Human mitochondrial variants influence on oxygen consumption

Mitochondrion 9 (2009) 27–30

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Human mitochondrial variants influence on oxygen consumption Ana Marcuello a,1, Diana Martínez-Redondo a,b,1, Yahya Dahmani a, José A. Casajús c, Eduardo Ruiz-Pesini a,b,d, Julio Montoya a,b, Manuel J. López-Pérez a,b, Carmen Díez-Sánchez a,b,* a

Departmento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain CIBER de Enfermedades Raras (CIBERER), ISCIII, Spain c Departamento de Enfermería y Fisiatría. Universidad de Zaragoza, Spain d Fundación Aragonesa para la Investigación y el Desarrollo (ARAID), Zaragoza, Spain b

a r t i c l e

i n f o

Article history: Received 11 June 2008 Received in revised form 25 September 2008 Accepted 7 October 2008 Available online 15 October 2008 Keywords: OXPHOS system mtDNA variants VO2max Mitochondrial haplogroups J Mitochondrial haplogroup

a b s t r a c t This work investigates if human mitochondrial variants influence on maximal oxygen consumption (VO2max). With this purpose we recruited, as a uniform population in term of nutritional habits and life style, 114 healthy male Spanish subjects that practiced fitness exercises 3-4 times a week. Once mtDNA haplogroups were determined, we found that J presents with lower VO2max (P = 0.02) than nonJ variants. J has been related with a lower efficiency of electron transport chain (ETC), diminished ATP and ROS production. Thus, the difficult to compensate the mitochondrial energetic deficiency could explain the accumulation of J haplogroup in LHON and multiple sclerosis. Furthermore, the lower ROS production associated to J could also account for the accrual of this variant in elderly people consequent to a decreased oxidative damage. Ó 2008 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction Maternal influence on the familial segregation of VO2max has been previously described (Lesage et al., 1985; Bouchard et al., 1998; Perusse et al., 2001) suggesting that mitochondrial DNA (mtDNA) is involved in this inheritance. The more paramount function of mitochondria is the generation of ATP through the oxidative phosphorylation system (OXPHOS) which protein constituents are encoded by two distinct genomes, the nuclear and the mitochondrial genomes. As it is well known, the mtDNA presents continent-specific polymorphisms that determine mtDNA haplogroups characteristic of the different geographic regions (Torroni et al., 1996; Brown, Sun et al., 1997; Macaulay et al., 1999; Finnila et al., 2001). Each haplogroup is defined as an individual group characterized by the presence of a particular set of polymorphisms, defined by the use of data from HVS-I (Richards et al., 1998), RFLPs (Torroni et al., 1996) of the coding region (Macaulay et al., 1999) and from HVS-II analysis (Helgason et al., 2000). During last years, some reports suggest that non-pathological mtDNA variability may have phenotypic consequences, as potential responsible for mild differences in OXPHOS activity, in neurodegenerative diseases (van der Walt et al., 2003), penetrance of some diseases (Brown, 1997; DiMauro and Schon, 2003), spermatozoa

motility in humans (Ruiz-Pesini et al., 2000b; Montiel-Sosa et al., 2006) and longevity in different population (Ivanova et al., 1998; De Benedictis et al., 1999; Rose et al., 2001; Ross et al., 2001; Niemi et al., 2003; Cacabelos et al., 2005; Santoro et al., 2006). Mitochondrial variability has been proposed as responsible for individual changes in OXPHOS system activity (Ruiz-Pesini et al., 2004) and, therefore, in oxygen consumption, which could account for the inheritable differences in VO2max previously reported (Bouchard et al., 1998). However, several studies carried out to find possible associations between mtDNA polymorphisms and VO2max have no reached conclusive results (Dionne et al., 1991; Rivera et al., 1998; Murakami et al., 2002). With the aim to demonstrate the influence of mitochondrial genetic background on VO2max, we recruited a male population with healthy life style (no-smoking, no-drinking or no-drug consumption) to take part in the study. After determining VO2max and mtDNA variants of participants, we found that J showed lower VO2max than nonJ variants. This result raises interesting questions about potential pathogenic events, ageing process and longevity in human. 2. Material and methods 2.1. Subjects

* Corresponding author. Tel.: +34 976761641; fax: +34 976761612. E-mail address: [email protected] (C. Díez-Sánchez). 1 These authors contributed equally to this work.

A total of 114 male with healthy nutritional habits and life style (non-smokers, non-alcoholic drinkers and non-drug consumers)

1567-7249/$ - see front matter Ó 2008 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2008.10.002

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A. Marcuello et al. / Mitochondrion 9 (2009) 27–30

that practiced fitness exercises 3–4 times a week participated in this investigation. They possessed a mean (±SD) age of 30.6 ± 7.2 years, body weight of 75.8 ± 7.3 kg, height of 178.9 ± 6.2 cm, maximal heart rate (HRmax) of 183.7 ± 10.3 beats min 1 and VO2max of 49.55 ± 7.05 mL kg min 1 (Table 1). The subjects were fully informed of the aims of the experiments before giving their informed written consent to participate. In order to test the mitochondrial variant frequency distribution in this group, we used 244 Spanish blood donors, randomly recruited, as control population. The study conforms to the code of Ethics of the World Medical Association (Declaration of Helsinki) and was approved by the Ethics Committee of the University of Zaragoza (approval number PI04/01).

tion at 600g and 4 °C, air dried to eliminate ethanol and resuspended in sterile water.

2.2. DNA extraction

2.4. Determination of cardio-respiratory variables

Total DNA was extracted from blood and buccal mucosa samples by conventional methods (Sambrook et al., 1989) with some modifications. Peripheral blood (3–5 ml in EDTA) was diluted with four volumes of TE buffer (20 mM Tris–HCl pH8, 5 mM EDTA). After gently mixing, tubes were kept on ice for 15 min and centrifuged at 600g, 4 °C for 15 min. The pellet was washed with 20 ml TE buffer and a further 15 min spin at 600g, 4 °C. The final pellet was then resuspended in 1.5 ml of 0.4% SDS, 200 lg/ml Proteinase K in TE buffer and incubated at 37 °C over night. The reaction was stopped with 1.5 M, ammonium acetate to facilitate the precipitation of nucleic acids later. The suspension was treated twice with 1.5 volumes of Phenol:Isoamyl Alcohol:Chlorophorm (25:25:1) (phenolIAC), vortex for 5 min and centrifuged at 200g, 5 min at RT. After this, 1.5 volume of chloroform:isoamyl alcohol (24:1) was added to the aqueous phase and centrifuged another 5 min at 200g. The aqueous phase was recovered and two volumes of cold 99% Ethanol were added for the precipitation of Total DNA at 20 °C overnight. Total DNA was recovered, air-dried to eliminate ethanol and finally resuspended in sterile water. The buccal mucosa was obtained with cotton swabs and stored at 20 °C until its DNA extraction. The cotton was introduced in a 1.5 ml eppendorf and 500 lL of TE (10 mM TRIS, 1 mM EDTA) was added. After vigorous mixing 0.25% SDS and 50 lg/lL proteinase K was added and incubated at 37 °C overnight. To start the extraction, ammonium acetate up to a concentration of 1.25 M was added. Afterwards, the reaction mixture was treated with 1.5 volumes of phenol-IAC, vortex for a few seconds and centrifuged at 600g for 5 min. The aqueous phase was vigorously mixed with 1.5 volumes of chloroform:isoamyl Alcohol (24:1) and centrifuged at 600g for 5 min. This last aqueous phase was recovered and two volumes of pure cold ethanol were added to precipitate total DNA. Finally, the clew of DNA pellet was recovered by 30 min centrifuga-

All cardio-respiratory variables were performed in subjects through a graded exercise stress test (GXT) at the same hour of the day (09:00–12:00 h) under similar environmental conditions (temperature, 20–22 ° C; relative humidity, 45–55%; barometric pressure, 720 mmHg). Respiratory gas-exchange data was measured breath-by-breath using open circuit spirometry (CPX/MAX MedGraphics, St. Paul, Minnesota, USA). Calibration against standard gases (12% O2 and 5% CO2) and volume (3000 mL) was performed immediately before each GTX. HR (b min 1) was continuously recorded via an ECG-CM5 bipolar derivation as control methodological reasons. Each subject performed a GXT to determine his VO2max. This variable was considered to be reached when two of the three criteria were satisfied: VO2 leveling off despite an increase in exercise workload, HRmax greater than 90% of the age-predicted (theoretical HRmax value = 220-age) and a respiratory exchange ratio (RER) greater than 1.15. During the previous 24 h period, subjects were refrained from performing intensive training.

2.3. Haplogrouping strategy The samples were haplogrouped by PCR amplification of short mtDNA fragments, followed by restriction enzyme analysis (RFLP) and confirmed by HVS-I and HVS-II sequencing (Macaulay et al., 1999). We used the haplogrouping strategy as previously described (Macaulay et al., 1999) with some modifications (Ruiz-Pesini et al., 2000; Montiel-Sosa et al., 2006). Phylogenetic relationships among the main mitochondrial lineages could be observed elsewhere (Kivisild et al., 2006).

2.5. Determination of fat weight from skinfold-thickness (SKT) measurements All SFT measurements were taken by the same trained member of staff, from identical positions on each subject, following the World Health Organisation (WHO) anthropometic guidelines (De Onis and Habicht, 1996). Holtain skinfold calipers (Holtain Ltd., Dyfed, Wales) were used with the subject in a standing position. Density was predicted from the sum of four skin-folds at the triceps, biceps, subscapular and suprailiac sites using the Durnin and Womersley equations (Durnin and Womersley, 1974) according to the age and sex-specific coefficients. Once density was calculated, the Siri equation (Siri, 1993) allowed to estimate the body fat percentage and, hence, the fat weight. 2.6. Statistical analysis

Table 1 Anthropometric, biochemical and physiological parameters of the healthy studied population (n = 114).

Age (years) Weight (kg) Height (cm) Fat weight (kg) Cholesterol (mg/dL) C-HDL (mg/dL) C-LDL (mg/dL) Triglycerides (mg/dL) IA(cholesterol/C-HDL) VO2Max HRMax

Mean

SD

30.64 75.77 178.85 8.56 178.82 55.09 109.62 68.59 3.25 49.60 183.70

7.24 7.26 6.19 1.91 31.23 10.27 28.27 38.49 0.92 7.05 10.28

The distribution of mitochondrial variants among populations was assessed by the v2-independence test from contingency tables and post-hoc analysis. Comparison of the mean level of VO2max and HRmax parameters among mitochondrial variants studied were assessed using a one-way analysis of variance (ANOVA) test. Normality of VO2max and HRmax parameters distribution was verified by Kolmogorov–Smirnov (K–S) one-sample test before performing ANOVA analysis. Results were expressed as mean and standard deviation (SD), and significant differences were assumed when P < 0.05. Statistical analysis was carried out with the statistical package Stat View 5.0 for Macintosh (SAS Institute, Inc) whereas K–S test was performed with SPSS 15.0 software for Windows.

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A. Marcuello et al. / Mitochondrion 9 (2009) 27–30

3. Results Mitochondrial DNA variants were determined in control and study populations (Table 2). After the genetic analysis, we observed that the mitochondrial variant frequency distribution in Spanish control (n = 244) and healthy male populations (n = 114), showed a non-significant difference (v2-independence test from contingency tables and post-hoc analysis; P > 0.05). On the other hand, VO2max and HRmax parameters normally distributed. Kolmogorov–Smirnov one-sample test to evaluate normality: VO2max (K–S value = 0.065; P > 0.05); HRmax (K–S value = 0.096; P > 0.05). All these results validated our healthy male population as adequate to the study. The analysis of cardio-respiratory variables, VO2max and HRmax, among subjects harboring different mitochondrial haplogroup showed non-significant difference (P > 0.05). Assuming that J is one of the less efficient mitochondrial haplogroup, as it has been previously proposed (Ruiz-Pesini et al., 2004), we split our population in two groups J and nonJ variants. Our results showed a significant lower value of VO2max in J mitochondrial group (P = 0.02) (Fig. 1a). Since oxygen consumption can depend, among other potential factors, on HR, we considered interesting to check the HRmax in both groups. The result (Fig. 1b) allowed discarding the heart beat frequency as responsible for the difference found in VO2max between mitochondrial DNA variants.

Fig. 1. VO2max (a) and HRmax (b) in J and nonJ mitochondrial variants. VO2max and HRmax parameters normally distribute. Kolmogorov–Smirnov one-sample test to evaluate normality: VO2max (K–S value = 0.065; P > 0.05); HRmax (K–S value = 0.096; P > 0.05). Values are expressed as mean ± SD. The Fisher’s PLSD analysis showed significant difference (P = 0.02) of VO2max in J respect to nonJ mitochondrial variants.

4. Discussion We have presently investigated whether the genetic mitochondrial background may have an influence on the maximal oxygen consumption. The key finding was that J mitochondrial haplogroup showed lower VO2max than nonJ variants, having to discard HRmax as responsible of this difference since J and nonJ variants showed similar values in this last parameter. To our knowledge, this is the first conclusive description that human mitochondrial background modifies oxygen consumption. The evidence already accumulated shows that different human mtDNA variants are also functionally different. It is currently accepted that some polymorphisms present in such variants could affect oxygen consumption by mitochondria and, hence, account for a different efficiency on mitochondrial ATP production. According with this, it has been reported that haplogroup H

shows higher complex I activity than the rest of haplogroups (Ruiz-Pesini et al., 2000a). Furthermore, numerous studies about mitochondrial diseases (Leigh and MELAS syndromes among others) showing diminished ETC accompanied by increased lactate concentration utterly confirm this idea. In particular, J mitochondrial haplogroup has been proposed as a variant with less efficient ETC activity (Ruiz-Pesini et al., 2004) and, therefore, with a minor ATP production. It has been repeatedly demonstrated that J haplogroup is more susceptible to LHON (Brown et al., 1997, 2001), multiple sclerosis (Kalman and Lublin, 1999; Houshmand et al., 2005) and both (Hanefeld et al., 1994; Kalman and Alder, 1998). In addition, LHON (Wong et al., 2002; Howell, 2003) and multiple sclerosis (Lev et al., 2006; van Horssen et al., 2006; Schreibelt et al., 2007; Sidoti et al.,

Table 2 MtDNA haplogroups and clades of study (n = 114) and control (n = 244) populations. The distribution of mitochondrial haplogroups did not show significant difference (v2independence test from contingency tables and post-hoc analysis; P > 0.05. CLADE

HG*

Studied population

Controls N

%

NTotal

%

HV

H V HV

56 5 0

49.12 4.39 0

61

53.51

114 13 2

46.72 5.33 0.82

129

52.87

IWX

I W X

1 3 0

0.88 2.63 0

4

3.51

0 2 3

0 0.82 1.23

5

2.05

JT

J T

9 10

7.89 8.77

19

16.67

20 17

8.2 6.97

37

15.16

U O*

U L M preHV preV O*

23 4 1 1 0 1

20.17 3.51 0.88 0.88 0 0.88

23 7

20.17 6.14

53 8 2 3 6 1

21.72 3.28 0.82 1.23 2.46 0.41

53 20

21.72 8.20

N

TOTAL *

HG, haplogroup; O, others.

114

%

NTotal

%

244

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A. Marcuello et al. / Mitochondrion 9 (2009) 27–30

2007; Ghafourifar et al., 2008; Mirshafiey and Mohsenzadegan, 2008) have been related to increased ROS production. Bearing in mind these reports, we think that the dropping of ATP production below pathological threshold could be easier in J haplogroup than in the other mitochondrial variants, that could account for the reported susceptibility of this variant to LHON and multiple sclerosis. On the other hand, as a toxic by-product of OXPHOS, the mitochondria generate most of the endogenous ROS. These free radicals could have toxic effects on proteins, lipids and DNA, especially mtDNA that lacks protein protection. In addition to nuclear factors, progressive damage to mtDNA could induce the loss of mitochondrial function and account for the ageing process (Beckman and Ames, 1998; Wallace, 2005). Assuming the precedent hypothesis, a decrease in ROS production by J mtDNA variant could explain a lower general cellular damage and, thus, the significant higher frequency of the J haplogroup in elderly individuals reported by different authors (Ivanova et al., 1998; De Benedictis et al., 1999; Rose et al., 2001; Ross et al., 2001; Niemi et al., 2003). In summary, the VO2max difference depending on mitochondrial variability described here raises interesting questions about potential pathogenic events, ageing process and longevity in human, either as a direct effect of mtDNA and/or nuclear mutations or as contribution to other primary causes. Acknowledgments This study was supported by grants from the Diputación General de Aragón (Grupos consolidados B33), Spanish Health Institute Carlos III (FIS-PI070045), CIBER de Enfermedades Raras is an initiative of the ISCIII. We thank S. Morales and Mª Jesús Torcal for technical assistance. References Beckman, K.B., Ames, B.N., 1998. The free radical theory of aging matures. Physiol. Rev. 78 (2), 547–581. Bouchard, C., Daw, E.W., et al., 1998. Familial resemblance for VO2max in the sedentary state: the HERITAGE family study. Med. Sci. Sports Exerc. 30 (2), 252– 258. Brown, G.K., 1997. Bottlenecks and beyond: Mitochondrial DNA segregation in health and disease. J. Inherit. Metab. Dis. 20 (1), 2–8. Brown, M.D., Sun, F., et al., 1997. Clustering of Caucasian Leber hereditary optic neuropathy patients containing the 11778 or 14484 mutations on an mtDNA lineage. Am. J. Hum. Genet. 60 (2), 381–387. Brown, M.D., Zhadanov, S., et al., 2001. Novel mtDNA mutations and oxidative phosphorylation dysfunction in Russian LHON families. Hum. Genet. 109 (1), 33–39. Cacabelos, R., Fernandez-Novoa, L., et al., 2005. Molecular genetics of Alzheimer’s disease and aging. Methods Find. Exp. Clin. Pharmacol. 27 (Suppl A), 1–573. De Benedictis, G., Rose, G., et al., 1999. Mitochondrial DNA inherited variants are associated with successful aging and longevity in humans. FASEB J. 13 (12), 1532–1536. De Onis, M., Habicht, J.-P., 1996. Anthropometric reference data for international use: recommendations from a World Health Organization Expert Committee. Am. J. Clin. Nutr. 64 (4), 650–658. DiMauro, S., Schon, E.A., 2003. Mitochondrial respiratory-chain diseases. N. Engl. J. Med. 348 (26), 2656–2668. Dionne, F.T., Turcotte, L., et al., 1991. Mitochondrial DNA sequence polymorphism, VO2max, and response to endurance training. Med. Sci. Sports Exerc. 23 (2), 177–185. Durnin, J.V., Womersley, J., 1974. Body fat assessed from total body density and its estimation from skinfold thickness: measurements on 481 men and women aged from 16 to 72 years. Br. J. Nutr. 32 (1), 77–97. Finnila, S., Lehtonen, M.S., et al., 2001. Phylogenetic network for European mtDNA. Am. J. Hum. Genet. 68 (6), 1475–1484. Ghafourifar, P., Mousavizadeh, K., et al., 2008. Mitochondria in multiple sclerosis. Front. Biosci. 13, 3116–3126.

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