Functional Differences between Mitochondrial Haplogroup T and Haplogroup H in HEK293 Cybrid Cells

<div><h3>Background</h3><p>Epidemiological case-control studies have revealed associations between mitochondrial haplogroups and the onset and/or progression of various multifactorial diseases. For instance, mitochondrial haplogroup T was previously shown to be associated with vascular diseases, including coronary artery disease and diabetic retinopathy. In contrast, haplogroup H, the most frequent haplogroup in Europe, is often found to be more prevalent in healthy control subjects than in patient study groups. However, justifications for the assumption that haplogroups are functionally distinct are rare. Therefore, we attempted to compare differences in mitochondrial function between haplogroup H and T cybrids.</p> <h3>Methodology/Principal Findings</h3><p>Mitochondrial haplogroup H and T cybrids were generated by fusion of HEK293 cells devoid of mitochondrial DNA with isolated thrombocytes of individuals with the respective haplogroups. These cybrid cells were analyzed for oxidative phosphorylation (OXPHOS) enzyme activities, mitochondrial DNA (mtDNA) copy number, growth rate and susceptibility to reactive oxygen species (ROS). We observed that haplogroup T cybrids have higher survival rate when challenged with hydrogen peroxide, indicating a higher capability to cope with oxidative stress.</p> <h3>Conclusions/Significance</h3><p>The results of this study show that functional differences exist between HEK293 cybrid cells which differ in mitochondrial genomic background.</p> </div

Cell survival after treatment with H₂O₂.

<p>Cell survival was measured 24 hours after H₂O₂ treatment and calculated as a percentage of the ratio between treated and untreated cells (% cell survival). (A) Comparison of HEK H (n = 3; gray bars) and HEK T (n = 3; black bars) cybrids at 250 µM to 475 µM H₂O₂ in glucose medium without serum and without sodium pyruvate. (B) Comparison of HEK H (n = 3; gray bars) and HEK T (n = 3; black bars) cybrids at 100 µM to 250 µM H₂O₂ in galactose medium without serum and without sodium pyruvate. Mean values of % cell survival are given; error bars: standard deviation; *p<0.05.</p

Growth curves of mitochondrial haplogroup-specific cybrid cells.

<p>The number of cells on the days given were normalized to the number of cells on day two and determined as growth rate. (A) Comparison of HEK H (n = 3; gray circles) and HEK T (n = 3; black squares) cybrids at days three to seven in glucose medium. (B) Comparison of HEK H (n = 3; gray circles) and HEK T (n = 3; black squares) cybrids at days three to seven in galactose medium. Mean values of growth rates are given; error bars: standard deviation; *p<0.05.</p

Enzymatic activities of citrate synthase and oxidative phosphorylation complexes I – V in haplogroup H and T cybrid cells.

a<p>Values are given as mean ± standard deviation (SD).</p>b<p>P-value: Independent samples t-test.</p>c<p>Reported previously in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052367#pone-0052367-g002" target="_blank">Figure 2</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052367#pone.0052367-Mueller2" target="_blank">[31]</a>.</p><p>Enzymatic activity measurements were made on isolated mitochondria of cells grown in glucose medium with antibiotics, and on cells with five to 15 passages after the cybridization process.</p

Results of TaqMan qPCR analysis of HEK H and HEK T cybrid competitive co-cultures.

<p>After 10, 20 and 30 days (d10, d20, d30) of co-culture, isolated DNA of the cell mixtures was analyzed using TaqMan qPCR. ΔC_t values were calculated by subtraction of the mean C_t value of the FAM signal (probe recognizing haplogroup T) from the mean C_t value of the VIC signal (probe recognizing haplogroup H). ΔΔC_t values were calculated by subtraction of the mean ΔC_t values of the original cell mixtures (n = 36; day zero) from the mean ΔC_t values of all co-cultures at days 10, 20 or 30 (n = 36; except for d30 in galactose: n = 35). Dominance of haplogroup H results in a negative ΔΔC_t value and is presented as gray bars, whereas dominance of haplogroup T results in a positive ΔΔC_t value and is presented as black bars. (A) ΔΔC_t values of competitive co-cultures cultivated in glucose medium, at days 10, 20 and 30. (B) ΔΔC_t values of competitive co-cultures cultivated in galactose medium, at days 10, 20 and 30. Mean ΔΔC_t values are given; error bars: standard deviation.</p

Figure 1. Phylogenetic tree of haplogroup H and T subsets.

<p>The phylogenetic tree was constructed according to phylotree.org <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052367#pone.0052367-vanOven1" target="_blank">[32]</a>. ^aC16296T did not appear in the mtDNA sequence of cybrid T1. ^bBases of the Revised Cambridge Reference Sequence that appear in HEK T cybrids as polymorphisms diagnostic for non-H haplogroups (m.73A>G, m.2706A>G, m.7028C>T, m.11719G>A and m.14766C>T).</p

Mitochondrial DNA copy number in cybrid cells.

<p>(A) Comparison of all cybrid clones cultivated in glucose medium and galactose medium. (B) Comparison of HEK H and HEK T cybrids cultivated in glucose medium. (C) Comparison of HEK H and HEK T cybrids cultivated in galactose medium. Mean values of copy numbers are given; error bars: standard deviation; *p<0.05.</p

Frequencies (%) of control region polymorphisms >5% in patients with CNV and in controls as well as the corresponding odds ratios and 95% confidence intervals.

a<p>mtDNA = mitochondrial DNA.</p>b<p>CNV = choroidal neovascularisation.</p>c<p>n: number of individuals with the respective polymorphism.</p>d<p>P-value: Pearson chi-square or Fisher's exact test.</p>e<p>CI = confidence interval.</p>f<p>adjusted for age and sex by logistic regression analysis.</p

Comparison of age-related macular degeneration case-control studies in the literature with the present study.

a<p>mtDNA = mitochondrial DNA.</p>b<p>According to PhyloTree.org <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030874#pone.0030874-vanOven1" target="_blank">[21]</a>.</p>c<p>P-values: present study: adjusted for age and sex. Jones et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030874#pone.0030874-Jones1" target="_blank">[12]</a>: adjusted for age, sex and current smoking. Canter et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030874#pone.0030874-Canter1" target="_blank">[13]</a>: adjusted for sex and three nuclear polymorphisms (CFH-Complement Factor H gene, rs1061170; LOC387715, rs10490924; APOE, ApoE2 allele). Udar et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030874#pone.0030874-Udar1" target="_blank">[14]</a>: no adjustment. SanGiovanni et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030874#pone.0030874-SanGiovanni1" target="_blank">[15]</a>: adjusted for age, sex and smoking.</p>d<p>CI = confidence interval.</p

Frequencies (%) of mitochondrial haplogroups in Caucasian patients with CNV and in controls.

a<p>CNV = choroidal neovascularization.</p>b<p>P-value: Pearson chi-square or Fisher's exact test.</p>c<p>Haplogroups that could not be assigned to one of the nine major European haplogroups by the SNP combination.</p