Pathogenesis And Tissue Specificity Of 15927G>A Mutation In Mitochondria Associated With Coronary Artery Disease

Cardiovascular disease is one of the serious diseases that plague humans all over the world.Among them,the prevalence and mortality of coronary heart disease are high.According to previous research of our lab,the mitochondrial m.15927G>A mutation altered the 42nd nucleotide of mitochondrial tRNATh1.which may cause coronary heart disease and showed typical maternal genetic characteristics in the BJH15 family.However,the pathophysiology underlying these tRNA mutations,specifcally the tissue specifc effect,remains poorly understoodIn this study,we obtained wild-type and Lutant tRNAThr by in vitro transcription to investigate the effect of the mutation on the structure and stability of tRNAThr.In the classic tRNA clover model,m.15927G>A mutation disrupted the 28C-42G base pair of the mitochondrial tRNAThr located in the anticodon arm.Using molecular dynamics simulations,we found that the substitution of 42nd nucelotide led to the instability of mitochondrial tRNAThr,which was verified by the reduction of melting temperature,as well as polyacrylamide gel electrophoresis.The decrease of the aminoacylation and t6A37 modification of mitochondrial tRNAThr were also found by[14C]-tagged threonine biochemical experiments in vitro.The results indicated that this mutant site had an effect on the structural stability of mitochondrial tRNAThr.To further investigate the role of this mutation in the development of coronary heart disease,we constructed a cybrid mitochondrial HUVEC(human umbilical vein endothelial cell)cell line carrying m.15927G>A mutation using immortalized lymphocytes from patient IV-3 in the BJH15 family and HUVEC.The rate of tRNAThr polyacrylamide gel electrophoresis extracted from the mutant was significantly lower than control group,which was consistent with the experimental results of in vitro transcription.Some proteins in the mitochondrial respiratory chain are translated by the mitochondrial tRNAiso the disturbed tRNATGL metabolism forces the mitochondria to have various physiological functions impaired,including mitochondrial oxygen consumption,membrane potential and the production of superoxide.Fluorescence immunostaining results also showed that a large amount of cytochrome c flowed out into the cytoplasm from mitochondria,indicating that m.15927G>A promoted cell apoptosis.Through the scratch healing experiment and angiogenesis experiment,we verified that the ability of proliferation and migration of the mutant cybrid was decreased.These tissue specificity studies provide a basis for the occurrence of coronary heart disease.