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Unmasking the causes of multifactorial
disorders: OXPHOS differences between
mitochondrial haplogroups
Aurora Go
´mez-Dura
´n1, David Pacheu-Grau1, Ester Lo
´pez-Gallardo1, Carmen Dı
´ez-Sa
´nchez1,
Julio Montoya1, Manuel J. Lo
´pez-Pe
´rez1and Eduardo Ruiz-Pesini1,2,∗
1
Departamento de Bioquı
´mica, Biologı
´a Molecular y Celular, Centro de Investigaciones Biome
´dicas En Red de
Enfermedades Raras (CIBERER), Instituto Aragone
´s de Ciencias de la Salud (I+CS) and
2
Fundacio
´n ARAID,
Universidad de Zaragoza, 50013 Zaragoza, Spain
Received April 27, 2010; Revised and Accepted June 9, 2010
Many epidemiologic studies have associated human mitochondrial haplogroups to rare mitochondrial dis-
eases like Leber’s hereditary optic neuropathy or to more common age-linked disorders such as
Parkinson’s disease. However, cellular, biochemical and molecular-genetic evidence that is able to explain
these associations is very scarce. The etiology of multifactorial diseases is very difficult to sort out because
such diseases are due to a combination of genetic and environmental factors that individually only contribute
in small part to the development of the illness. Thus, the haplogroup-defining mutations might behave as
susceptibility factors, but they could have only a small effect on oxidative phosphorylation (OXPHOS) func-
tion. Moreover, these effects would be highly dependent on the ‘context’ in which the genetic variant is
acting. To homogenize this ‘context’ for mitochondrial DNA (mtDNA) mutations, a cellular approach is avail-
able that involves the use of what is known as ‘cybrids’. By using this model, we demonstrate that mtDNA and
mtRNA levels, mitochondrial protein synthesis, cytochrome oxidase activity and amount, normalized oxygen
consumption, mitochondrial inner membrane potential and growth capacity are different in cybrids from the
haplogroup H when compared with those of the haplogroup Uk. Thus, these inherited basal differences in
OXPHOS capacity can help to explain why some individuals more quickly reach the bioenergetic threshold
below which tissue symptoms appear and progress toward multifactorial disorders. Hence, some population
genetic variants in mtDNA contribute to the genetic component of complex disorders. The existence of
mtDNA-based OXPHOS differences opens possibilities for the existence of a new field, mitochondrial phar-
macogenomics.
New sequence accession nos: HM103354–HM103363.
INTRODUCTION
Mitochondrial DNA (mtDNA) accumulates mutations much
faster than nuclear DNA (nDNA) (1). Very severe mtDNA
mutations are rapidly removed from the female germline by
purifying selection, thereby minimizing their impact on
population fitness. Most of the mtDNA mutations that
cause disease are moderately deleterious and persist in
human populations for a small number of generations (2).
On the other hand, the population survival of functionally
neutral mutations is a matter of chance. Mutations that lie
elsewhere on the spectrum with mild phenotypic effects
will increase their population frequencies depending on a
combination of randomness and selective advantage (3).
These mutations could be advantageous in some environ-
ments but detrimental in other conditions (4). Thus, accord-
ing to the common disease–common variant hypothesis (5),
mutations of the latter category can be part of the genetic
basis underlying complex disorders, such as age-linked
diseases.
∗
To whom correspondence should be addressed at: Departamento de Bioquı´mica, Biologı´a Molecular y Celular, Universidad de Zaragoza, C/Miguel
Servet, 177, 50013 Zaragoza, Spain. Tel: +34 976761640; Fax: +34 976761612; Email: eduruiz@unizar.es
#The Author 2010. Published by Oxford University Press. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Human Molecular Genetics, 2010, Vol. 19, No. 17 3343–3353
doi:10.1093/hmg/ddq246
Advance Access published on June 21, 2010
at Facultad MedicinaHemeroteca on June 20, 2011hmg.oxfordjournals.orgDownloaded from
mtDNA encodes very important subunits of the oxidative
phosphorylation (OXPHOS) system and the RNAs required for
their expression. Through this system, cells obtain most of the
ATP necessary to live. The importance of mitochondrial
energy makes mtDNA an interesting candidate to study in
relation to multifactorial diseases. In fact, many disease pheno-
types have been related to groups of phylogenetically related
mtDNA genotypes or haplogroups (Supplementary Material,
Table S1), and biochemical evidence obtained from individuals
with these associations has been provided in epidemiologic
studies (6). However, progress in this area has been limited
because the phenotypic effects produced by variation in
mtDNA are difficult to isolate due to confounding variations of
the nuclear genome and to environmental factors. Nevertheless,
by using rat or mouse conplastic strains, with identical nuclear
genomes but divergent mtDNA genomes, it has been shown
that natural mtDNA variation can promote OXPHOS differences
that are relevant to the pathogenesis of common diseases (7–10).
Obviously, this model cannot be applied in human beings.
However, phenotypic differences due to mtDNA variation have
also been confirmed by using a different experimental model,
mouse trans-mitochondrial cell lines or cybrids (11). These
cybrids share the same nuclear genetic background and environ-
mental conditions but differ in their mtDNA.
Thus, before one can seriously consider the role of human
mtDNA haplogroups in terms of clinical applications, evi-
dence of cellular, biochemical and molecular-genetic differ-
ences between the groups must be obtained. Because the
haplogroup Uk has been found to be under-represented in
patients with Parkinson’s or Alzheimer’s diseases, thus behav-
ing as a resistance factor, and the haplogroup H has been
found to be over-represented in individuals with neurodegen-
erative diseases, thus being a potential susceptibility factor
(Supplementary Material, Table S2), we used the cybrid
model to compare the OXPHOS function of these hap-
logroups. Our results showed that the mtDNA haplogroup
differentially contributes to OXPHOS functionality and can
therefore be a risk factor that contributes to the development
of some of these age-linked diseases.
RESULTS
Nuclear- and mitochondrially convenient cybrids for
analysis of mtDNA population genetic variants
To compare the OXPHOS function between the haplogroups
H and Uk, we built cybrids with an osteosarcoma 143B
TK2nuclear background. Before we used this cell line in
our experiments, we considered several aspects of the
model. It is known that this cell line is aneuploid, and it was
previously shown that cybrids from this background could
have different chromosomal numbers (12). Therefore, to rule
out major nuclear influences, we took two approaches. First,
to avoid clonal effects, we constructed five cybrid cell lines
per haplogroup and confirmed that we had introduced the
correct mtDNAs (Supplementary Material, Fig. S1). Second,
we karyotyped all our cybrids and observed that the chromo-
somal number was not significantly different between them
[H, 66.3 +3.8 (5); Uk, 64.6 +2.1 (5)] (Fig. 1A and Sup-
plementary Material, Table S3).
Because we were interested in the functional effects of
haplogroup-defining polymorphisms, we sought to discard the
existence of non-haplogroup-defining mutations with potential
phenotypic effects, so we built our cybrids (9 out of 10) from
donors in their 20s or 30s, well-below the age when somatic
mtDNA mutations tend to accumulate (13). Moreover, although
this cell line is thymidine kinase negative (TK2), cells also
contain a mitochondrial TK (TK2). Thus, it could be possible
that during the selection of the cybrids with 5-bromo-
2′-deoxyuridine, the mtDNA may accumulate mutations (14).
Therefore, we sequenced the entire mtDNA from the cybrids
after selection instead of directly from the donors. In addition,
it was recently shown that cybrids could accumulate mtDNA
mutations during culture (15). To have a gross estimation of
the culture mutation rate, we compared control region
sequences that consisted of the mtDNA region with a higher
mutational rate between cybrids and donor blood, and we did
not find any sequence differences between them.
These trans-mitochondrial cell lines included haplotypes
from two subhaplogroups of Uk (Uk1, 2 cybrids; Uk2, 3
cybrids) and three subhaplogroups of H (H1, H5 and H13
with 3, 1 and 1 cybrids, respectively) (Fig. 1B). To exclude
private variants (those occurring at the tips of individual
branches within the phylogenetic tree) with a possible pheno-
typic effect, mtDNA mutations were analyzed in a large
mtDNA database that included more than 3000 human
sequences (16). We found 43 mutations (16 in the control
region, 14 synonymous and 5 non-synonymous in protein
genes and 8 in RNA genes), but 42 had already been
described. Only one, the m.10428A .G/MT-TR mutation in
the cybrid 110K, had not been reported previously. This
mutation was a heteroplasmic transition (Supplementary
Material, Fig. S2A), a condition frequently associated with
pathologic mutations, and it broke a Watson – Crick base
pair in the tRNA-Arg anticodon stem (Supplementary
Material, Fig. S2B). In this cell line, however, a posterior
analysis of mitochondrial protein synthesis and other
OXPHOS parameters did not show any significant difference
with cybrids from the same haplogroup (Supplementary
Material, Fig. S2C).
Therefore, these results allowed us to consider these 10
cybrid cell lines nuclear and mitochondrially convenient for
posterior analysis of the functional effect of haplogroup-
defining polymorphisms.
mtDNA levels were lower in cybrids from the
haplogroup Uk
OXPHOS complex activities are under tight control by
mtDNA levels (17). To be sure that these levels had been
recovered after the cybridization process, we determined
the mtDNA amount by qRT-PCR in different culture pas-
sages and observed that at least 20 passages were necessary
to get the steady-state levels. Therefore, all the experiments
were performed in cybrids with a passage number higher
than 20. When we compared the mtDNA levels between
cybrids H and Uk, we found that cybrids Uk had 7.3%
less mtDNA than those in the haplogroup H and this differ-
ence was significant (Fig. 2A). The existence of differences
in mtDNA levels was also recently shown for cybrids from
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the haplogroups H and J (18). These differences were appar-
ently due to a single-nucleotide polymorphism (SNP) in a
sequence of the mtDNA control region that was important
for its replication. However, our cybrids did not have
SNPs in sequences involved in mtDNA replication
(Fig. 1B). Consequently, mtDNA levels had to be explained
by another functional difference that is due to mitochondrial
genotype. Another explanation could involve the fact that it
has been reported that high reactive oxygen species (ROS)
levels enhance mtDNA replication (19).
Because high ROS levels due to mtDNA pathologic
mutations could affect aconitase activity (20) or trigger antiox-
idant responses, such as manganese superoxide dismutase
(MnSOD) overexpression (21), we measured these parameters
but did not find any significant difference between both hap-
logroups. Moreover, we were not able to find differences in
either cellular hydrogen peroxide or mitochondrial superoxide
anion levels (Supplementary Material, Fig. S3). This could be
because our cybrids did not have pathologic mutations but
only had population SNPs.
Figure 1. Nuclear and mitochondrial genome analysis in cybrids H and Uk. (A) Spectral karyotyping (SKY) analysis of the cybrids. Metaphase after DAPI stain
(a.1), SKY hybridization (a.2) or classified colors (a.3). In this karyotype of one cybrid cell 48K (a.4), structurally rearranged chromosomes are grouped accord-
ing to the chromosomal type of their major component. Every chromosome from pictures a.1 to a.3 is aligned in this karyotype. (B) Phylogenetic tree of the
cybrids’ mtDNA. Black, green, red and blue colors define control region, protein synonymous, protein non-synonymous and RNA mutations, respectively.
The affected mtDNA gene or sequence is showed in parenthesis. For non-synonymous mutations, amino acid substitution and position in the protein is also
indicated. rCRS means revised Cambridge reference sequence (45).
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On the other hand, in a recent study, it was shown that
mouse cybrids producing more ROS had higher mtDNA
levels, which decreased after treatment with the antioxidant
N-acetyl-cysteine (NAC). However, the mtDNA amount was
unaffected in those cybrids producing less ROS (11). Simi-
larly, although both cybrids H and Uk decreased their
mtDNA levels after the NAC treatment, the effect was
larger for cybrids H (20.2% versus 8.3%). Very interestingly,
the mtDNA levels after treatment were not significantly differ-
ent between both haplogroups (Fig. 2A), as though they
reached a basal level.
Mt-rRNA levels were lower in cybrids from the
haplogroup Uk
To check whether the mtRNA levels were related to mtDNA
amount, we studied mtDNA gene expression by qRT-PCR
and found that, despite the lower mtDNA levels in cybrids
Uk compared with cybrids H, there were no significant
differences in the RNA levels of mtDNA-encoded complex
I (CI), IV (CIV) and V (CV) subunits (Fig. 2B). Because
most of the mt-mRNAs (except that for p.MT-ND6) are
part of the same polycistronic transcript, the lower levels
of cytochrome bmRNA found (Fig. 2B) could be related
to differences in the molecule’s half-life or in the hybridiz-
ation process due to the presence of particular MT-CYB
SNPs in the mtDNA Uk (Fig. 1B). On the other hand, the
levels of the rRNAs were significantly lower in the cybrids
Uk (Fig. 2B). Given that there were not genetic differences
between the mtDNA sequences of both haplogroups that
related to the control of the transcription process (Fig. 1B),
these lower rRNA levels are likely due to other
mtDNA-related factors, one of which might be the mitochon-
drial ATP amount.
Human mtRNA synthesis starts at three different locations,
one for the L-strand (L) and two for the H-strand (H1 and H2).
Mt-rRNAs are mainly synthesized when transcription starts at
H1, whereas most of the mRNAs are produced when H2 tran-
scription begins (22). It was shown that the pattern of mtRNA
synthesis changes dramatically depending upon the level of
ATP available. mRNA synthesis was stimulated at low ATP
levels, whereas at high intra-mitochondrial ATP levels,
rRNA synthesis and L-strand transcription were strongly
stimulated (23). Because the decrease in the amount of
mtRNA from cybrids Uk was larger for L and H1 than for
H2 transcripts (14.1%, 11.6% and 7.0% for L, H1 and H2,
respectively), and because it was recently reported that ATP
levels were higher in CD4
+
cells from haplogroup H versus
non-H patients suffering from Huntington disease (24), we
measured the levels of ATP in our cybrids. We found that
these levels were significantly higher in cybrids Uk growing
in glucose (Fig. 3). If ATP levels mirror cell energy require-
ments, then cybrids Uk had higher ATP necessities. By
growing the cells with 2-deoxy-glucose, a glycolytic inhibitor,
and pyruvate, a respiratory substrate, we observed that cybrids
H and Uk produced the same ATP amount, although this level
was around 30% lower than that obtained by growth in glucose
(Fig. 3). This is probably because these are very glycolytic
cells and by inhibiting the OXPHOS function with oligomy-
cin, we found that the difference previously described was
due to ATP produced by glycolysis (Fig. 3). Therefore,
because it was not the intra-mitochondrial ATP, another
mtDNA-related functional parameter likely accounts for the
difference in the rRNA levels.
Figure 2. Nucleic acid levels in cybrids H and Uk. White and black bars rep-
resent mean values for cybrids H and Uk, respectively. (A) mtDNA levels. The
mean value for cybrids H without NAC has been set to 100%. H, 100.0 +
2.6% (5); H (NAC), 79.8 +7.0% (5); Uk, 92.7 +3.6% (5); Uk (NAC),
85.0 +5.2 (5). ∗P≤0.026. Striped bars represent the mtDNA levels after
treatment with 5 mMNAC. (B) mtRNA levels. The mean value for cybrids
H has been set to 100%. 12S: H, 100.0 +7.8% (5); Uk, 89.9 +5.0% (5);
16S: H, 100.0 +7.2% (5); Uk, 86.9 +2.9% (5); ND4-ND4L: H, 100.0 +
10.2% (5); Uk, 95.5 +12.6% (5); ND6: H, 100.0 +9.4% (5); Uk, 84.3 +
17.5% (5); Cytb: H, 100.0 +7.9% (5); Uk, 85.9 +8.9% (5); COI: H,
100.0 +7.9% (5); Uk, 96.0 +5.9% (5); COII: H, 100.0+9.0% (5); Uk,
87.2 +9.4% (5); COIII: H, 100.0 +9.2% (5); Uk, 94.0 +9.0% (5);
ATP6-ATP8: H, 100.0 +13.9% (5); Uk, 99.2 +6.7% (5). ∗P≤0.041.
Figure 3. ATP levels in cybrids H and Uk. G, DGP, DGPO and GO are
abbreviations for glucose, 2-deoxy-glucose plus 1 mMpyruvate, 2-deoxy-
glucose plus 1 mMpyruvate and 2.5 mg/ml oligomycin and glucose plus
2.5 mg/ml oligomycin, respectively. The mean value for cybrids H in
glucose has been set to 100%. G: H, 100.0 +8.3% (5); Uk, 132.9 +18.3%
(5); DGP: H, 71.1 +33.2% (5); Uk, 66.2 +10.9% (5); DGPO: H, 5.9 +
2.6% (5); Uk, 4.3+0.6% (5); GO: H, 96.2 +10.7% (5); Uk, 138.3 +
22.8% (5). ∗P¼0.009. White and black bars represent the mean values for
cybrids H and Uk, respectively.
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Mitochondrial protein synthesis and respiratory complex
IV levels and activities were lower in cybrids from the
haplogroup Uk
To test whether different mtRNA levels could affect
OXPHOS function, we determined the enzymatic activities
of CII (nDNA-encoded) and CIV (nDNA&mtDNA-encoded)
as electron transport chain (ETC) markers and normalized
these values for citrate synthase (CS) enzymatic activity, a
matrix enzyme (nDNA-encoded) that reflects the mitochon-
drial number or volume. The results showed that there
were no significant differences for CS and CII/CS.
However, the CIV/CS ratio was significantly lower in the
Uk cybrids (Fig. 4A). Except for the synonymous poly-
morphism m.7028C.T, there were no other SNPs in mito-
chondrial CIV genes that differed between these
haplogroups (Fig. 1B). To explain this lesser activity, we
determined the CIV levels and found that they were signifi-
cantly lower in cybrids Uk (Fig. 4B). Moreover, there was a
statistically significant correlation between CIV activities and
levels (Fig. 4C). Therefore, the reduction in CIV activity was
due to a lower CIV quantity.
The decline in the CIV amount could be due to decreased syn-
thesis efficiency because of lesser rRNA levels or SNPs in
mtDNA protein synthesis genes. There were two polymorphisms
in the MT-RNR2 (m.1811A.G and m.2706A.G) gene and one
in the MT-TL2 (m.12308A.G) gene that could be responsible
for these differences (Fig. 1B). Very interestingly, the analysis
of mitochondrial translation products showed a decrease in
mitochondrial protein synthesis in cybrids Uk (Fig. 4D and E).
The 29.4% decline in CIV levels in cybrids Uk was
accompanied by a 29.0% reduction in mitochondrial protein
synthesis.
Oxygen consumption and mitochondrial inner membrane
potential were different in cybrids from haplogroups
H and Uk
Subsequently, we measured oxygen consumption by using
high-resolution respirometry and we did not find significant
differences in the endogenous, leaking or uncoupled
respiration between cybrids H and Uk when expressed as
fmole/min/cell (data not shown). However, oxygen consump-
tion rate, when expressed relative to cell number, tends to
decrease with increasing cell density due to a decrease in
the size of the cells (25) and, probably, in the number of
mitochondria. To avoid this problem, we measured oxygen
consumption again and related it to the CIV/CS ratio in the
cybrid cell lines, as a surrogate of the oxygen consumption
per ETC unit (26). We showed that endogenous, leaking and
uncoupled respiration was significantly higher in cybrids Uk
(Fig. 5A).
Oxygen consumption rate is inversely related to the mito-
chondrial inner membrane potential (MIMP) (27). Thus, our
results on oxygen consumption suggested that cybrids Uk
had lower MIMP than cybrids H. The determination of
MIMP showed that this potential was significantly lower in
the cybrids Uk (Fig. 5B). To rule out the differences in the
mitochondrial inner membrane surface (MIMS), we used a
Figure 4. Mitochondrial enzyme and protein synthesis analysis. White and black bars represent the mean values for cybrids H and Uk, respectively. The mean value
for cybrids H has been set to 100% in A,B,Cand E. (A) Enzyme activities. CS: H, 100.0 +12.6% (5); Uk, 123.5 +27.3% (5); CII/CS: H, 100.0 +7.8% (5); Uk,
90.2 +23.7% (5); CIV/CS: H, 100.0+24.6% (5); Uk, 57.2 +15.9% (5); ∗P¼0.001. (B) CIV study. CIV specific activities: H, 100.0 +14.4% (5); Uk, 61.1+
14.7% (5); CIV levels: H, 100.0+10.9% (5); Uk, 70.6+14.8% (5). ∗P≤0.007. (C) % Activity/% amount relationship. y¼1.68 +0.141 x. R
2
¼0.96, P¼
0.0001. (D) Mitochondrial translation products. Gels showing the loading control and electrophoretic patterns of mitochondrial translation products from the
cybrid cell lines. M, molecular weight marker. (E) Quantification of mitochondrial protein synthesis. H: 100.0+14.5% (5) and Uk: 71.0 +21.4% (5); ∗P¼0.037.
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probe with high affinity for cardiolipin, and we found no
differences in MIMS (Fig. 5B).
The cybrids Uk grew more slowly than the cybrids H
To analyze how these OXPHOS differences finally affect cell
performance, we analyzed cybrid viability and growth capa-
bility. Cell viability was not different between cybrids (Sup-
plementary Material, Fig. S4A). Growth capability was
measured in two ways. First, we analyzed the cell doubling
times (DTs) in glucose or galactose medium, and we observed
that the growth capability in glucose was the same for cybrids
of both haplogroups but that the growth in galactose was
slower for cybrids Uk, though not significantly (Supplementary
Material, Fig. S4B). Secondly, we used competitive mix exper-
iments to estimate cell growth and found that there were signifi-
cant deviations in the percentage of each genotype H and Uk.
After 10 days, the percentage of the genotype H was signifi-
cantly higher than that at the initial mix, in both glucose and
galactose medium. Moreover, there was a significant difference
in the percentage of genotype H of cells growing in galactose
versus those growing in glucose (Fig. 6), thus suggesting a
growth advantage for this haplogroup.
DISCUSSION
A specific combination of diverse genetic (including nuclear
and mitochondrial genetic variants) and environmental
factors may be involved in a multifactorial disorder, but
most of the factors involved are often still unknown. Different
combinations of these factors can hamper the analysis of the
contribution of any particular factor. mtDNA haplogroups
have been epidemiologically associated with different dis-
eases. To analyze the phenotypic effects of human variation
in mtDNA and to remove confounding nuclear and environ-
mental influences, a cybrid approach is necessary. Because
individuals from the haplogroup T had been found to be
over-represented in moderate asthenozoospermia and other
phenotypes, whereas those from the haplogroup H were over-
represented in the normal sperm motility group (6), cybrids
were used to investigate mitochondrial function in mtDNA
haplogroups H and T (28). Researchers did not find differences
between cybrids H and T in terms of the percentage of basal,
leaking and uncoupled respiration (28). We also did not find
differences in endogenous, leaking and uncoupled respiration
between cybrids H and Uk when expressed as fmole/min/
cell. However, we observed that cybrids Uk had lower
mtDNA and mt-rRNA levels. These levels were accompanied
by a decrease in mitochondrial protein synthesis and CIV
activities and levels. Therefore, lower CIV levels per cell or
per mitochondrion means that the same level of cell oxygen
consumption was carried out by a lower amount of ETC.
Thus, each ETC unit consumed more oxygen. Because nor-
malized oxygen consumption was significantly different
between uncoupled cybrids from both haplogroups, meaning
that the proton gradient was not affecting this rate, the electron
transport rate through the ETC should be higher in the cybrids
Figure 5. Oxygen consumption and mitochondrial inner membrane potential (MIMP) and surface (MIMS) in cybrids H and Uk. White and black bars represent
the mean values for cybrids H and Uk, respectively. (A) Corrected oxygen consumption (oxygen consumption per ETC unit). E, L and U code for endogenous,
leaking and uncoupled respiration, respectively. The mean value for E respiration in cybrids H has been set to 100%. E: H, 100.0 +36.9% (5); Uk, 148.3 +
26.5% (5); L: H, 28.7 +9.3% (5); Uk, 51.7 +11.9% (5); U: H, 132.5 +39.1% (5); Uk, 190.2 +34.1% (5); ∗P≤0.045. (B) MIMP and MIMS. The mean value
for cybrids H has been set to 100%. MIMP: H, 100.0 +18.6% (5); Uk, 74.4 +15.5% (5); ∗P¼0.046. MIMS: H, 100.0 +4.4% (5); Uk, 106.0 +6.4% (5).
Figure 6. Growth capacity of cybrids H and Uk. White and striped bars rep-
resent the genotype H shift, compared with its initial situation, when growing
in glucose and galactose, respectively. Glucose and galactose indicate glucose
minus initial and galactose minus initial genotype H percentage, respectively.
DDC
t
codes for threshold cycle differences. Glucose: 0.56 +0.85 (16); galac-
tose: 1.13+0.47 (16). ∗P¼0.023.
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Uk. Moreover, by using oligomycin to measure leaking
respiration, ATP synthase is inhibited, the proton gradient
increases and the oxygen consumption decreases. Under these
conditions, the higher leaking respiration rate might indicate a
lower MIMP, as was shown for cybrids Uk, due to proton
leakage or inefficiency in the proton pumping of cybrids Uk.
The faster electron flow and lower MIMP in cybrids Uk
probably means that electron slippering was happening (i.e.
non-coupled or decoupling respiration in which electrons are
transported without formation of a potential) (29).
Because only two non-synonymous SNPs (m.14766C.T
and m.14798T.C) were present in OXPHOS subunits that
are involved in electron flow and proton pumping (Fig. 1B),
only these could be responsible for the differences in oxygen
consumption and inner membrane potential. The first
(p.MT-CYB:Thr7Ile) defines the cluster HV, and the Thr7 is
only conserved in 4 of 276 mammalian species (conservation
index, CI ¼1.4%), but it has been hypothesized that this sub-
stitution has an impact on the efficiency of the CIII Q cycle
(30). The second one (p.MT-CYB:Phe18Leu) was found
twice at internal branches of an mtDNA phylogenetic tree
built with more than 3000 complete mtDNA sequences (16)
and defines genetic backgrounds J1c and Uk. Interestingly,
both haplogroups were found to be over-represented in cente-
narians and LHON patients and under-represented in patients
with Parkinson’s disease. Phe18 is conserved in 220 of 276
mammal species (CI ¼79.7%), thus hinting at its functional
importance. This position was located ,3.5 A
˚from the
inner ubiquinone binding (Qi) site (31), and it was shown
that a similar change alters the susceptibility to diuron in
yeast, an ETC inhibitor (32). Moreover, this position was situ-
ated in a helical region parallel to the plane of the membrane
and might participate in relaying conformational information
between the cytochrome bmonomers (33).
ETC is a metabolic pathway involved in many cell functions.
The proton gradient that originates from electrons passing
through the ETC complexes is used for many different purposes
such as protein and substrate import toward the mitochondria,
thermogenesis, apoptosis, maintenance of the cytosolic
calcium levels and production of ROS and ATP. Moreover,
OXPHOS is important for adaptation to the environment. In
fact, external signals, in the form of nutrients and oxygen, inter-
act at the OXPHOS level and trigger intracellular retrograde
responses mediated by second messengers such as cell redox
state or levels of ATP, ROS and Ca
2+
. We did not find differ-
ences between cybrids H and Uk in ROS production. In fact,
our results were similar to those obtained with the mtDNA
mutator mice that accumulated mtDNA mutations and suffered
premature aging phenotypes. In these mice, the amount of ROS
was normal and the aconitase activity or expression levels of
antioxidant enzymes indicated no oxidative stress in their
tissues (34). However, we observed that the antioxidant NAC
decreased mtDNA levels. Thus, it is possible either that we
experienced methodological problems from the fluorescent
dyes being unable to distinguish small differences in ROS
levels (35) or that NAC affected another process that is quanti-
tatively different in cybrids H and Uk (36). In any case, if
differences are not a result of ROS, another difference in
cybrids H and Uk due to distinct OXPHOS capacities must
be responsible for the mitochondrial phenotypes that we
observed. Considering this, it has been shown that mitochon-
drial matrix pH and intracellular calcium dynamics were differ-
ent in cybrids from mtDNA macrohaplogroups N and non-N
(37). ROS, calcium or other second messengers can modify
the expression of many nuclear and mitochondrial genes.
HSP60 mRNA and protein levels have also been found to be
different in cybrids H (38). These nuclear compensations
might hide true differences in OXPHOS function (28), as has
recently been shown in mouse cybrids (11). Thus, the signifi-
cant surplus of glycolytic ATP observed in cybrids Uk might
be an attempt to perform nuclear compensation for their
lower MIMP. Curiously, it had been previously shown that
osteosarcoma 143B.TK2cells treated with dinitrophenol for
3 days did not change in oxidative capacity but increased
their glycolytic metabolism. It was suggested that glycolytic
ATP in these cells supplied energy for maintaining mitochon-
drial membrane potential (39,40).
Thus, along with time and with the cumulative effects of
mtDNA somatic mutations and other nuclear and environmental
factors, the mtDNA inherited basal differences in OXPHOS
capacity reported here can help to explain why some individuals
take longer to reach a certain threshold below which tissue
symptoms appear and progress toward multifactorial disorders
(41). This finding could aid in understanding the overre-
presentation of haplogroup Uk in individuals who become
centenarians and the underrepresentation in individuals with
age-related neurodegenerative disorders such as Parkinson’s
and Alzheimer’s diseases. Moreover, slight modifications of
the cybrid model can contribute to an unraveling of the particu-
lar combination of nuclear, mitochondrial and environmental
factors that cause a particular multifactorial disease.
MATERIALS AND METHODS
Biological samples
After winning the approval of the Ethical Committee of the
Government of Aragon (Acta n817/2008) and securing
signed informed consent, blood from 165 healthy volunteers
was obtained. To homogenize nuclear and environmental
factors, we used trans-mitochondrial cell lines or cybrids
with the osteosarcoma 143B rho0 nuclear background (42).
Ten cybrids (five from mtDNA haplogroup H and five from
Uk) were built by fusing platelets from selected individuals
with this rho0 cell line (43).
Growth conditions, DTs and cell mix experiments
Most of the experiments were performed with cell lines grown
in Dulbecco’s modified eagle medium (DMEM) containing
glucose (4.5 g/l), pyruvate (0.11 g/l) and fetal bovine serum
(FBS) (5%). When cells were grown with an antioxidant,
5m
Mof NAC was used in the culture medium (11). To
avoid undesired phenotypic effects, we grew our cybrid cell
lines without any antibiotics. DTs of 10 cybrid cell lines
growing in DMEM as previously reported or DMEM with
galactose (0.9 g/l), pyruvate (0.11 g/l) and FBS (5%) were
determined by using the Z2 Beckman Coulter. Initially,
1.5 ×10
5
cells were plated. Three to six growth curves were
performed for every cell line, and each time point (0, 24,
Human Molecular Genetics, 2010, Vol. 19, No. 17 3349
at Facultad MedicinaHemeroteca on June 20, 2011hmg.oxfordjournals.orgDownloaded from
48, 72 and 96 h) was counted in triplicate. Only those curves
with R
2
≥0.9 were considered.
To perform the competitive mix experiments, we combined
each cybrid cell line from the mitochondrial haplogroup H
with each cybrid cell line from the mitochondrial haplogroup
Uk. We grew them in galactose or glucose medium for 10
days, and then we estimated the percentage of every genotype
(H and Uk) by qRT-PCR at the final and initial (just after the
mix) time points.
Genetics analysis
Samples from the volunteers were genetically characterized by
performing PCR-RFLP for mitochondrial haplogroup-defining
SNPs in the coding region and sequencing the hypervariable
regions I and II (HVRI and HVRII) (6,44).
For molecular cytogenetic analysis of cybrids, cells were
exposed to colchicines (0.5 mg/ml) for 4 h at 378C and har-
vested routinely. Metaphases were prepared from the cybrids
following a conventional cytogenetic protocol for methanol-
acetic acid (3:1)-fixed cells. Slides were prepared from the
fixed material and hybridized using the SKY method accord-
ing to the manufacturer’s protocol (Applied Spectral
Imaging, Migdal Ha’Emek, Israel). Images were acquired
with an SD300 Spectra Cube (Applied Spectral Imaging)
mounted on a Zeiss Axioplan microscope using a custom-
designed optical filter, SKY-1 (Chroma Technology, Brattle-
boro, VT, USA). Around 20 metaphase cells were captured
and analyzed for each cell line.
The mtDNA sequence was obtained by using the BigDye
Terminator v 3.1 Cycle Sequencing Kit (Applera Rockville,
MD, USA) and an ABI Prism 3730xl DNA analyzer
(Applied Biosystems, Foster City, CA, USA). To locate
mutations, the human revised Cambridge reference sequence
was used (GenBank NC_012920) (45). The mtDNA content
was measured by the qRT-PCR method using an Applied Bio-
systems StepOneTM Real-Time PCR System Thermal Cycling
Block (Applied Biosystems), as described elsewhere (46). The
mtDNA levels were determined in triplicate in three to five
independent experiments.
The genotype shifting quantification in the mix experiments
was performed by qRT-PCR, using TaqMan reagents. It
includes two specific primers around the m.7028 position
and two probes: one labeled with the fluorophore VIC that is
specific for m.7028C; and another labeled with the fluorophore
FAM that is specific for m.7028T. DNA was amplified in a
final volume of 25 ml, using 12.5 ml of TaqMan Gene
Expression Master Mix (Applied Biosystems), a final concen-
tration of 0.9 mMof each primer, a final concentration of
0.2 mMof each probe and 10 ng of total DNA. The amplifica-
tion was performed under universal conditions.
To assess the mtRNA levels, total RNA was isolated from
exponentially growing cells using a RNA isolation kit
(NucleoSpin
w
RNA II) from Macherey-Nagel according to
the manufacturer’s protocol; 2.5 mg of total RNA was
reversed-transcribed into cDNA with the High capacity
cDNA reverse transcription kit (Applied Biosystems), using
the manufacturer’s conditions. The levels of MnSOD mRNA
and mtRNAs were determined in triplicate in two independent
experiments by qRT-PCR using the One-Step Real-Time
system (Applied Biosytems). The expression levels were nor-
malized using the 18S rRNA. The comparative C
t
method was
used for relative quantification of gene expression as described
by the real-time PCR machine manual. Differences in the C
t
values (dC
t
) of the transcript of interest and the reference
gene were used to determine the relative expression of the
gene in each sample. The dC
t
method was used to calculate
fold expression. StepOne software version 2.0 (Applied Bio-
systems) was used for data analysis.
Measurement of ROS production
The production of the mitochondrial superoxide anion was
measured in triplicate with a Cytomics FC 500 flow cytometer
(Beckman Coulter, Fullerton, CA, USA) by using MitoSOX
Red
w
(Invitrogen, Carlsbad, CA, USA) as described pre-
viously (47), with slight modifications. The production of
cell hydrogen peroxide was measured in triplicate in 4–5 inde-
pendent experiments with the same flow cytometer by using
2′,7′-dichlorodihydrofluorescein diacetate (2,7-DCFH
2
-DA)
(Invitrogen) as described previously (48), with slight modifi-
cations. Aconitase activity was measured in triplicate in 3 – 5
independent experiments as described previously (49,50),
with slight modifications. The values were expressed as
mU/mg protein.
Determination of ATP levels
ATP levels were measured four times in three independent
experiments as described previously (51), with some modifi-
cations, using the CellTiter-Glo
w
Luminiscent Cell Viability
Assay (Promega) according to the manufacturer’s instructions.
Briefly, 20 000 cells/well were seeded 10 – 12 h before
measurement. Then, cells were washed twice with PBS and
incubated for 6 h in record solution with either 5 mM
glucose, 5 mMglucose plus 2.5 mg/ml oligomycin (glycolytic
ATP generation), 5 mM2-deoxy-D-glucose plus 1 mMpyruvate
(oxidative ATP production) or 5 mM2-deoxy-D-glucose plus
1m
Mpyruvate plus 2.5 mg/ml oligomycin. Cells were lysed,
and lysates were incubated with the luciferin/luciferase
reagents. Samples were measured using a NovoStar MBG
Labtech microplate luminometer, and the results referred to
the protein quantity.
Oxygen consumption and respiratory complex activities
and levels
Oxygen consumption was analyzed using the high-resolution
oxygraph OROBOROS
w
. Exponentially growing cells were
collected by trypsinization, washed, counted and resuspended
at 1.5 ×10
6
cells/ml. Endogenous, leaking (with oligomycin
added at 49 nM) and uncoupled (with FCCP added at
1.2 mM) respiration analyses were performed. To correct for
the oxygen consumption that is not due to the ETC, respiration
inhibition by KCN was performed. Each cell line was
measured three to four times in DMEM glucose. Respiration
was measured at 378C with chamber volumes set at 2 ml.
The software DatLab (Oroboros Instrument, Innsbruck,
Austria) was used for data acquisition (1 s time intervals)
and analysis (52).
3350 Human Molecular Genetics, 2010, Vol. 19, No. 17
at Facultad MedicinaHemeroteca on June 20, 2011hmg.oxfordjournals.orgDownloaded from
The enzymatic activities of OXPHOS CII and CIV and CS
were assayed following previously described protocols (53 –
55) in a Unicam UV 500 spectrometer (Unicam Instruments,
Cambridge, UK). Mitoprofile
w
Human Complex IV Activity
and Quantity from Mitosciences (Invitrogen) was used accord-
ing to the manufacturer’s instructions for the determination of
CIV activity and levels. A NovoStar MBG Labtech microplate
instrument was used for analysis.
Determination of MIMP and MIMS and cell viability
The determination of the MIMP was done in triplicate in three
independent experiments using 3,3′-dihexyloxacarbocyanine
[DiOC
6
(3)] as published previously (56,57). The MIMS was
measured, based on the quantity of cardiolipin, four times in
three independent experiments by using NAO (nonyl-
acridine-orange) (58). The cell viability was measured in tri-
plicate in two independent experiments by using propidium
iodide (PI) as described previously (59). A Beckman Coulter
Cytomics FC500 cytometer was used for measurements of
intracellular fluorescence.
MtDNA-encoded protein synthesis
The mitochondrial protein synthesis was analyzed as described
previously (60) with minor modifications. Electrophoresis was
performed with a Protean II xi system (BIORAD). As a load
control, we dyed the gel for 15 min with fixing solution
(30% methanol, 10% acetic acid) plus 0.025% of Brilliant
Blue R (Coomassie Blue) (Sigma). Then, the gel was
washed several times with a 50% methanol, 10% acetic acid
solution and left overnight in fixing solution. Finally, it was
treated for 20 min with Amplify solution (AMERSHAM),
dried and used for autoradiography. The band intensities
from appropriate exposures of the fluorograms from two inde-
pendent gels were quantified by densitometric analysis with
the Gelpro analyzer v 4.0. Three bands, corresponding to
p.MT-ND5 (upper part of the gel), p.MT-ND1 (middle part
of the gel) and p.MT-ND3 (lower part of the gel) polypeptides
were selected for quantification.
Statistics analysis
The statistical package StatView 6.0 was used to perform all the
statistics. Data for mean, standard deviation and sample size
[M+SD (N)] are presented. The normal distribution was
checked by the Kolmogorov–Smirnov test. For those normal
variables, the unpaired two-tailed t-test was used to compare
parameters. Those variables that were not normally distributed
(ATP in glucose and in glucose plus oligomycin; genotype
shifting) were analyzed by the non-parametric Mann–
Whitney U-test (ATP in glucose and in glucose plus oligomy-
cin) or the Wilcoxon signed-rank test (genotype shifting).
P-values of ,0.05 were considered statistically significant.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG online.
ACKNOWLEDGEMENTS
We would like to thank Belen Revilla, Magdalena Carreras,
Santiago Morales and Dolores Herrero-Martı´n for their help
in the laboratory; and Mamen Martı´n, Francesco Acquadro
and Dr Juan Cigudosa from the Centro Nacional de Investiga-
ciones Oncolo´gicas (CNIO) for the karyotyping analysis.
Conflict of Interest statement. None declared.
FUNDING
This work was supported by grants from Instituto de Salud
Carlos III-FIS (PI07-0045 and PI08-0264) and Diputacio´n
General de Arago´n (Grupos Consolidados B33, PM-083/
2008 and PIPAMER0901). The CIBERER is an initiative of
the ISCIII.
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