buffer tor 20 rain. To facilitate desegregation, the tissue and cell suspension was intermittently forced through a pipette with progressively smaller sized tip diameters. The suspension was then filtered through a nylon mesh and resuspended in buffer containing 30 mM potassium chloride and 2% albumin and centrifuged at 30 x g for 2 min. The supernatant was aspirated

Endogenous Antioxidants in Hypertrophied Myocytes and the remaining cell pellet was resuspended in buffer containing 1.25 mM calcimn.

Hypoxia-reoxygenation Cardiac myocytes isolated from sham and hypertrophied hearts were superfused in a specially designed air-tight perfusion bath with a total capacity of 12 ml. The cells were superfused with either hypoxic or normoxic buffer (pH 7.4) in a non-recirculating mode at a flow rate of 4 m l / rain as described earlier (Kirshenbaum and Singal, 1992b). Glucose free buffer was pregassed with 95% nitrogen and 5% carbon dioxide lbr 60rain and was used for hypoxic perfusion. Previous studies have shown that this pregassing procedure is adequate to produce a buffer POx of <5 m m Hg and is sufficient to produce hypoxic injury in isolated myocytes as well as whole hearts (ttearse et al., 1973: Kirshenbaum and Singal, 1992a,b). Normoxic buffer with glucose was used for control as well as reoxygenation and was gassed with 95% oxygen and 5% carbon dioxide for 45 rain. Myocytes were subjected to hypoxia for 30 rain with glucose free Joklik buffer and subsequently reoxygenated for 115 min with the normoxic buffer containing glucose. Serial samples of myocytes were taken at 0 m i n control, after 3 0 m i n hypoxia and alter 15 min reoxygenation. These timings for hypoxiareoxygenation were based on a study published earlier (Kirshenbaum and Singal, 1992b). Myocytes were studied for morphological changes, antioxidant enzymes, lipid peroxidation and cations.

Morphology Each myocyte preparation was examined as a fresh suspenskm under phase light microscope. Length and width of myocytes were determined from several different isolates (>500 cells) using a standard precalibrated stage micrometer and ocular insert set. Trypan blue exclusion method was used to determine cell viability after each myocyte isolation procedure.

Biochemical assays

Antioxidant enzymes

Superoxide dismutase (SOD). Myocytes were homogenized with a Brinkmann Polytron (1:10) in

265

50raM Tris-HCl, pill 8.20, containing 1 mM diethylenetriamine pentaacetic acid al 4°C. Cell homogenates were centrifuged at 20 000 x g for 20 min. The supernatant was aspirated and assayed lbr total SOD activity by following the inhibition of pyrogallol autoxidation (Marklund, 1985). Pyrogallol (24raM) was prepared in lOmM ttCl and kept at 4°C prior to use. Catalase, 3()#M stock solution was prepared in an alkaline bufIbr (pH 9.0). Aliquots of cell supernatant (150 #g protein) were added to Tris HC1 buffer containing: 25 #1 pyrogallol and 20 #1 catalase. The linal volume of 3 ml was made up with the same buflier. Changes in absorbance at: 42(i)nm were recorded at: 1 rain intervals tbr 5 min. SOl) activity was determined from a standard curve related to % inhibition of pyrogallol autoxidation with commercially available SO/). This assay was highly reproducible and the standard curve was linear up to 250 ctg myocyte protein with a correlation coefficient of r = 0 . 9 9 8 . Data are expressed as SOD U/mg protein as compared to the standard.

Glutathione Peroxidase (GSHPx). Enzyme activity was expressed as nanomoles Of reduced nicotinamide adenine dinucleotide phosphate (NADPIt) oxidized to nicotin amide adenine dinucleotide phosphate (NAI)P) per minute per milligram protein, with a molar extinction coefficient of 6.22 x 10 ~'for NA1)PH (Paglia and Valentine, 1967). Myocytes were homogenized at 4°C (1:10) in 75 mM phosphate buffer pH 7.(i) and centrifuged at 3() 000 x g for 45 min and supernatant was used to measure GSttPx activity. Myocyte GSHPx was assayed in a 3-ml cuvette containing 2.4 ml of phosphate buffer. The tbllowing solutions were then added: 50 #1 of 60 mM glutathione, 100 #l glutathkme reductase solution (30 units/ml), 50 #1 of 0.12 M NAN,, 1 O0 #1 of 15 mM Na2 EDTA, 100#1 of 3.0raM NADPtt, 100 ¢d aliquot of supernatant and 100/~1 of 7.5 mM H20> Conversion of NADPH to NADP was monitored by a continuous recording of the change in absorbance at 340 nm at 1 min intervals for 5 min. Catalase (CAT). Myocytes were homogenized at 4°C (1:10) in 50raM potassium phosphate buffer pH 7.0. Homogenate was centrifuged at 30 000 x g for 45 min and 50 #1 of the supernatant was added to the cuvette containing 2.95 ml of 19 mM hydrogen peroxide solution prepared in potassium phosphate buffer. The decrease in absorbance was followed at 240 n m a l l min intervals for 5 rain. Specific activity of the enzyme was expressed as U/mg myocyte protein (Claiborne, 1985).

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Lipid peroxidation Lipid peroxide tbrmation was determined by examining the content of thiobarbituric acid reactive substances (3?BARS) as described earlier (Lee et al., 1991). Recently it has been suggested that study of TBARS using a colorimetric method provides better estimation of lipid peroxidation (Lapena and Cuccurullo, 1993) as compared to the HPLC analysis of malondialdehyde (MDA) content (Ceconi et al., 1991). Myocytes were homogenized at 4°C (1:10) in 0.2M Tris-O.16M KC1 buffer, pH 7.4 and the homogenate was incubated for l h at 37°C in a water bath. A i ml aliquot was withdrawn from the incubation mixture and pipetted into a 8 ml Pyrex tube. This was tbllowed by the addition of 1.0 ml of 40% trichloroacetic acid and after mixing, 1 . 0 m l of 2% sodium thiobarbiturate was immediately added. The tubes were heated for 15 min and cooled on ice for 5 rain. Two millilitres of 70% trichloroacetic acid was added. Tubes were allowed to stand for 20 min and were centrifuged at 800 × g for 15rain. The colour was read at 5 3 2 n m . In order to minimize peroxidation during the assay procedure, 2 % butylated hydroxytoluene (BI IT) was added to the TBA reagent, mixture. Commercially available malondialdehyde was used as standard.

Cation content For the study of myocyte cation content, cell aliquots were digested in 1:] 0 volumes of acid mixture containing 70% perchloric acid and 70% nitric acid in equal volumes (Willis, 1961). Lucite tubes containing myocytes and acid mixture were incubated at 40°C for 24 h in a shaking water bath. The digestion was continued until the solution was clear. The acid extract was then analysed for total myocytic Na ~ and Ca a~ content using atomic absorption spectrometer (Perkin-Elmer, 2380). Lant h a n u m chloride (1%) was added to samples as well as to known sta:ndards in order to minimize interference by phosphates. Standards were processed in a m a n n e r similar to samples so that they also contained acid in amounts equivalent to that :in the sample (Khatter et al., 1978).

Protein estimation and statistics Protein content of myocytes was determined using bovine serum albumin (fraction V, Sigma) as the protein standard (Lowry et al., 1955). Since the number of myocytes tbr a given aliquot of cell suspension undoubtedly would vary, all values were expressed in terms of myocyte protein. Data are

given as mean+s.E.M, of (8-9) individual experiments unless mentioned otherwise. One-way analysis of variance (ANOVA) was used to determine the significance of differences among groups and Tukey's test was used to identify differences between groups. Data were considered to be statistically significant with a P value of <0.05.

Results Hypertrophy characteristics Hypertrophied hearts, after 10 weeks of pressure overload, showed about a 40% increase in heart to body weight ratio and about a 61% increase in left ventricular peak systolic pressure with no difference in body weight and heart rate as compared to sham control hearts (Table 1). In the hypertrophy group, left ventricular end diastolic pressure was unchanged and none of the animals showed any other signs of heart failure such as ascites and cyanosis. Cardiac myocytes obtained from sham control and hypertrophied hearts were examined for cell morphology, antioxidant enzymes, lipid peroxidation, cation content and high energy phosphates.

Cell morphology Based on tl~eir morphological appearance, the cells from sham and hypertrophied hearts were grouped into three categories: rod, rounded and hypercontracted. The rod cells were readily distinguished by phase light microscopy from both round and hypercontracted cell types, because of lheir striated appearance and high length to width ratio. The hypercontracted cells also showed distinguishable sarcomere pattern and excluded trypan blue. They were cubical in appearance with lower length-to-width ratio. The rounded cells did not show striations, failed to exclude trypan blue and appeared ball-shaped and granular. Freshly isolated pre-hypoxia cell preparations from sham hearts contained on average 70% of viable rodshaped cells which excluded trypan blue dye and tolerated 1.25 mM Ca 2~ . Remaining cell population was comprised of round (25%) and hypercontracted cells (6%) respectively (Table 2). The rod shape myocytes isolated from s h a m control hearts, had an average length of 131.4_+2.1tim and width 40.3 _+2.4 #m. The rod shape myocytes isolated from hypertrophied hearts had an average length of

Endogenous Antioxidants in Hypertroplaied Myocytes

267

'lhble / Body weight, heart weight and hemodynamic data lrom 10 week sham control and banded animals, 1;VSP, left: ventricular systolic pressure; LVEDI), left: ventricular end diastolic pressure; HR, heart rate, tlemodynamic data are mean ± s.v. of at least 9 animals in each group. Heart and body wt data are mean !s.~;:. from 20-23 animals in each group. Parameter

Sham

Body wt (g) Heart/Body wt (rag/g) LVSP(mmftg) LVEDP(mmHg) l:[R(beats/min)

Hypertrophied

5116.2±12.1 511,6± 9.3 2.9:j- 0.4 4.1 ± 0.4* t10.4± 5.7 J77.9-~ 13.2" 3.2q 1.2 4.7:[~ 1.~ 321.3±19.2 362.3~:t3.7

'I)<0.05 in comparison Io the sham conl.ro| value. ~l?able 2 Effects of 30rain hypoxia (HYPX) and 15 rain reoxygenation (I~.EOXY)on the percenl distribution of cellinjury in myocytes isolated from lO week sham control and hypertrophied hearts. I)ata are expressed as mean ±s.r. from 8-9 different myocyte preparations, P
Rod Hypercon tracted Round

Sham Hypertrophied ....................................................................................................... CTL ttYPX REOXY CTL HYPX REOXY 69 ± 8 6± 2 25 .± 3

39 ± 4* 22 t- 5" 39 + 4*

23 ± 3"-131 ± 2"I46 ± 5*

70 ±:9 1() ! 3 20 + 5

52 ± 6*" 26 7" 22 ± 5"

47 ~-_ 8*" 30 I 7" 23 4: 10"

*Different ti'omthe. prehypoxiccontrol (CTL)group wfluc; l"l)ifli:rent fi'omhypoxicgroup P
change, as compared to the hypoxia group, in the number of any cell types in the hypertrophied myocyte populations.

Antioxidant enzymes

Hypoxia-reoxygenationchanges Morphology Myocytes exposed to glucose-l~ee hypoxia for 30 min at 3 7°C showed about 43% reduction in the number of rod-shaped cells compared to prehypoxic control values (Table 2). Hypertrophied myocytes showed significantly less (P
Antioxidant enzymes SOD, GSHPx and CAT were examined in sham and hypertrophied myocytes during control, hypoxia and reoxygenation and the data are shown in Figures l(a), (b) and (c) respectively. SOD activity in the hypertrophied myocytes was increased by 24% compared to age matched sham myocytes and hypoxia or reoxygenation showed no effect on this activity [Fig. l(a)], Hypoxia caused about a 51% reduction in the total SOD activity in sham myocytcs compared to prehypoxic controls. Reoxygenation of sham myocytes, however, resulted in a significant recovery of the SOD activity. SOD activity in the hypertrophic myocytes was significantly higher than sham myocytes after hypoxia as well as reoxygenation. Tile prehypoxic value for GSHPx activity was about 45% higher in the hypertrophied myocytes than the corresponding value in the sham myocytes [Fig. l(b)]. Sham cardiac myocytes exposed to

268

L.A. Kirshenbaum et al. 40 (a)

1.5

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30 b~

20

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[ ] Sham [ ] Hyper

I0

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CTL

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140 120 --~ 100

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Figure 2 Lipidperoxidation in sham and hypertrophied myocytes as indicated by the content of thiobarbituric acid substances (TBARS). Control (CTL), after 30rain hypoxia (Hypoxia) and 15 rain reoxygenation (Hypoxiareoxygenation). Data is mean ± S.E. of 8 individna/ myocyte preparations. TDifferent from its prehypoxic control; **l)ifferent from the corresponding value in the sham group. P
60 40 20

0

CTL

HypoxiaReoxygenation

(b)

80

o

Hypoxia

HYPX HYPXIREOX

4O (e)

peffusate was analyzed tbr the enzyme activities and the findings were negative.

Lipid peroxidafion rO CTL

HYPX HYPX-REOX

Figure 1 (a) Superoxide dismutase (SO1)), (b) (;lutathione peroxidase (GStlI'x) and (c) Catalase (CAT)activities in sham and hypertrophied myocytes lbr control (CTL), 30rain hypoxia (HYPX) and 15rain reoxygenation (HYPXdlEOX). *l)iffe:centfrom its own prehypoxic control group; rl)itlbrent fcom its hypoxic group; **l)iffcrent from corresponding value in sham group. Data is mean:#s.t~, of 8-9 individual myocyte preparations. P
hypoxic conditions showed a 37% reduction in GSHPx activity compared to prehypoxic control values and no further change was seen upon reoxygenation, llypoxia as well as reoxygenation did not influence GSHPx activity in hypertrophied myocytes at any of the time points and this activity remained significantly higher than sham controls at all points. There was no observable difference in the base line prehypoxic CAT activity between the sham and hypertrophied myocytes [Fig. 1(c)]. The hypoxiareoxygenation protocol had no effect on CAT activity in either group. In order to test if the observable loss of antioxidant enzyme activities in sham myocytes was due to membrane leakage, the

The TBARS content of sham and hypertrophied myocytes were determined to assess the level of lipid peroxide content and relative oxidative injury during hypoxia/reoxygenation (Fig. 2). Myocardial TFIAP,S content of prehypoxic sham control myocytcs was significantly higher than hypertrophied myocytes (P
Cations In order to further characterize hypoxia and reoxygenation inj[try in hyperlrophied myocytes, Na ~ and Ca-' + contents in the sham and hypertrophied myocytes were measured and these data are shown in (Table 3). Cation (Na + and Ca ~ ) contents in sham control and hypertrophied myocytes prior to hypoxia-reoxygenation were no different from each other, and the values were in the range previously reported (Cheung et aI., ] 982; 1984). During hypoxia, there was some but statistically insignificant

Endogenous Antioxidants in Hypertrophied Myocytes lhble 3 l'ercent changes in the cation conlenl in sham and hyperlrophied myocytes during 30rain hypoxia (HYPX) and 15 rain reoxygenation (REOXY). Data are from 8-9 different myoeyte preparations and expressed as Fs.IG pelx;e:H{ el c{}ni:r{}[ values. Base line control values for cellular Na ~{0.039 ±0.0075 #reel/rag) and Ca2 ~(0.035 ± {}.12 #reel~rag) in sham and hyperl:rophied myocytes were nut different. Group

Cation

tIYPX

RI~;(}XY

Na ~ Ca2~

117±16 20{}± 15"

1512!£22* 324± 12"1

Na ~ Ca2~

118¢:16 143:~14""

] 4 6 + 8"I" 231 --}--£1(}*'-~'I

Sham Hypertrophied

*Dillbrent l~om prchypoxiccontrol value. "iI)iflZ~.renlfi'om the hypoxic myocytes. q)iffcrenl t]x}m lhe corresponding value in sham myocytc group. P<0.O5.

increase in Na ~-content of sham and hypertrophied myocytes. There was almost a 100% and 43% increase in the Ca 21 content of sham and hypertrophied myocytes respectively. This increase in Ca-' + content in the hypertrophied myocytes, however, was significantly less than the corresponding sham group (P
Discussion Increase in endogenous antioxidants in response to a variely of chronic work overload on rat heart has been reported before (Gnpta and Singal, 1989; Kanter et al., 1985; Kirshenbaum and Singal, 1993). The present study On isolated myocytes show for the first time that this increase is indeed taking place at the level of cardiac myocyte and is also accompanied by a concomitant decrease in the oxidative stress as indicated by a decrease in TBARS. Whether TBARS strictly represent lipid peroxidation, is still debatable. However, there is some agreement that the technique reflects total biomolecule oxidant stress (Ceconi et al., 1991; Gutteridge, 1982: Lapena and Cuccurullo, 1993). Althougt] in heart multiple stinmli including pressure overload have been suggested to induce proto-oncogenes' mRNAs and proteins (Izumn et al., 1988; Parker and Schneider,

269

1991; Schunkert et al., 1991), molecular basis of endogenous antio×idant enzyme changes in hypertrophied heart remains to he established. Cardiac myocytcs from sham hearts exposed {o hypoxic conditions showed a depression in SOl) activity similar to that seen in the whole heart (l)ha/iwal et aL, 1991 ; Guarnieri et al., ] 980; Kirshenbaum and Singal, 1992a). In a hypertrophied rat heart, ex rive exposure to lnypoxia-reoxygenation has also been shown to cause a signiticant reduction in SO/) activity (Kirshenbaum and Singal, 1992a). This change in the hypertrophied heart appears t:o bc specific with respect to the SOl} aclivity while GS[tPx activity in the whole heart was not in/luenced by hypoxia-reoxygenation (Kirshenbaum and Singal, 1992a). In contrast, no change in the SOl) activity was detected during either hypoxia or reoxygenation of hypertrophied myocytes. Absence of a similar response in SOD activity in isolated hyperlrophied myocyles may suggest that wtscular components, present in the whole heart but absent in the isolated myocyte preparation, m a y be important determining lhctors in this diflbrence. In this regard, an increase in endothelial cell superoxide productkm and increased vascular cell permeability during hypoxia and reoxygenation has been reported (Lure et al., 1992). 1)ata on morphological changes, antioxidants, TBARS and cation contents clearly indicate less injury in hypertrophied myocytes subjecled to ihypoxia-reoxygenation. Greater resistance to oxidative injury in the hypertrophied myocy/es than in sham myocytes is also consistent wilh the data from whole heart studies (Kirshenbaum and Singal, 1992a; Kirshenbaum and Singal, 1993). Although content of TBARS was increased in both sham and hypertrophied myocytes, the increase was significantly less in the hypertrophied myocytes. Since SOD, GSt IPx and catalase activities dkt not change during hypoxia/reoxygmmtion in the hypertrophied myocytes and since increase in TBARS was still signilicantly higher in this group, it appears that high levels of endogenous antioxidants were either inadequate and/or other mechanisms m a y also be operative during oxidative stress injury. This m a y also explain the lack of a complete protection against hypoxia-reoxygenation injury in hypertrophied myocytes. Although the occurrence of Ca 2+ overload was apparent in the present study and has been suggested to mediate myocardial cell injury (Dhalla et al., 1982), a direcl cause and effect relationship has not been established. DelL'cts in the energy dependent Ca ~+ efllux, Na+/Ca 2~ exchange and Ca 2~ entry secondary to free radical-induced mere-

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L.A. Kirshenbaum et al.

brane changes, have been offered as some of the mechanisms to explain Ca ~÷ overload (Dhalla et al., 1982; Kaneko et al., 1990; Kirshenbaum et al., 1.992) and occurrence of hypercontracted and round cell types (Lambert et al., 1986). It should be noted that the increase in Ca 2+ content was considerably less in the hypertrophied myocytes than in the corresponding sham myocytes. Since high energy phosphate levels are known to be depressed during hypoxia, it is possible that energy dependent transport systems such as the Na+-K +ATPase and Ca 2 ~ - A T P a s e may be impaired, resulting in Na ~ and Ca 2+ accumulation during hypoxia with subsequent cell injury. Inhibition of these enzyme activities during hypoxia-reoxygenation as well as ischemia-reperfusion has been reported (Dixon et al., 1987; Hess et al., 1983). Accumulatkm ofNa f during hypoxia have been suggested to account for the rise in intracellular Ca 2~ upon reoxygenation through increased Na+-Ca 2~ exchange (Tani and Neely, 1989). Since Na ÷ content of the sham and hypertrophied myocytes was not different, relatively less Ca 2+ accumulation in the hypertrophied myocytes must have some other explanation. It has been suggested that free-radical mediated membrane changes through oxidative-injury do contribute to the occurrence of Ca 2 ~ overload (Noronhaq)url,a and Steen, 1982). In a previous study, exogenous supply of an antioxidant was shown to attenuate hypoxia-reoxygenation induced Ca -~ overload in the sham myocytes (Kirshenbaum and Singal, 1992). In the present study, a significant decrease in the accunmlation of Ca -'~ in hypertrophied myocytes may also have been due to reduced free radical injury subsequent to increase in endogenous antioxidants. We have recently reported a time-dependent increase in myocyte Na + and Ca 2+ upon exposure to oxygen-radicals which correlated with an increase in the content of TBARS as well as hypercontracted and round cells (Kirshenbaum et al., 11992). Only partial protection against these changes in tiypertrophied myocytes indicated that in addition t o these mechanisms, a high energy deficit may also contribute in Ca 2+ overload and cell injury due to hypoxia-reoxygenation. In conclusion, myocytes isolated from hypertrophied hearts show higher endogenous antioxidant enzyme activities and lower levels of lipid peroxidation as compared to the sham myocytes. Data also show specific antioxidant changes in isolated sham but not in hypertrophied myocytes, independent of non-myocytic and vascular components, as a direct consequence of oxidative stress. Maintenance of SOD and GSHPx activities during

hypoxia as well as reoxygenation in the hypertrophied myocytes along with a better preservation of cell morphology and intracellular calcium; suggests less vulnerability of these cells to oxidative stress.

Acknowledgements This research was supported by the Medical Research Council Group Grant in Experimental Cardiology (PKS). Dr L. A. Kirshenbaum was supported by an MRC Studentship Award.

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