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Cardiovascular Research 2000 47(3):624-631; doi:10.1016/S0008-6363(00)00122-X
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Copyright © 2000, European Society of Cardiology

Aging is associated with increased lipid peroxidation in human hearts, but not with mitochondrial respiratory chain enzyme defects

Òscar Miróa,*, Jordi Casademonta, Elena Casalsb, Milagrosa Pereaa, Álvaro Urbano-Márqueza, Pierre Rustinc and Francesc Cardellacha

aMuscle Research Unit, Department of Internal Medicine, Hospital Clìnic, August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Villarroel 170, 08036 University of Barcelona, Barcelona, Catalonia, Spain
bClinical Biochemistry Laboratory, Hospital Clìnic, IDIBAPS, Barcelona, Catalonia, Spain
cUnité des Recherches sur les Handicaps Génétiques de l’Enfant, INSERM U393, Département de Pédiatrie, Hôpital des Enfants-Malades, Paris, France

* Corresponding author. Tel./fax: +34-93-227-5539 omiro{at}medicina.ub.es

Received 7 December 1999; accepted 27 April 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 References
 
Background: Aging is associated with increased oxidative damage at multiple cellular and tissular levels. A decrease in mitochondrial function has repeatedly been advocated as a primary key event, especially on the basis of analysis of skeletal muscle mitochondria. However, some doubts on this issue have arisen when confounding variables (such as physical activity or smoking habit) have been taken into account in the analysis of mitochondrial respiratory chain (MRC) enzyme activities or when additional analytical parameters such as enzyme ratios have been considered. Objective: To determine whether oxidative damage and enzyme activities of the MRC are influenced by the aging process in human hearts. Patients and methods: We studied cardiac muscle obtained from 59 organ donors (age: 56±12 years, 75% men). Oxidative membrane damage was evaluated through the assessment of lipid peroxidation. Absolute and relative enzyme activities (AEA and REA, respectively) of complex I, II, III and IV of the MRC were spectrophotometrically measured. Stoichiometric relationships among MRC complexes were also assessed through calculating MRC ratios. Linear regression analyses were employed to disclose any potential correlation between mitochondrial dysfunction and aging. Results: We found a progressive, significant increase of heart membrane lipid peroxidation with aging (P<0.05). Conversely, neither AEA nor REA decreased with age (P=n.s. for all complexes). Similarly to observations in other tissues, we found that stoichiometry of the MRC enzymes is maintained within a narrow range in human hearts. When the effects of aging on MRC ratios were explored, we failed again in demonstrating any subtle disarray. Conclusion: MRC enzymes remain preserved in heart with aging, and thus they cannot be considered the main cause of the increased oxidative damage associated with aging.

KEYWORDS Aging; Energy metabolism; Free radicals; Mitochondria; Oxidative phosphorylation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Material and methods
 3 Results
 4 Discussion
 References
 
During the last decades, much research has been directed towards establishing correlations between oxidative damage, antioxidant defense systems, mitochondrial function, and aging. Under normal physiological conditions, about 1–5% of oxygen consumed by mitochondria is converted into reactive oxygen species. Several sites of the mitochondrial respiratory chain (MRC) are involved in the generation of such reactive oxygen species, which have been claimed to be maintained at a relatively high steady-state level in mitochondrial matrix [1]. Due to their location in the mitochondrial matrix, mitochondrial DNA molecules as well as several structural and functional mitochondrial proteins are exposed to an increased risk of suffering from oxidative reactions, eventually resulting in mitochondrial dysfunction. In this regard, several investigators have reported a progressive, although very limited, accumulation of deleted mitochondrial DNA (mtDNA) molecules with aging in a great variety of human tissues [2–5], an increase in protein oxidation [6,7], and enhanced membrane lipid peroxidation [8,9]. Accordingly, a decay in MRC activity has been proposed as a cause and simultaneously as a consequence of oxidative damage. This MRC dysfunction has been advocated to play an essential role in human aging [10–12], although after including some confounding variables in the analysis, we found no significant modification in mitochondrial function with age in skeletal muscle [13].

The heart is one of the organs in which the potential effects of oxidative damage would be more readily detectable due to its high dependence on oxidative phosphorylation to derive energy. In keeping with this, small amounts of mtDNA deletions in cardiac muscle have been reported with increasing age [2,13–17], although studies on the effect of aging on MRC function in human cardiac muscle are scarce. Therefore, the aim of the present study was to determine the potential increase in the oxidative damage of human heart with age, as well as to determine whether enzyme activities of the heart MRC complexes decrease during the aging process.


   2 Material and methods
Top
 Abstract
 1 Introduction
 2 Material and methods
3 Results
 4 Discussion
 References
 
2.1 Hearts samples
We studied hearts obtained from organ donors which were not suitable for transplantation because of the advanced age, the absence of heart recipients with matching, or size inadequacy. Only patients who did not exhibit clinical, radiological or electrocardiographic signs of myocardial diseases were included. Ventilatory and hemodynamic requirements of the donor patients were properly maintained and, accordingly, no patient suffered from hypoxia (paO2 in intensive care unit was always greater than 90 mmHg) and none required cardiopulmonary resuscitation maneuvers.

A biopsy of the left ventricular free wall (3–5 g) was obtained in situ from the beating heart and was immediately snap frozen into liquid nitrogen until biochemical analysis. Heart samples were collected from June 1995 until January 1999. The present protocol was approved by the Ethical Committee of our Hospital.

2.2 Assessment of lipid peroxidation of cardiomyocyte membranes
All biochemical and enzymatic studies were performed consecutively during a 3-week period in March 1999. Cardiac muscle was homogenized as previously described [18], and the protein content in the supernatant was measured according to Bradford [19]. cis-Parinaric acid fluorescence was used to determine the chemical process of lipid peroxidation. cis-Parinaic acid is a fatty acid that contains four conjugated double bonds, which render it naturally fluorescent. The double bonds are broken in lipid peroxidation reactions. Since cis-parinaric readily incorporates into membranes, a decay in fluorescence is used to indirectly monitor the degree of membrane lipid peroxidation. For this purpose, a sample of myocardial homogenate containing 100 µg of protein was labeled with 5 µM cis-parinaric acid (Molecular Probes, Eugene, OR, USA) in a cuvette containing 2 ml of PBS. The samples were incubated in the dark at 37°C and fluorescence (excitation: 318 nm; emission: 10 nm; bandpass: 10 nm) was measured every 3 min for 30 min as described [20,21]. Loss of parinaric acid fluorescence was used as an index of lipid peroxidation and, accordingly, the greater lipid peroxidation, the less fluorescence was detected.

2.3 Assessment of antioxidant status
2.3.1 Superoxide dismutase
The method was based on the superoxide dismutase (SOD)-mediated increase in the rate of autoxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzofluorene in aqueous alkaline solution to yield a cromophore with maximum absorbance at 525 nm. The method determines both Cu–ZnSOD and MnSOD activity (Bioxytech®, Oxis Health Products, Portland, OR, USA).

2.3.2 Glutathione peroxidase
In the assay, oxidized glutathione produced upon reduction of an organic peroxide by glutathione peroxidase (GPx) is recycled to its reduced state by the enzyme glutation reductase. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm (Bioxytech).

2.4 Assessment of enzyme activity of MRC complexes
Measurement of the specific activity of the individual complexes of the MRC (absolute enzyme activity, AEA) was performed spectrophotometrically (Uvikon 922, Kontron, Zurich, Switzerland) on heart homogenates. A total of 10–40 mg of homogenate protein was required to determine the activity of each complex, except for complex IV, for which we used 2–4 mg [22]. Measurements of complex I (rotenone-sensitive NADH-decylubiquinone oxidoreductase, EC 1.6.99.3 [EC] ), complex II (succinate decylubiquinone DCPIP reductase, EC 1.3.99.1 [EC] ), complex III (ubiquinol-cytochrome c reductase, EC 1.10.2.2) and complex IV (cytochrome c oxidase, EC 1.9.3.1 [EC] ), as well as citrate synthase (EC 4.1.3.7) were performed at 37°C in a total volume of 1 ml as reported elsewhere [23–25]. Enzyme activity was expressed as nmolmin–1mg of homogenate protein–1.

2.5 Data handling
AEA were corrected by the citrate synthase activity, used as a marker of tissue mitochondrial content. This allows obtaining the relative enzyme activities (REA), which are not influenced by the variable mitochondrial content of cardiac muscle.

Alternatively, because a balanced proportion among complexes is required for the normal functioning of MRC, the ratios between the activities of the different MRC complexes were calculated. These ratios, which represent the stoichiometric relations among complexes of MRC, have been demonstrated to be more sensitive in detecting a partial deficiency of any particular MRC complex than AEA or REA [23]. In this study, the ratios showed a normal distribution, which is not always the case for crude enzyme activities, thus allowing the use of parametric statistical tests more appropriately.

2.6 Statistical analysis
Mean±1S.D. was used to describe quantitative variables, while percentages were utilized for qualitative variables. To detect association between quantitative variables, we employed linear regression analysis. For cis-parinaric acid experiments, two-way ANOVA for repeated measures was used to test differences in the fluorescence curves over time among the different groups of age. Values of P less than 0.05 were considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
4 Discussion
 References
 
We included 59 patients. The mean age was 56±12 (range 8–86) years. Forty-six (75%) were males and 16 (29%) were current smokers before death. Causes of death were cerebral hematoma in 31 patients (53%), cranial trauma in 22 patients (37%), and subarachnoidal hemorrhage in 6 patients (10%).

Time-sequential determination of cis-parinaric acid fluorescence is presented in Fig. 1 dividing the sample in low (8–40 years), middle (41–65 years) and advanced (66–86 years) aged individuals. A greater loss of fluorescence (i.e., a higher membrane lipid peroxidation) is observed in older individuals. When the effects of aging on lipid peroxidation were assessed through regression analysis at the end of experiment (30 min), a significant relationship between aging and lipid peroxidation was also found. The relationship between aging and antioxidant status is presented in Fig. 2. Unlike cis-painaric acid fluorescence the activity of SOD and GPx in heart homogenates did not disclose any variation with increasing age (Fig. 2).


Figure 1
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  		Fig. 1 Time-sequential cis-parinaric acid fluorescence recording in human hearts showing an increased lipid peroxidation in older individuals. This is confirmed by linear regression analysis of remaining cis-parinaric acid fluorescence at the end of the experiments.

 

Figure 2
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  		Fig. 2 Dot diagrams and regression lines showing the absence of correlation between aging and superoxide dismutase or glutathione peroxidase.

 
The mean activity of citrate synthase was 463±165 nmolmin–1mg of protein–1. Enzyme activity was not influenced by age (r2=0.00; P=0.83; n=56), which suggests that mitochondrial content in the myocardium did not change with age in human hearts.

AEA for complexes I–IV were 122±45, 319±104, 551±221 and 532±170 nmolmin–1mg of protein–1, respectively. In Fig. 3 activity of MRC enzyme complexes (AEA and REA) have been plotted as a function of age. No correlation was found for any complex, either assessed in absolute values or corrected by citrate synthase activity.


Figure 3
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  		Fig. 3 Dot diagrams and regression lines showing that absolute (left panels, in nmolmin–1mg–1) and relative (right panels, no units) mitochondrial respiratory chain enzyme activities of heart muscle do not decrease during aging. NQR (C1): NADH-coenzyme Q-reductase (or complex I); SDDR (C2): Succinate-decylubiquinone-DCPIP-reductase (or complex II); QCCR (C3): Coenzyme Q-cytochrome c reductase (or complex III); COX (C4): Cytochrome c oxidase (or complex IV); CS: citrate synthase.

 
We found a strong stoichiometric relationship between the different MRC activities (Fig. 4). Since these relationships have been shown to be the more sensitive method in detecting subtle MRC deficiencies and/or imbalances [23,26], MRC ratios were plotted against age. As presented in Fig. 5, there was no apparent disarray in the stoichiometric relationships between MRC complexes.


Figure 4
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  		Fig. 4 Dot diagrams and regression lines showing that stoichiometry of mitochondrial respiratory chain enzymes of heart maintain a strong interdependence. For abbreviations, see Fig. 3.

 

Figure 5
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  		Fig. 5 Dot diagrams and regression lines showing that stoichiometry of mitochondrial electron transport chain enzyme activities of heart do not decay during aging. For abbreviations, see Fig. 3.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
References
 
We have studied the potential effect of age on oxidative damage of cardiomyocyte membranes, and on MRC enzyme activities of hearts obtained from a large cohort of individuals. The results show an age-related increase in membrane lipid peroxidation but unaltered MRC enzyme activities. The lack of biochemical dysfunction of the MRC also persisted when normalized by citrate synthase activity, allowing the exclusion of any hypothetical influence of mitochondrial content of the myocardium. Additionally, stoichiometry of MRC complexes did not change with age, ruling out even subtle imbalance between MRC complexes.

Our findings of enhanced membrane lipid peroxidation agree with previous reported data obtained in laboratory animals [27–30]. Heart mitochondria isolated from senescent animals exhibit higher rates of oxygen radical production than those from young animals [27–29]. Furthermore, myocardium of senescent animals appeared to be more susceptible to insults, including ischemia–reperfusion-induced oxidative damage [31]. Other signs attributed to oxidative damage such as lipofuscin accumulation, decreased phospholipid unsaturation index and increased formation of both hydrogen peroxide and/or 8-hydroxy-2'deoxyguanosine have been detected in aged hearts of some animal species [30,32,33] and also in human hearts [13,34,35]. The activities of the main oxygen-radical scavenger enzymes have been found increased in the mitochondria of such aged hearts and also in human liver mitochondria, possibly as a compensatory reaction [30,36]. In our case, the combined cytosolic and mitochondrial activities of both SOD and GPx, did not seem to change with age. This finding has to be interpreted with caution. A possible, though unlikely explanation is that an increase of antioxidants in mitochondria is masked by a simultaneous decrease in cytosolic activities. An alternative explanation is that the protective effect against reactive oxygen species depends upon the simultaneous induction of several proteins with antioxidant properties which include, besides SOD and GPx, catalase, thioredoxin oxidoreductase and others. The balance among them is still poorly understood up to the point that even the overexpression of one of them does not necessarily mean a protective effect against oxygen free radicals [37]. A lack of increase of antioxidant activity may be interpreted as an absence of oxygen radical production, or as a defect in activation of antioxidant enzymes.

Interestingly enough, the data reported in the present study do not support the view that increased oxidative damage contributes to aging process through a decrease in MRC complex activity in humans. Our findings partly agree with those of Marin-Garcia et al. [38] in the only previous study dealing with the relationship between MRC enzyme activities and age in the human heart. In their study, these authors also observed no change of AEA of MRC complexes, but an age-related decrease of MRC activity when enzyme activities were corrected by the citrate synthase activity. It is possible that cardiomyocytes exhibit mitochondrial proliferation which effectively compensates for the putative decrease in MRC activities observed with aging. Alternatively, as pointed out by Marin-Garcia et al., citrate synthase may not be a reliable indicator for the total mitochondrial number and therefore, their main conclusions appear to be very similar to the present study concerning the preservation of the MRC enzyme activity in aging [38].

Our findings on cardiac muscle mitochondria enzyme activities are similar to those we recently reported in skeletal muscle mitochondria [23,26,39]. Although we [10] and others [11,12] had previously reported a decrease of mitochondrial efficiency with aging, more recently some doubts in respect to that issue have arisen. For example, failure in recognizing factors that have been reported as influencing mitochondrial function such as gender [12], smoking habit [21,40], physical activity [41] or the kind of anesthesia employed during tissue sampling [25], may have contributed to wrong conclusions. In this regard, we investigated the relationship between age and respiratory chain function of skeletal muscle mitochondria in 132 control individuals and found that the apparent inverse correlation between age and mitochondrial function disappeared after correction for physical activity and tobacco consumption, suggesting that human skeletal muscle mitochondria remain largely undamaged during the aging process [39]. On the other hand, during last years other analytical parameters, as MRC enzyme ratio analysis, have been reported to be better tools than the proper AEA for detecting subtle MRC deficiencies [23,26,39] due to the enormous variation of normal MRC enzyme activities, that expand over one to almost two orders of magnitude and frequently overlap with results found in patients with MRC defects. Conversely, most of the ratios between activities of the different MRC complexes show narrow ranges and abnormal results can be unambiguosly identified. When these parameters were used to investigate mitochondrial activities as a function of age in the present study, evidence of mitochondrial dysfunction were not found [23]. Fig. 6 depicts the variability of complex IV for one age interval (50–59 years) together with values measured in the oldest and youngest subjects included in the study. The variation within a range of age is greater than the differences seen between these two extreme subjects.


Figure 6
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  		Fig. 6 Distribution of cytochrome c oxidase (COX) activity (in nmolmin–1mg–1) for patients in the 50–59 years of age class-interval. Open circles represent subjects and the horizontal line the mean. The wide variability in this class-interval is greater than that observed between the youngest and the oldest individuals of the present study.

 
The preservation of normal MRC enzyme activities with aging does not rule out the existence of mitochondrial dysfunction at other levels than MRC complexes, such as reduced metabolic fluxes, decreased cell phosphorylation potential, protein synthesis and/or import, or reduced intracellular metabolite exchanges. Alterations at any of these or other levels may cause the generation of free radicals and contribute to the observed increase in lipid peroxidation. It is possible that MRC complexes are more resistant to oxidative damage than other molecules and need longer exposition to oxidative attack until its effects become evident. In any case, if lipid peroxidation contributes or is simply a consequence of senescence remains an open question. Our data show that MRC function remains essentially unaffected with age and therefore cannot contribute as a central factor in heart aging process. This confirms our previous data obtained on skeletal muscle tissue [13,23] and the conclusions reached several years ago by Hansford [28] in its still up-to-date review on mitochondria and aging in mammals.

Time for primary review 28 days.


   Acknowledgements
 
We are indebted with Dr. J. Orús (Department of Cardiology, Hospital Clìnic, Barcelona, Catalonia, Spain) and Dr. C. Cabré (Transplantation Coordination Unit, Hospital Clìnic, Barcelona, Catalonia, Spain) for providing heart samples. Present work has been supported, in part, by grants from DGICYT (PM95/0105), FIS (00/0927), and Fundació La Marató de TV3 (2102/97).


   References
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 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 References
 

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