Age-dependence of free radical-induced oxidative damage in ischemic-reperfused rat heart

E L S E V I E R Archives of Gerontology and Geriatrics

22 (1996) 297-309 297

ARCHIVF.,S OF GERONTOLOGY AND GERIATRICS

Age-dependence of free radical-induced oxidative damage in ischemic-reperfused rat heart

Katalin Nagy a,*, Ildik6 E. Tak~ics b, Csaba Pankucsi b

aF. Verz~r International Laboratory for Experimental Gerontology (VILEG), Hungarian Section, University Medical School, Debrecen 4012, Hungary

bBIOGAL Pharmaceutical Works, Department of Pharmacology, Debrecen, Hungary

Received 25 September 1995; revised 4 January 1996; accepted 8 January 1996

Abstract

Oxygen free radical-induced oxidative damage is involved in both aging and ischemia- reperfusion. The purpose of this study was to determine the aging-induced oxidative alterations in rat heart as well as the age-dependence of heart injury following ischemia- reperfusion. A comparative study was performed on young and old ischemic-reperfused rat hearts. Protein oxidation and the ascorbyl radical level in heart tissue were determined in order to characterize the oxidative stress. Comparing the control conditions, old hearts have 31% more oxidized proteins as measured by protein carbonyl content, and 18% lower ascorbyl radical level as determined by ESR, than young ones. The extent of increase of protein oxidation and ascorbyl free radical depletion induced by ischemia-reperfusion is less pronounced in the old hearts (7 and 8% respectively), as compared to the young ones (55 and 21% respectively). Pre-treatment with a free radical scavenger, such as centrophenoxine, diminished the ischemia-reperfusion injury in both young and old rat hearts.

Keywords: Heart ischemia-reperfusion; Peroxidative damage; Protein oxidation; ESR spec- troscopy; Ascorbyl radical; Centrophenoxine

* Corresponding author.

0167-4943/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII 0167-4943(96)00700-5

298 K. Nag), et al./ Arch. Gerontol. Geriatr. 22 (1996) 297 309

1. Introduction

Reperfusion of the ischemic heart plays an important role in the morbidity and mortality of patients undergoing cardiac surgery. Several biochemical and physiological studies have directly or indirectly demonstrated the role of oxygen free radicals in myocardial tissue injury during myocardial ischemia and particularly re-oxygenation (Pietri et al., 1989; Poston and Parenteau, 1992; Powell and Tor- tolani, 1992; Tavazzi et al., 1992; Wang et al., 1992).

Old people have more frequently cardiovascular diseases than young ones, but it would be interesting to know, whether there is an age-dependent difference in the response of heart to an ischemia-reperfusion insult. To approach this question, an animal model experimental system was studied.

In an earlier experiment, Tak~ics et al. (1993) have measured lipid peroxidation and antioxidant enzyme alteration after myocardial ischemia-reperfusion in young rats. They have demonstrated that the activities of superoxide dismutase, glutathione peroxidase and the amount of reduced glutathione increased, while the degree of lipid peroxidation and the activity of catalase remained unchanged in the first minute of reperfusion, following a 15 min global, reversible ischemia.

In the present study protein oxidation and ascorbyl radical level were used as markers for the severity of oxidative damage.

The study of oxidative damage of proteins has been applied only in the last few years to demonstrate the oxygen free radical-induced damage to tissue. Oxidative modification of cellular proteins by metal-catalyzed oxidation during reperfusion leads to the generation of carbonyl derivatives. Carbonyl content can be used as an index of oxygen radical-mediated protein damage. Protein carbonyl level of rat heart increased markedly at 5 min of reperfusion compared to the initial value (Poston and Parenteau, 1992).

Ascorbate is a naturally occurring antioxidant in biological systems. In aqueous solution, there is a redox equilibrium from ascorbate to ascorbyl neutral or anion radicals and dehydroascorbic acid (Pietri et al., 1990). Because of the low reduction potential of ascorbyl radical/ascorbate monoanion couple, ascorbate reacts as a reductant with oxidizing free radicals, forming ascorbyl free radicals (Buettner and Jurkiewicz, 1993). The steady-state concentration of ascorbyl radicals can be used as a measure of oxidative stress in biological systems (Pietri et al., 1990; Buettner and Jurkiewicz, 1993). Dimethyl sulfoxide (DMSO) was used by Pietri et al. (1990) to stabilize ascorbyl radicals for ESR detection. Pietri et al. (1990) and Nohl et al. (1991) have demonstrated, that the damaged ischemic and reperfused myocardium releases ascorbate (i.e. ascorbate ion and ascorbyl radical in equilibrium). Parallel with it, hearts following ischemia-reperfusion exhibited significantly lower tissue ascorbate level than control hearts (Pietri et al., 1990; Tavazzi et al., 1992), according to ESR detection of ascorbyl radicals. A correlation was found between the severity of ischemic insult and the extracellular release or endogenous depletion of ascorbate and/or ascorbyl radical.

K. Nagy et al. / Arch. Gerontol. Geriatr. 22 (1996) 297-309 299

Centrophenoxine (CPH) has been found to slow down certain phenomena associated with aging (Zs.-Nagy, 1989). CPH treatment of old animals reduced lipofuscin deposits, activated antioxidant enzymes (Roy et al., 1983) and exerted a protective effect on proteins against oxygen free radical-induced cross-linking (Nagy and Zs.-Nagy, 1984). It has been established, that CPH is a O H free radical scavenger (Zs.-Nagy and Floyd, 1984; Nagy et al., 1994).

The aim of the present study was to perform a comparative study of young and old rat hearts. Data are presented on the level of protein oxidation and ascorbyl free radical content in the pre-ischemic control and ischemia-reperfused hearts of both age groups, and on the influence of CPH-pre-treatment of animals on these levels. We did not perform the CPH-treatment of young control rats, because the only available data regarding the effect of CPH on myocardium were reported on old guinea pigs (Spoerri et al., 1974).

2 . M a t e r i a l s a n d m e t h o d s

2.1. Chemicals

Phenylmethylsulfonyl fluoride (PMSF) and leupeptin were purchased from Sigma Chemical Co., and HEPES from Calbiochem. Centrophenoxine was used as a lyophylized powder from Helfergin 500 ampoules provided as a gift from Promonta Chemische Fabrik GmbH (Hamburg, Germany). All other reagents were the products of Reanal (Budapest, Hungary).

2.2. A n i m a l s

Young and old male CFY rats were used at 2 and 20.5 months of age, respectively. Heart weight and body weight data are seen in Table 1. Heart weight

Table 1 Heart weight and body weight characteristics of male CFY rats

Age (months)

2 20.5

n = 21

HW, mg 963 + 29 p < 0.01 BW, g 336 + 12 p < 0.01 HW/BW, mg/g 2.92 + 0.09 p NS

n = 23 1309 + 40

461 + 18

2.88 + 0.08

Values are means + S.E.M.; n, number of animals; HW, heart weight; BW, body weight; HW/BW, heart weight/body weight ratio; NS, not significant. Significance was calculated with the two-tailed Student's t-test.

300 K. Nagy et al. / Arch. Gerontol. Geriatr. 22 (1996) 297-309

and body weight undergo a significant age-related increase, however, the heart weight/body weight ratios were not different.

CPH-pre-treatment was carried out as follows: rats were injected intraperi- toneally 5 days a week for 4 weeks with 100 mg CPH/kg body weight. CPH was dissolved in physiological saline immediately prior to injection.

2.3. Experimental procedure

For the comparative study of young and old hearts, samples were taken at the pre-ischemic period - it serves as control sample-, and in the first minute of reperfusion, because hearts show the worst physiological functions: ventricular fibrillation and decreased myocardial contractility. Samples were frozen immediately in liquid nitrogen and stored at - 70°C until use. The effect of centrophenoxine was also tested in both animal groups (6 animals in each group).

2.4. Perfusion technique

All the experiments were performed in isolated perfused rat heart preparation according to Langendorff (1895).

Rats were treated with heparin, then anesthetized with sodium pentobarbital. The hearts were rapidly excised and quickly perfused with Krebs solution through the aorta with a constant perfusion pressure of 9.3 kPa. (The composition of Krebs solution in mM was: 118.1 NaC1; 4.7 KC1; 25.0 NaHCO3; 0.5 MgClz; 0.9 NaH2PO4; 1.9 CaC12; 11.1 glucose equilibrated with 5% CO2 and 95% 02, giving a pH of 7.4 at 37°C.

Hearts were allowed to stabilize by perfusion at 10-12 ml/min for 15 min at 37°C while the mechanical and electrophysiological functions of hearts showed physiological values, but no significant difference was found between young and old rat groups, not even after CPH-pre-treatment. After equilibration the hearts were subjected to 15 min of global, zero-flow ischemia followed by reperfusion.

2.5. Measurement of protein carbonyl groups

Heart samples were minced and homogenized in 10 mM HEPES buffer, pH 7.4 (137 mM NaC1, 4.6 mM KC1, 1.1 mM KH2PO4, 0.6 mM MgSO4) supplemented with protease inhibitors: 0.5/~g/ml leupeptin, 40/~g/ml PMSF, and 1.1 mM EDTA, using a teflon-glass homogenizer. Insoluble cell debris was pelleted at 15 000 g for 20 min. The protein concentration of the supernatant was determined according to Markwell et al. (1978).

The method of Levine et al. (1990) was used to determine the protein carbonyl content. Oxidative damage of proteins is accompanied by the generation of protein carbonyl derivatives which react with dinitrophenylhydrazine (DNPH) to form hydrazone derivatives. Parallel samples were treated with 2 M HC1 or with an equal volume of 10 mM DNPH in 2 M HC1, and allowed to react at room temperature for 30 min. The samples were then precipitated with 10% trichloroacetic acid and


centrifuged at 4000 g for 10 min. The pellets were washed 3 times with ethanol-ethyl acetate (1:1) solution. The resulting precipitate was dissolved in 6 M guanidine hydrochloride, and the absorbance of the samples treated with DNPH in HC1 was determined versus the sample treated with HC1 alone. The concentration of carbonyl groups was calculated from the absorbance at 366 nm, using the extinc- tion coefficient of 22.0 mM - ~cm- ~ for aliphatic hydrazones (Levine et al., 1990).

2.6. Tissue preparation for ESR measurements

DMSO was used for tissue preparation as a chemical stabilizer for the ascorbyl radical and extracting agent for tissues (Pietri et al., 1990). Seven-hundred-and-fifty milligrams of heart tissue was minced, homogenized and then extracted by vortex- ing in 2 ml of DMSO. The homogenates were cent~'uged at 3000 g for 4 min and the supernatant was used for ESR measurement. The intensity of the doublet of ascorbyl radical (g = 2.0052) in the samples prepared by DMSO is stable for at least 2 h at room temperature (Pietri et al., 1990). The ESR spectra were recorded with a JEOL JES-RE1X spectrometer. Typical instrumental parameters were as follows: magnetic field 337.4 ___ 5 mT, modulation frequency 100 kHz, modulation amplitude 0.1 mT, time constant 0.3 s, receiver gain 5 x 103 at 4 mW power level. All the recordings were carried out at room temperature.

2. 7. Data analysis

Statistical analysis for significance between the absolute values of means was carded out with the one-way analysis of variance of SPSS/PC + 4.01 program, wherever appropriate.

3. R ~

3.1. Protein oxidation

The change of protein carbonyl content resulted from aging and ischemia-reperfusion in the rat hearts, and the influence of CPH-pre-treatment were determined.

Fig. 1 shows that the oxidative damage, that is the protein carbonyl content, was 55% higher in the hearts of young animals after ischemia-reperfusion, compared to the control value (p < 0.001). CPH-pre-treatment of young rats diminished significantly the carbonyl level following reperfusion compared to the untreated reperfused young hearts ( p < 0.001).

Old control, pre-ischemic hearts (Fig. 2) had 31% more oxidized proteins than young control ones (p < 0.001). CPH-pre-treatment of old rats reduced considerably the protein carbonyl content of hearts compared to untreated old control value (p < 0.05). Following ischemia-reperfusion the protein carbonyl level of old hearts was only 7% higher relative to the old control value, but this difference is not significant. CPH-pre-treatment protected somewhat, but not significantly the old

302 K. Nag), et al. / Arch. Geromol. Geriatr. 22 (1996) 297 309

6

5 r -

4-, 0

CX

~ 4 E

73 r.-

v

4.o E

~ 2 O O

r" Q

( .3

CPH-pretreated

0 Ischemia 0 15 15 minutes

Reperfusion 0 1 l

Fig. 1. Protein carbonyl contents in young control rat hearts as well as after ischemia-reperfusion with and without CPH-pre-treatment of young rats. Each bar represents the mean _+ S.E.M. for 6 individual heart samples.

heart proteins from the oxidative injury during ischemia-reperfusion compared to the carbonyl content of untreated reperfused hearts.

3.2. E S R measurement o f tissue ascorbyl j~'ee radical

According to the ESR study of Pietri et al. (1990), the ascorbate depletion was followed in the control and reperfused heart of young and old, or CPH-pre-treated rats, by the ESR measurement of tissue ascorbyl radical intensities. Fig. 3 shows typical examples of representative ESR spectra obtained from hearts of each experimental groups. Peak height changes in the ESR spectra reflect the tissue ascorbyl radical content of hearts exposed to ischemia-reperfusion. The ESR signal intensity of ascorbyl radicals was expressed in arbitrary units, i.e. in the mean height of the two signals under identical recording conditions.

As can be seen in Fig. 4, during the first minute of reperfusion, the ascorbyl radical content of heart of young animals decreased 21% (p < 0.001) compared to the young control value. CPH-pre-treatment of young rats reflected an efficient protective effect during ischemia and reperfusion, the ascorbyl radical content


remained much higher in these hearts following ischemia-reperfusion compared to the untreated reperfused young hearts (p < 0.001).

There was an 18% lower ascorbyl radical level in old control, pre-ischemic hearts (Fig. 5) than in young ones (p < 0.001). CPH-pre-treatment increased significantly by 12% the ascorbyl radical content of old control hearts (p < 0.001). At the beginning of reperfusion the level of ascorbyl radicals decreased only 8% compared to the old control value (p < 0.05). CPH-pre-treatment of old animals completely eliminated the decrease of ascorbyl radicals following ischemia-reperfusion, there was no difference in the ascorbyl radical content of CPH-pre-treated control and CPH-pre-treated reperfused hearts.

4. Discussion

It has been demonstrated using the level of protein carbonyl groups as an index of the oxidative damage that oxidized proteins accumulate during aging. The amount of oxidized protein increases with age: in cultured human fibroblasts

~ 4 0

O .

~3

i - v

,,,2 t - g~

t- o o

0

CPH-pretreated CPH-pcetceated

0 Ischemia 0 0 15 i5 minutes

Reperfusion 0 0 i i

Fig. 2. Changes of protein carbonyl content in the old control and CPH-pre-treated old control rat hearts, as well as after ischemia-reperfusion with and without CPH-pretreatment of old rats. Each bar represents the mean _ S.E.M. for 6 individual heart samples.

304 K. Nagy et al. / Arch. Gerontol. Geriatr. 22 (1996) 297-309

1 mT

A

CPH-pt~t~at~

CIPH-pretreated

B CPH-pretreeted

Ischemte 0 0 15 15 minutes

Reperfuelon 0 0 1 1

Fig. 3. Typical ESR spectra of tissue ascorbyl radicals obtained from heart of young (A) and old (B) rats. In both age groups curves are shown before and after ischemia-reperfusion, as well as following CPH-pre-treatment.


(Oliver et al., 1987), in human lenses (Garland et al., 1988), in rat hepatocytes (Starke-Reed and Oliver, 1989), in gerbil brains (Carney et al., 1991) and now in rat heart. As our results show, oxidized proteins accumulated considerably in the heart of old animals. This age-related increase in the level of rat heart protein carbonyls may be due in part to the decreasing rate of oxidized protein degradation. Old gerbils and rats have significantly decreased alkaline protease activity compared to young ones (Starke-Reed and Oliver, 1989; Carney et al., 1991).

CPH-treatment of old rats reduced significantly the m o u n t of oxidized proteins in the heart.

Oxygen free radicals are implicated in numerous biological phenomena like cellular aging (Zs.-Nagy, 1994). Aging is not necessarily accompanied by a more intense free radical formation, but the effect of oxygen free radicals on organic molecules is dependent on the dry mass content (physical density) of the system. Because of the age-dependent increase of intracellular dry mass content, even fewer free radicals may result in a higher rate of cellular oxidative damage (Zs.-Nagy, 1994). CPH, a free radical scavenger, not only protects cellular proteins against free radical induced damage, but improves the overall physical condition of the cells

140 T

r - m

120

= 100 g ¢ -

80 g ¢ • r , , l ~

60

m o

20

0 Ischemia 0 15 15

Reperfusion 0 1 1

mlnutes

CPH-pretreated

Fig. 4. Tissue ascorbyl free radical contents in young control rat hearts as well as after ischemia-reperfusion with and without CPH-pre-treatment of young rats. Each bar represents the mean + S.E.M. for 6 individual heart samples.

3 0 6 K. Nagy et al. / Arch. Gerontol. Geriatr. 22 (1996) 297-309

120 CPH-pretreated CPH-pretreated

C t~

100 T

o3 C

- ~ 80 C .r-I

r - t s,-~

m C

°,4

~ 40 "O .I-~

O

" ~ 20 O -,'-'t O m m r -

0 Ischemia 0 0 15 15 minutes

Reperfusion 0 0 1 1

Fig. 5. Changes of tissue ascorbyl free radical level in the old control and CPH-pre-treated old control rat hearts, as well as after ischemia-reperfusion without and with CPH-pre-treatment of old rats. Each bar represents the mean + S.E.M. for 6 individual heart samples.

(Roy et al., 1983; Zs.-Nagy, 1994). It also increases the rate of elimination of damaged proteins.

Ascorbic acid as an antioxidant plays a protective role against free radicals. Ascorbate levels decrease significantly during aging in most tissues (Cutler, 1984). This indicates a decline in the defense system with advancing age. Our results also show an age-dependent decrease of ascorbyl radical content in the heart of old rats. However, the CPH-treatment of old animals reverses this phenomenon. As it has already mentioned in the Introduction, CPH-treatment of old animals also activated some antioxidant enzymes (Roy et al., 1983).

Several papers have reported the generation of oxygen-derived free radicals during post-ischemic reperfusion, using spin trapping or salicylate trapping tech- niques (Arroyo et al., 1987; Pietri et al., 1989; Oliver et al., 1990; Powell and Tortolani, 1992). They also showed that free radical-mediated reactions lead to oxidative damage of tissues.

Our results confirmed that protein oxidation, as measured by protein carbonyl content, increased significantly at the beginning of reperfusion following ischemia, but the extent of the oxidative damage was much higher in the heart of young rats


compared to old ones. Since the old control hearts had originally higher amount of oxidized proteins than young controls, and since the cross-linking of proteins increases during aging (Nagy and Zs.-Nagy, 1984), it may be that the number of available amino acid residues, which can be converted to carbonyl derivatives, decreased in the heart of old rats. That could be one of the reasons why we measured lower ratio of increase of oxidized proteins in the old hearts than in the young ones, following ischemia-reperfusion.

The oxygen-derived free radical generation induced by ischemia and reperfusion was followed by extracellular release and intracellular depletion of ascorbyl radical as detected by ESR (Pietri et al., 1990). Ascorbyl radical, as an endogenous protector, can scavenge oxygen-derived free radicals via its self-disproportionation. The depletion of ascorbyl radicals can serve as a marker of free radical induced post-ischemic injury.

Our results are in agreement with some earlier ones that hearts following ischemia-reperfusion exhibit significantly lower tissue ascorbyl radical levels than control hearts (Pietri et al., 1990; Tavazzi et al., 1992), but the extent of decrease is much stronger in the young hearts than in the old ones.

Administration of SOD, catalase, free radical scavengers or iron chelators reduced the formation of oxygen-derived free radicals and ischemia-reperfusion injury in tissues (Arroyo et al., 1987; Simpson and Lucchesi, 1987; Omar et al., 1991; Mao et al., 1993; Sun et al., 1993; Tosaki et al., 1993). A free radical spin trap administered in the pre-ischemic period partially prevented protein oxidation during reperfusion (Oliver et al., 1990), and perfusion of SOD during low-flow ischemia greatly inhibited both extracellular release and endogenous depletion of ascorbate (Pietri et al., 1990). In our study the pre-treatment of rats with the free radical scavenger CPH, also protected the hearts of both young and old animals from the oxidative injury during ischemia-reperfusion. The amount of oxidized proteins and the depletion of ascorbyl free radicals were diminished in the reperfused hearts.

The data presented here indicate that intracellular protein oxidation and ascorbyl radical depletion are in correlation, and characterize simultaneously the oxidative damage induced by aging or ischemia-reperfusion in rat hearts.

Acknowledgements

The help of Dr. M~tria Mahunka (Laboratory of Informatics of University Medical School) in the statistical analysis is acknowledged.

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Age-dependence of free radical-induced oxidative damage in ischemic-reperfused rat heart

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