Coronary heart disease is associated with a mutation in mitochondrial tRNA

Abstract

Coronary heart disease (CHD) is the leading cause of death worldwide. Mitochondrial genetic determinant for the development of CHD remains poorly explored. We report there the clinical, genetic, molecular and biochemical characterization of a four-generation Chinese family with maternally inherited CHD. Thirteen of 32 adult members in this family exhibited variable severity and age-at-onset of CHD. Mutational analysis of their mitochondrial genomes identified the tRNA(Thr) 15927G>A mutation belonging to the Eastern Asian haplogroup B5. The anticipated destabilization of a highly conserved base-pairing (28C-42G) by the 15927G>A mutation affects structure and function of tRNA(Thr). Northern analysis revealed ≈80% decrease in the steady-state level of tRNA(Thr) in the mutant cell lines carrying the 15927G>A mutation. The 15927G>A mutation changed the conformation of tRNA(Thr), as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. In addition, ∼39% reduction in aminoacylated efficiency of tRNA(Thr) was observed in mutant cells derived from this Chinese family. An in vivo mitochondrial protein labeling analysis showed ∼53% reduction in the rate of mitochondrial translation in mutant cells. The impaired mitochondrial protein synthesis leads to defects in overall respiratory capacity or malate/glutamate-promoted respiration or succinate/glycerol-3-phosphate-promoted respiration, or N,N,N',N'-tetramethyl-pphenylenediamine/ascorbate-promoted respiration in mutant cells. An increasing production of reactive oxygen species was observed in the mutant cells carrying the 15927G>A mutation. These results provide the direct evidence that the tRNA(Thr) 15927G>A mutation is associated with CHD. Our findings may provide new insights into pathophysiology and intervention targets of this disorder.

Figure 1.

A Han Chinese pedigree with CHD. Affected individuals are indicated by filled symbols. An arrowhead denotes proband (BJH15-III-7).

Figure 2.

Identification and qualification of the 15927G>A mutation in the mitochondrial tRNA^Thr gene. (A) Partial sequence chromatograms of tRNA^Thr gene from an affected individual (III-7) and a married-in-control (II-8). An arrow indicates the location of the base changes at position 15927. (B) The location of the 15927G>A mutation in the mitochondrial tRNA^Thr. Cloverleaf structure of human mitochondrial tRNA^Thr is derived from Suzuki et al. (22). Arrow indicates the position of the 15927G>A mutation. (C) Quantification of 15927G>A mutation in the tRNA^Thr gene of mutants and controls derived from the Chinese families. PCR products amplified from total DNA isolated from whole blood of subjects were digested with HpaII and analyzed by electrophoresis in a 7% polyacrymide gel stained with ethidium bromide.

Figure 3.

Northern-blot analysis of mitochondrial tRNA. (A) Equal amounts (2 μg) of total mtRNA samples from the various cell lines were electrophoresed through a denaturing polyacrylamide gel, were electroblotted and were hybridized with DIG-labeled oligonucleotide probes specific for the tRNA^Thr, tRNA^Lys, tRNA^Leu(CUN), tRNA^Ser(AGY) and tRNA^His, respectively. (B) Quantification of mitochondrial tRNA levels. Average relative tRNA^Thr content per cell, normalized to the average content per cell of tRNA^Lys, tRNA^Leu(CUN), tRNA^Ser(AGY) and tRNA^His in cells derived from five affected subjects carrying the 15927G>A mutation and three Chinese controls (A6, IV-4 and III-8) lacking the mutation. The values for the latter are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations of each tRNA content in each cell line and three determinations of the content of reference tRNA marker in each cell line. The error bars indicate two standard errors of the mean (SEM). P indicates the significance, according to the t-test, of the difference between mutant mean and control mean.

Figure 4.

In vivo aminoacylation assay for mitochondrial tRNA. (A) Equal amounts (2 µg) of total mitochondrial RNA purified from various cell lines under acid conditions were treated with electrophoresis at 4°C through an acid (pH 5.1) 10% polyacrylamide /7 m urea gel, electroblotted onto a positively charged nylon membrane and hybridized with DIG-labeled oligonucleotide probes specific for mitochondrial tRNA^Thr. The blots were then stripped and rehybridized with DIG-labeled probes for tRNA^Lys, tRNA^Leu(CUN) and tRNA^Ser(AGY), respectively. (B) In vivo aminoacylated proportion of tRNA^Thr in the mutant and controls. The calculations were based on three independent determinations. The error bars indicate two SEs.

Figure 5.

Mitochondrial translation assay. (A) Electrophoretic patterns of the mitochondrial translation products of lymphoblastoid cell lines and of 143B.TK^– cells labeled for 30 min with [³⁵S]methionine in the presence of 100 µg/ml of emetine. Samples containing equal amounts of total cellular protein (30 µg) were run in SDS/polyacrylamide gradient gels. COI, COII and COIII indicate subunits I, II and III of cytochrome c oxidase; ND1, ND2, ND3, ND4, ND4L, ND5 and ND6, subunits 1, 2, 3, 4, 4L, 5 and 6 of the respiratory chain reduced nicotinamide-adenine dinucleotide dehydrogenase; A6 and A8, subunits 6 and 8 of the H⁺-ATPase; and CYTb, apocytochromeb. (B) Quantification of the rates of the mitochondrial translation labeling. The rates of mitochondrial protein labeling, as detailed elsewhere (26), were expressed as percentages of the value for 143B.TK^– in each gel, with error bars representing 2 SEMs. A total of three independent labeling experiments and three electrophoretic analyses of each labeled preparation were performed on cell lines. Graph details and symbols are explained in the legend to Figure 3.

Figure 6.

Respiration assays. (A) Average rates of endogenous O₂ consumption per cell measured in different cell lines are shown, with error bars representing two SE. A total of four to six determinations were made on each of lymphoblastoid cell lines. (B) Polarographic analysis of O₂ consumption in digitonin-permeabilized cells of the various cell lines using different substrates and inhibitors. The activities of the various components of the respiratory chain were investigated by measuring on 1 × 10⁷ digitonin-permeabilized cells the respiration dependent on malate/glutamate, on succinate/G3P and on TMPD/ascorbate. A total of three to six determinations were made on each of the lymphoblastoid cell lines. Graph details and symbols are explained in the legend to Figure 3. mal/glu, malate/glutamate-dependent respiration; succ/G-3-P, succinate/G3P-dependent respiration; and asc/TMPD, TMPD/ascorbate-dependent respiration. Graph details and symbols are explained in the legend to Figure 3.

Figure 7.

The ROS production assays. The rates of production in ROS from five affected matrilineal relatives and three control individuals were analyzed by BD-LSR II flow cytometer system with or without H₂O₂ stimulation. The relative ratio of intensity (stimulated versus unstimulated with H₂O₂) was calculated. The average of three determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure 3.