Mitochondrial activities of rat heart during ageing

Mechanisms of Ageing and Development 76 (1994) 73-88

ELSEVIER

Mitochondrial activities of rat heart during ageing Cinzia Castelluccio a, Alessandra Baracca a, Romana Fato ~, Francesco Pallotti a, Magda Maranesi a, Vanni Barzanti a, Antonella Gorini b, Roberto F. Villa b, Giovanna Parenti Castelli ~, Mario Marchetti a, Giorgio Lenaz *a aDipartimento di Biochimica 'G. Moruzzi', University of Bologna, Via Irnerio 48, 40126 Bologna, Italy blstituto di Farmacologia, UniversitiJdi Pavia, 27100 Pavia, Italy

Received 7 February 1994; revision received 15 June 1994; accepted 5 July 1994

Abstract Some analytical and functional parameters of rat heart mitochondria have been investigated at six different periods of ageing from 2 to 26 months. The fatty acid composition of the mitochondrial membranes reveals a percentage increase of polyunsaturated fatty acids (20:4 n-6, 22:6 n-3) up to 12 months, followed by a decrease; however, fluorescence polarization of the membrane probe diphenylhexatriene is not changed, revealing that membrane fluidity is not significantly affected. No major change in ubiquinone-9 and in cytochrome content is apparent, indicating that the relative ratio of the respiratory chain components is unmodified. Nevertheless, significant changes in enzyme specific activities are detected: NADH cytochrome c reductase and cytochrome oxidase activities increase up to 12 months, then decrease at 18-26 months; ubiquinol cytochrome c reductase exhibits a peak at 18 months, followed by a decrease. All these activities follow a similar trend during the whole life span of the rat, even though the 'maximum' is different. No significant changes have been found in ATP synthase. Succinate-cytochrome c reductase steadily increases over the whole life span. The results, showing activity decreases in the respiratory enzymes having subunits encoded by mitochondrial DNA, are compatible with the 'mitochondrial' theory of ageing. Keywords: Ageing; Mitochondria; Coenzyme Q; Respiratory chain

* Corresponding author. 0047-6374/94/$07.00 © 1994 Elsevier Science Ireland Ltd, All rights reserved SSDI 0047-6374(94)01482-2

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1. Introduction

It is well known that mitochondria are deeply involved in the ageing process. There is some evidence that increased oxygen radical production during ageing may impair mitochondrial respiratory chain function [1]. This damage could involve membrane lipids, respiratory chain proteins and mitochondrial DNA (mtDNA). In particular, mitochondrial DNA somatic mutations in postmitotic tissues were suggested to play an important role in the ageing process [2-4]. Mitochondrial DNA is more exposed to oxidative damage than nuclear DNA, and both deletions and base oxidative changes have been described upon ageing [5-9]. Mitochondrial DNA codes for a limited number of polypeptides of the inner mitochondrial membrane, including seven subunits of Complex I (NADH ubiquinone reductase), the cytochrome b subunit of Complex III (ubiquinol cytochrome c reductase), three subunits of Complex IV (cytochrome c oxidase) and two subunits of the ATPase-ATP synthase complex, besides for the tRNAs and rRNAs involved in mitochondrial protein synthesis [10]. The possibility that progressive loss of transcription from mutated or deleted DNA leads to impaired electron transfer and ATP synthesis, resulting in defective energy conservation as the basis of ageing [2-4], is gaining support. A decreased level of mitochondrial mRNA has been described [11,12] and changes have been documented in mitochondrial activities (in both experimental animals and humans) during ageing (cf. Refs. [13-16]). Histochemical studies in skeletal muscle have provided evidence for a patch loss of cytochrome oxidase in the elderly [17]. Much of the work so far accomplished on experimental animals has compared 'young' and 'old' individuals; the correct choice of age groups to be compared is quite important, particularly if we consider that changes may take place continuously over the entire life span, and not all of them are necessarily ascribed to ageing. The present investigation represents a study of the composition and functional activities of rat heart mitochondria in six groups of animals in the interval between 2 and 26 months of age, in order to find out an eventual definite pattern during the whole life span. 2. Materials and methods 2.1. Materials

All chemicals used were purchased from Sigma Chemical Co., St. Louis, MO, and all solvents were pure reagents from Merck (Darmstadt, Germany) and Carlo Erba (Milan, Italy). Ubiquinone-2 was a kind gift from Eisai Co., Tokyo, while ubiquinol2 was obtained by reduction of ubiquinone-2, using sodium dithionite as reducing agent, according to Rieske [18]. 2.2. Animals

Male albino rats (Wistar Kyoto from Charles River) aged 2, 6, 12, 18, 24, and 26 months were used for this experiment. They were kept under constant environment conditions (temperature, 22 ± I°C; relative humidity 60 ± 5; circadian rhythm, 12h light, 12-h dark) and fed a normal laboratory diet.

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Each group comprised 8-15 rats, except the 26-month group, which consisted of five rats; at that age, most rats develop tumors and die. Only individuals which were apparently healthy were used.

2.3. Preparation of rat heart mitochondria The mitochondria were isolated from frozen rat hearts using differential centrifugation, as described by Fleischer and Kervina [19,20] for mitochondrial preparation from frozen organs: rat hearts, excised immediately post-mortem, were weighed in a tared beaker and placed in 1 volume of the medium 0.21 M mannitol, 0.07 M sucrose, pH 7.5, containing 20% (w/v) dimethyl sulfoxide (DMSO), a cryoprotective substance; afterwards, the organs were frozen in liquid nitrogen, and kept in this for 24 h before preparing mitochondria. The yield and state of mitochondria were comparable to those from fresh tissues: no significant changes in respiratory activities were found using mitochondria from fresh and frozen organs. Mitochondrial protein was determined by a biuret method [21] with bovine serum albumin as standard. Lipid phosphorus was assayed by the method of Marinetti [22] and the amount of phospholipids was calculated by multiplying the phosphorus content by 25. 2.4. Enzymatic activities Oxidative enzymatic activities, both of each single complex and of integrated electron transfer, were assayed after freezing and thawing of the isolated mitochondria and after a swelling treatment, according to Brown and Raison [23], in order to remove any permeability barriers to impermeable substrates. Each assay was performed at 25°C in a 50 mM KCI, 10 mM Tris-HCl, 1 mM EDTA buffer, pH 7.4, with 2 mM KCN, except for cytochrome oxidase activity, in which 1/zM antimycin A was added. Ubiquinol cytochrome c reductase activity was assayed by titration of ubiquinol-2 (0.4-4 #M) keeping oxidized cytochrome c at a quasi-saturating concentration of 45/zM. The values reported a r e Vmax obtained from each titration. For cytochrome c oxidase activity, ferrous cytochrome c (0.3-3 #M), reduced by dithionite and purified on a Sephadex G-25 column [241, was used and the data reported are the Vmax extrapolated from these titrations. NADHcytochrome c reductase and succinate cytochrome c reductase activities were assayed using either 75 /zM NADH or 12.5 mM succinate in the presence of 45 /~M cytochrome c. All the activities were evaluated by monitoring the absorbance change of cytochrome c upon reduction or oxidation at 550 minus 540 nm in a double wavelength spectrophotometer [24]. The extinction coefficient used for cytochrome c was 19 mM- l era- i I24]. ATPase activity was measured at 25°C and at pH 7.5 using an ATP-regenerating system [25]: the reaction mixture (1 ml) contained 25 ttmol Tris/acetate, 25 ttmol KOH, 0.3 mmol sucrose, 5/zmol MgCI2, 160 nmol NADH, 1.5 ttmol Na-phosphoenol-pyruvate, 5 units of lactate dehydrogenase, 3.5 units of pyruvate kinase and 1 t~g of rotenone. The reaction was started by addition of 20/zg of mitochondrial protein to the medium. The decrease in NADH, which is a measure of ADP formation

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from ATP, was followed at 340 nm on a Zeiss PMQ Ill spectrophotometer equipped with a Sergovor recorder and a thermostating system.

2.5. Coenzyme Q evaluation An aliquot of mitochondria (0.5 mg of protein) was mixed with 2.5 ml of a 60% (v/v) methanol/light petroleum (b.p. 40-60°C) mixture in a centrifuge tube with glass stopper, according to Takada et al. [26]. After centrifugation at 2500 x g for 5 min, the upper layer was collected by aspiration and the residue was reextracted twice with 1 ml of light petroleum. The dry residue of combined extracts was diluted in the HPLC mobile phase (ethanol/3% water/0.1% HCIO4, with the addition of 7 g of NaCIO4) [27], and measured in a Waters Data Module M730-Model 721 HPLC system, using a Spherisorb ODS I 25 x 0.46 cm reverse phase C18 column with a guard column containing the same material as the main column. The flow rate was 1 ml/min at 25°C. Determination was performed using a ubiquinone-9 calibration curve, obtained by injection of different quantities of a standard solution (1 pmol/#l) into the HPLC system. The ubiquinone was detected spectrophotometrically at 275 nm.

2.6. Content of cytochromes The content of cytochromes was evaluated by the differential spectra (dithionite reduced minus ferricyanide oxidized) in a sample of 5-6 mg of mitochondrial protein diluted with 1 ml of 25 mM KH2PO4, 1 mM EDTA, pH 7.4 in the presence of 1% deoxycholate in a Jasco (Uvidec B 10) double-beam spectrophotometer, according to Vanneste [28] and Nicholls [29].

2. 7. Fatty acid composition Fatty acid composition of the mitochondrial lipids was determined by gas-liquid chromatography, after lipid extraction according to Folch et al. [30]. The extracted lipids were methylated by using a mixture of methanol/5% H2SO 4 at 70°C for 12 h. The fatty acid methyl esters were assayed in a Varian 3700 gas chromatograph at 195°C, using N2 as gas carrier, injected with a flow rate of 30 ml/min, and equipped with a flame ionization detector.

2.8. Fluorescence polarization Fluorescence polarization of the membrane probe diphenylhexatriene (DPH), as an empirical assessment of membrane order and rigidity, was determined in a Jasco FP 777 spectrofluorimeter, equipped with polarization filters, using 356 and 430 nm as the excitation and emission wavelength, respectively. The samples were prepared with 0.2 mg of mitochondria in 1 ml of 10 mM Tris-HCl, 1 mM EDTA, pH 7.4, to which the probe was added at a 1/~M concentration. The measurement was performed after 40 min of incubation in the dark.

2.9. Statistical analysis Data are presented as means • S.E.M., except for fatty acids measurements, which are presented as means ± S.D. The significance of differences was evaluated

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by the unpaired t-test, at P < 0.001 for enzymatic activity, except for the relation between 12 and 26 months in cytochrome oxidase activity, showing P < 0.002. As for fatty acid changes, the significance of differences was taken at P < 0.05. In every plot reported, the same letters indicate no significant difference between the age pairs considered. 3. Reselts The fatty acid composition of the mitochondrial membrane lipids is reported in Table 1. Analysis of the data reveals a percentage increase in polyunsaturated fatty acids of both the linoleate and linolenate families (20:4 n-6, 22:6 n-3) up to 12 months of age, followed by a decrease; the 20:4/18:2 ratio follows a similar trend, and the unsaturation index also exhibits a maximum at intermediate ages (Fig. l a,b). Notwithstanding the variations in lipid unsaturation, the 'fluidity' of the mitochondrial membrane, expressed in terms of diphenylhexatriene fluorescence polarization, is not changed according to a definite pattern during ageing (Fig. 2). This is not in contrast with the results reported above, considering the high protein/lipid ratio in the mitochondrial membranes (0.3 mg phospholipid/mg protein, not shown). At high polarization values induced by the high protein content, relatively small fluidity differences cannot be detected. Table 1 Fatty acid composition of rat heart mitochondrial membrane Fatty acid composition

2 months

16:0 16:1 18:0 18:1 18:2 n6 20:4 n6 22:4 n6 22:6 n3 U.I. a E D n6 b E D n3 b Db 20:4/18:2 ¢ 18:1/18:0 d

17.69 3.27 19.23 15.45 20.61 8.98 1.54 4.76 136.58 31.13 5.46 37.09 0.44 0.80

± 4. ± ± 4. 4. 4. 4. 4. 4. 4. 4. 4. 4.

6 months 1.55 17.13 ± 1.80 0.70 1.82 4. 0.35 1.85 17.86 4. 2.00 1.60 12.26 4. 1.75 2.10 23.13 ± 2.40 1.40 12.93 ± 0.65 0.20 1.50 4. 0.18 0.36 6.80 ± 0.75 10.77 167.11 ~,- 16.13 1.95 37.56 4. 1.94 1.30 8.45 ± 2.11 2.50 46.01 ± 2.86 0.11 0.56 4. 0.04 0.16 0.69 4. 0.12

12 months

18 months

24 months

16.70 2.40 16.00 11.50 21.60 14.63 2.15 6.86 174.63 38.38 8.71 47.51 0.68 0.72

17.00 2.00 19.50 12.00 22.40 14.60 1.25 6.00 165.20 38.25 7.40 45.65 0.65 0.62

16.09 3.20 15.88 14.10 24.00 12.40 1.50 4.50 154.40 37.90 5.80 43.70 0.50 0.89

± 4. 4. ± 4. 4. 4. 4. 4. 4. 4. 4. 4. 4.

0.65 0.20 0.40 1.00 0.38 1.11 0.20 0.48 5.12 1.35 0.67 1.58 0.04 0.12

4. 4. 4. ± 4. 4. ± 4. 4. 4. 4. 4. 4. 4.

2.16 0.40 2.40 0.35 1.60 0.75 0.30 0.36 11.05 2.55 1.09 3.27 0.05 0.06

4. 4. 4. ± 4. 4. ± ± ± ± 4. 4. ± 4.

1.50 1.00 1.40 0.85 0.70 1.35 0.25 0.85 10.00 2.05 1.29 2.57 0.07 0.13

Values are expressed as means ± S.D. from pools of two or three individuals. Only major fatty acids are reported in the table. au.I.: unsaturation index, defined as ~ moi % of each fatty acid x number of double bonds of the same fatty acid. bD n6, D n3, D indicate the amount of linoleate, linolenate families and total polyunsaturated fatty acids, respectively. CRatio 20:4/18:2 is an indirect measurement of A-5, and A-6 desaturases. dRatio 18:i/18:0 is an indirect measurement of A-9 desaturase.

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a 200

J ab 160

120

80 12 AGE (months)

18

b 0.8 b

0.6-

°

L

~ o.4J

0.2

2

6

12 AGE (months)

18

24

Fig. 1. (a) Unsaturation index, defined as r. tool % of each fatty acid x number of double bonds of the same fatty acid and (b) ratio 20:4/18:2 in rat heart mitochondrial membrane during ageing. The reported values are expressed as means ± S.D. of pools of 2-3 individuals.

The enzymatic activities determined are all significantly changed during ageing, except for A T P synthase activity (Fig. 3). Both rotenone-sensitive N A D H - c y t o chrome c reductase and cytochrome oxidase activities present a peak at 12 months, whereas ubiquinol-cytochrome c reductase activity has a peak at 18 months; the following decrease at 2 4 - 2 6 months, shown by all the activities reported, makes the

C Castelluccio et al./Mech. Ageing Dev. 76 (1994) 73-88

79

04

b

t.

~

0.2

2

ad .c d

6

12

18

24

26

A G E (months)

Fig. 2. Fluorescence polarization of dyphenylexatriene (DPH) in rat heart mitochondria during ageing. Values are expressed as means ± S.E.M. of pools of two or three individuals. S.E.M. lies in the range 0.001-0.009.

1.5

g

.;~ 0.5

6

12

AGE

t8onths)

24

26

Fig. 3. Age-dependence of Vmax of ATP synthase activity in rat heart mitochondria. The reported values are means of 5-15 different individuals ± S.E.M.

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C. Castelluccio et al./ Mech. Ageing Dev. 76 (1994) 73-88 0.4

0.3

ih

0.2

a

g 0.1

2

6

12 A G E (months)

18

24

26

Fig. 4. NADH-cytochrome c reductase (complex I + Ill) activity in rat heart mitochondria during ageing. The reported values are means of 5-15 different individuals ± S.E.M.

behavior of all these enzymes similar during the entire life range (Figs. 4-6). On the other hand, succinate-cytochrome c reductase activity steadily increases over the whole life span (Fig. 7). The decrease in enzymatic activity could be due to a change in concentration of respiratory complexes. This parameter may be quantified by measuring the contents

1.5

--=

~bc

1

o

0.5

2

6

12

18

24

26

AGE (months)

Fig. 5. Cytochrome oxidase (complex IV) activity (Vmax, see text) in rat heart mitochondria during ageing. The reported values are means of 5-15 different individuals ± S.E.M.

C. Castelluccio et al./ Mech. Ageing Dev. 76 (1994) 73-88

81

4.5

3

b ac

bc

a¢ ~. 1.5

6

6

12

18

24

26

AGE (months)

Fig. 6. Ubiquinol-2 cytochrome c reductase (complex III) activity (Vmx, see text) in rat heart mitochondria during ageing. The reported values are means of 5 - 1 5 different individuals ± S.E.M.

of cytochromes. The contents of both cytochromes b and a of heart mitochondria at different ages, however, do not reveal significant changes (Fig. 8a,b). Also, the levels of Coenzyme Q, reported as a function of mitochondrial protein content, although exhibiting some significant changes during the lifespan, do not show any major change ascribed to ageing (Fig. 9a). On the other hand, its level as a function of mitochondrial phospholipid content (a measure of the quinone effective concen0.2

0.15

'i 0.1

O.05

6

12

18

24

26

AGE (months) Fig. 7. Succinate-cytochrome c reductase (complex II + I I I ) activity in rat heart mitochondria during ageing. The reported values are means of 5 - 1 5 different individuals ± S.E.M.

82

C. Castelluccio et al./Mech. Ageing Dev. 76 (1994) 73-88 0.6

a

.~ 0.4 a ------.___.______

~

~ 0.2

2

6

12

18

24

26

AGE ( m o n t h s ) 0.2

-

b

a ac

ac

0.15

[ ~

0.1 ~

,o

0.05 !

2

6

12

18

24

26

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Fig. 8. Content of (a) cytochrome a a 3 and (b) cytochrome b in rat heart mitochondria during ageing• Values are expressed as means ± S.E.M. of pools of 2-3 individuals.

tration in the membrane) is not significantly different at any age (Fig. 9b). Only the contents of CoQ9 are shown, since the homolog CoQ]0 is present at concentrations around 1/10 of CoQ9 and closely follows its variations. 4. Discussion This study supports previous findings [30-33] that heart mitochondrial membranes are significantly modified during the ageing process, but no simple pattern emerges from the results obtained. As for the lipid composition of the membrane,

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C. Castelluccio et al. / Mech. Ageing Dev. 76 (1994) 73-88

a

r ~2

2

6

12

18

24

26

AGE (months)

15

b

10

8

e~

a

g

a

r

--

5

2

6

12

18

24

26

AGE (months)

Fig. 9. CoenzymeQ9content in rat heart mitochondria during ageing, reported (a) as nmol/mgprotein; (b) as nmol/mg phospholipids (raM in the lipid phase). The reported values are expressed as means ± S.E.M.S.E.M. lies in the range 0.058-0.283 for a, and 0.133-0.945 for b, respectively.

we observed an increase in polyunsaturated fatty acids of both the linoleate and linolenate families up to 12 months, followed by a decrease; the same trend was found both in the 20:4/18:2 ratio, the indirect measurement of the activity of the desaturases involved in the process, and in the unsaturation index. Nevertheless, no changes in membrane fluidity were noticed; it is possible that the fluorescence polarization method is not sensitive enough to reveal small fluidity changes due to variations in lipid unsaturation. Moreover, static fluorescence polarization represents

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only an empirical evaluation of membrane fluidity, containing both static and dynamic components [341. Modifications of polyene fatty acids in myocardial phospholipids were previously reported during the neonatal and postneonatal period [31] and during ageing [32]. Recently, a decrease in cardiolipin, followed by a loss of unsaturated fatty acids in the mitochondrial membrane, has been reported in rat heart mitochondria during ageing [33]. The rate of NADH cytochrome c reductase is the integration of NADH CoQ reductase and ubiquinol cytochrome c reductase through the CoQ pool [35]; its changes during development and ageing cannot be the result either of a variation of the CoQ pool (which is unchanged) or of the observed changes of ubiquinol cytochrome c reductase, since this activity is not rate-limiting. In fact, according to the CoQ pool equation of Kr6ger and Klingenberg [35], a decrease in the less limiting enzyme would have negligible consequences on the integrated activity. It is therefore evident that the changes in NADH cytochrome c reductase must be the consequence of changes in Complex I activity. We have not assayed NADH-CoQ reductase activity directly, because the assays routinely employed for this enzymatic activity have yet to be clarified. Use of short chain quinone analogs or homologs such as Ubiquinone-1 (Q0, Ubiquinone-2 (Q2), Duroquinone (DQ), Decyl-benzoquinone (DB) [36], may have strong limitations due to the existence of quinone reduction sites in Complex III [36,37], inhibition of Complex I activity [37], or nonlinear kinetics, as we have found for DB [36]. So far, the best quinone acceptor appears to be Ql, although we have reason to believe that it is not reduced as fast as the endogenous Ql0. For this reason, we believe that NADH-cytochrome c reductase is paradoxically a better assay of Complex I in mitochondrial membranes than NADH-Q reductase. The extent of decrease in complex I activity in ageing is probably higher than that detected in the NADH-cytochrome c reductase assay, as a kinetic result of the pool equation [35]. Since previous studies on mitochondrial respiratory chain in toto [38] had not shown marked decreases with ageing, the separate analysis of different complexes could add some information to the investigation of mitochondrial ageing. In spite of the high individual variability already observed by others [15] during ageing, some significant changes were observed in all activities investigated, except ATPase. The results obtained both for ubiquinol-cytochrome c reductase and cytochrome oxidase activity show a change in the real Vma x extrapolated from titrations of the respective substrates (see Materials and methods section). Moreover, the lack of changes in the content of both cytochrome a and cytochrome b found in our experiments and agreeing with the results reported by Manzelmann and Harmon [38], let us conclude that these changes in activity are not due to a decrease in the concentration of these individual complexes. On the contrary, other authors [39] reported some changes in both cytochrome b and cytochrome c+cl. These findings are not in contrast with a lack of decrease in mitochondrial respiratory function or energy production, but suggest an independent involvement of each individual complex in ageing, as a result of its impaired function. Such functional impairment of the enzymatic proteins could be the result of either phenotypic

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changes due to DNA mutations, or direct protein damage, or finally to changes in the lipid environment. The changes of activity linked to complexes having subunits coded by mitochondrial DNA (NADH-coenzyme Q reductase, ubiquinol cytochrome c reductase, cytochrome oxidase) support the mitochondrial DNA theory of ageing. In particular Complex I, with priority over the others, seems to be susceptible to mutations in mitochondrial genome, in view of the fact that the mitochondrial genes coding for Complex I subunits are 7, in comparison with 1-3 for each of other complexes (III, IV and ATPase). It is to be noted that NADH-cytochrome c reductase was decreased to some extent in the skeletal muscles of the same rats at 24-26 months of age (unpublished observations from our laboratory). No decrease in ATPase activity was observed in our experiments, in spite of its content of mitochondrially coded subunits. As for ATPase, if a change in the relative subunit content is present, this is not rate-limiting at any stage of ageing. The activity of the adenine nucleotide carrier cannot represent a rate-limiting step, because the mitochondria are used after freezing and thawing and therefore do not have permeability barriers. The lack of changes in ATPase contrasts with the results of Guerrieri et al. [40] who found a strong decrease in ATPase activity and content of F1 subunits in rat hearts upon ageing. The reasons for the discrepancy are not known, although the use of EDTA submitochondrial particles in the results of Guerrieri et al. [40] may remove a rate-limiting step, as the endogenous inhibitor protein. Coenzyme Q is synthetized by a complex pathway not involving mitochondrial gene products [41]; the lack of any major change in aged rats is in agreement with the working hypothesis of a mitochondrial genetic defect. Our finding, however, differs from those of Beyer et al. [42] and Appelkvist et al. [41] who reported decreased CoQ levels in the hearts of rats and humans, respectively, upon ageing. An explanation for this discrepancy could be found in a possible different exposure to free radical damage as a secondary effect of hampered respiration (T. Ozawa, personal communication); we have observed that in perfused rat livers, an oxidative stress leads to a decrease in the CoQ content [43], possibly as the direct result of oxidative damage on the quinone molecule. It may be reasoned that factors indirectly related to ageing lead to oxidative damage of CoQ, aggravating the consequence of hampered electron transfer in a cyclical fashion. It is likely that this extreme stage has not been reached in our experimental animals, since the decrease in activity observed by us is not a major one. It has to be noted, however, that our study is the only one exhibiting Coenzyme Q levels of isolated mitochondria, whereas those quoted above [40,41] deal with whole tissues. The high content of extramitochondrial coenzyme Q [44] makes it difficult to extrapolate mitochondrial coenzyme Q levels from tissue levels. Since mitochondrial CoQ appears to be less easily modified than the extramitochondrial quinone (G. Dallner, personal communication), one possible explanation for the discrepancy could be in age-dependent changes in extramitochondrial CoQ pools. Although the decrease in activities of enzymes containing mt-DNA encoded subunits suggests the involvement of mt-DNA damage in ageing, it must be considered that a decrease occurs e.g. between 12-18 months and 24-26 months of age,

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after a previous increase. This means that an activity may be identical e.g. at 6 and 24 months of age, throwing doubts on the significance of the changes observed with respect to the ageing process. Nevertheless, we feel that the lack of extreme bioenergetic changes in rat heart during ageing, observed in this study, does not contrast the mitochondrial theory [2-4], but stresses the possible existence of intrinsic problems in studying senescence. First of all, events of opposite direction could occur concomitantly to ageing as a result of developmental phenomena unrelated to ageing itself. This hypothesis might be supported by the observation that succinate cytochrome c reductase steadily increases during the lifespan, in accordance with complex II having no mitochondrially encoded subunits (and complex III not being rate limiting). Furthermore, in most postmitotic tissues, an eventual decline in energy charge due to mitochondrial failure would be detectable only as a transient state between the state when the cell is healthy and the occurrence of cell death. In such a case, the ratio of cells existing in this transient state to total cells in the tissue may be small enough to prevent detection of statistical changes as an average. On the other hand, the extent of change detected could be a function of additional factors, including lipid composition (see before), exposure to free radical attack and extent of antioxidant defenses. Acknowledgments

This work was supported by the CNR Target Project on Ageing (Rome), Code number 921182. Coenzyme Q homologs were kind gifts from Eisai Co., Tokyo. References Ill D. Harman, Free radical theory of ageing: consequences of mitochondrial ageing. Age, 6 (1983) 86-94. [2] J. Miquel, A.C. Economos, J. Fleming and J.E. Johnson, Mitochondrial role in cell ageing. Exp. Gerontol., 15 (1980) 575-591. [3] J.E. Fleming, J. Miquel, S.F. Cottrell, L.S. Yengoyan and A.C. Economos, Is cell ageing caused by respiratory-dependent injury to the mitochondrial genome? Gerontology, 28 (1982) 44-53. [4l J. Miquel, An integrated theory of aging as result of mitochondrial DNA mutation in differentiated cells. Arch. Gerontol. Geriatr., 12 (1991)99-117. [5] S. Sugiyama, K. Hattori, M. Hayakawa and T. Ozawa, Quantitative analysis of age associated accumulation of mitochondrial DNA with deletion in human hearts. Biochem. Biophys. Res. Commun., 180 (1991) 894-899. [6l K. Asano, M, Nakamura, A. Asano, T. Sato and H. Tauchi, Quantitation of changes in mitochondrial DNA during aging and regeneration of rat liver using non-radioactive DNA probes. Mech. Ageing Dev., 62 (1992) 85-98. [7] J.M. Cooper, V.M. Mann and A.H.V. Schapira, Analyses of mitochondrial respiratory chain function and mitochondrial DNA deletion in human skeletal muscle: effect of ageing. Z Neurol. Sci.. 113 (1992) 91-98. [8] B. Kadenbach and J. Miiller-H6cker, Mutations of mitochondrial DNA and human death. Naturwissenschaften, 77 0990) 221-225. [9] C. Miinschen, T. Rieger, J. Miiller-H6cker and B. Kadenbach, The point mutation of mitochondrial DNA characteristic for MERRF disease is found also in healthy people of different ages. FEBS Left., 317 (1993) 27-30.

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