The proband (Q5-III-3) was first diagnosed with CHD upon presenting at the Cardiology Clinic of affiliated hospital of Qingdao university at age 40, after which he underwent a full medical evaluation. The patient was diagnosed with hypertension (159/99 mmHg), significant ischemia (65% narrowing was evident upon coronary angiography), and high cholesterol (LDL-C = 159 mg/dL, TC = 232 mg/dL). The patient was not affected by any other comorbid conditions such as diabetes or neurological disease. When family members of this patients were evaluated for these same conditions, 5 were diagnosed with all three of these conditions (Figure 1a and Table 1). In each case, any fathers with CHD had not transmitted it to their children, whereas mothers did transmit it, suggesting matrilineal inheritance consistent with mitochondrial involvement in this inherited CHD risk.
Analysis of mitochondrial mutations
To explore potential mitochondrial mutations linked with inherited CHD risk, we sequenced the mitochondrial genome of this proband patient Q5-III-3. A total of 45 mutations were evident in their mitochondrial genome upon comparison with the Cambridge consensus sequence, and the mitochondrial haplogroup for this patient was identified to be M7b’c (Figure 2). As shown in Table 2, of these 45 variants, 19 were known silent variants, 14 were known D-loop variants, 8 were known missense mutations affecting protein-coding genes, 2 were known 12S rRNA variants, 1 was a known 16s rRNA variant, and one was a novel homoplasmy 15910C>T mutation in the tRNAThr gene (Figure 1B). The detected missense mutations were as follows: 5460G>A (Ala331Thr) in the ND2 gene, 7853G>A (Val90Ile) in the CO2 gene, the 8701A>G (Thr59Ala) in the ATP6 gene, the 10398A>G (Thr114Ala) in the ND3 gene, 12811T>C (Tyr159His) in the ND5 gene, the 14766C>T (Thr>Ile), 14978A>G (Ile78Val) , and m.15326A>G (Thr>Ala) in the CYTB gene. We compared the variance at these mutated RNA residues phylogenetically across 16 different primate species, revealing this tRNAThr 15910C>T mutation to have a 100% conservation index across species, making it more likely to have functional significance when mutated as in this patient. This mutation was also not detected when 136 Chinese control subjects were analyzed.
Mutation leads to decreased mitochondrial tRNAThr levels
We next assessed how this 15910C>T mutation altered the metabolism of tRNAThr, subjecting cybrid cell lines bearing this mitochondrial mutation to Northern blotting using probes specific to this and 3 other tRNAs. We found that tRNAThr levels in these mutant cybrid lines were significantly reduced relative to control wild type cells (Figure 2), with baseline tRNAThr levels in these mutant cells being 65.25% of those in control cells, with 5S RNA used for normalization purposes. In contrast, baseline tRNAHis, tRNAAla, and tRNAGlu levels in these mutant cell lines were unchanged relative to control cells (102.13%, 98.89%, and 107.91%, respectively).
Mutation leads to reduced mitochondrial protein levels
We next performed Western blotting to assess levels of the mtDNA-encoded components of the respiratory complex in cells bearing the 15910C>T mutation or controls. As shown in Fifure 3, e found that mutant cells expressed mitochondrial protein levels that were 19.31% to 31.55% of those in control cells (average =24.96%; P<0.05). These mutated cells also showed clear reductions (18.71%, 26.56%, 37.52%, 33.00% and 39.48%) in 5 polypeptides (ND4, ND1, ND6, CYTB and ATP6), while CO2 levels were not significantly reduced (0.15%) relative to control cells.
Mutation led to decreased complex I and III activity
We further assessed the consequences of this m.15910C>T mutation on oxidative phosphorylation using isolated mitochondrial from our mutant and control cybrid cell lines. We found that complex I and III activity in the 15910C>T mutant mitochondria was 66.72%, and 75.48% that of the activity observed in control cells, whereas no changes in complex II/IV activity were observed(Figure 4A).
Mutation leads to reduced mitochondrial ATP generation
We further assessed the generation of ATP by wild type and mutant cells in an effort to better gauge how this mutation influenced oxidative phosphorylation. To test this, either glycolysis or oxidative phosphorylation were selectively induced in cells via culture with glucose, glucose + oligomycin, or 2deoxy-D-glucose + pyruvate. When cells could only engage in oxidative phosphorylation, mutant cells bearing the 15910C>T mutation ,exhibited ATP production that was 65.68 -67.98% (average: 67.37%) of that in control cells(Figure 4B).
Mutation leads to increased ROS production
We next assessed ROS production in our mutant cybrid cell lines via flow cytometry, comparing baseline staining intensity for each cell line to that upon oxidative stress in order to obtain a ratio corresponding to ROS generation. We observed somewhat increased ROS generation for our mutant cybrid lines bearing the 15910C>T mutation, with ROS production 118.45 - 123.98%, (average: 121.04%) that of control cells(Figure 4C).