Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy

Abstract

Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by Tars2), disruption of which accumulated Ser-tRNA^Thr and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.

Keywords: aminoacyl-tRNA synthetase; cardiomyopathy; editing; mitochondria; tRNA.

Fig. 1.

mmtThrRS editing defects lead to Thr-to-Ser misincorporation during mitochondrial translation in vivo. (A) Schematic showing the NIH3T3-MU cell line construction by CRISPR/Cas9-mediated homologous recombination. DNA sequencing of the mmtThrRS gene in NIH3T3-MU cells is shown. Codons His¹³⁸ and His¹⁴² (CAC and CAT) have been changed to GCC and GCT Ala codons, respectively. (B) The amount of each amino acid moiety attached to mtRNA^Thr in the two cell lines was determined by LC–MS/MS. The results are reported as the averages of two independent trials with the SD indicated. (C) LC–MS analysis showing Thr-to-Ser misincorporation during mitochondrial translation in the NIH3T3-MU cell line.

Fig. 2.

Mitochondrial translation, biogenesis, and morphology were impaired in the NIH3T3-MU cell line. (A and B) Relative level of several proteins in various OXPHOS complexes. (C) Enzymatic activity levels of mitochondrial oxidative respiratory chain complexes. (D) OCR of NIH3T3-WT (blue) and NIH3T3-MU (red) cells. Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; R+A, rotenone + antimycin A. (E) Relative level of proteins in mitochondrial replication, transcription, and translation. (F) Relative level of mitochondrial morphology-associated proteins. (G) Representative TEM images (Left) and quantification (Right) of mitochondria in NIH3T3-WT and NIH3T3-MU cells. Swollen mitochondria are marked by red arrowheads. The data are presented as the mean ± SEM (n = 3 to 12, ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗∗P < 0.0001; NS, not significant). Student’s t test was performed.

Fig. 3.

NIH3T3-MU cell lines showed reduced cell proliferation due to cell cycle arrest. (A) Cell growth determination of NIH3T3-WT (blue) and NIH3T3-MU (red) cells over time. (B) Cell cycle analysis performed with flow cytometry. (C) GO enrichment analysis of the subset of dysregulated genes in the RNA-seq data. Cell cycle relative BP subsets were marked with red. (D) The cell cycle pathway was enriched by ES enrichment. (E) Relative level of p21. (F) A higher level of p21-Cdk2 interaction in NIH3T3-MU cells was observed by coimmunoprecipitation with an anti-Cdk2 antibody. (G) Relative level of pAtm and p53. The data are presented as the mean ± SEM (n = 3 to 4, ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; NS, not significant). Student’s t test was performed.

Fig. 4.

Oxidative stress contributed to NIH3T3-MU cell cycle arrest. (A) Fluorescence intensity (Left) and level (Right) of ROS measured using a DCF fluorescent probe in vivo. (B) Relative level of the ROS-related proteins Nox4 (Left) and Sod2 (Right). (C) Number of NIH3T3-WT and NIH3T3-MU cells before and after NAC treatment. (D) Analysis of the NIH3T3-MU cell cycle in the absence or presence of NAC treatment, as determined via flow cytometry. Ratios of cells in various phases to the total number of cells were calculated and compared with those of NIH3T3-WT cells. (E) Relative protein level of p21 and p53 in NIH3T3-WT and NIH3T3-MU cells before and after NAC treatment. (F) OCR in cells before [NIH3T3-WT (blue), NIH3T3-MU (red)) and after (NIH3T3-WT + NAC (green), NIH3T3-MU (yellow)] NAC treatment. (G) Activities of Complexes I and III in NIH3T3-MU cells after NAC treatment. The data are presented as the mean ± SEM (n = 3-12, ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001; NS, not significant). Student’s t test was performed.

Fig. 5.

Cardiomyocyte-restricted mmtThrRS-H138A/ H142A mutations resulted in cardiac dysfunction. (A) mmtThrRS-H138A/H142A was expressed in cardiomyocytes by flipping the floxed mmtThrRS-WT cassette between the mmtThrRS gene promoter and mmtThrRS-MU and crossing the mice carrying this mutation with mice harboring Myhc6 promoter-driven Cre recombinase. Myhc6-Cre; Mice expressing mmtThrRS-H138A/H142A in cardiomyocytes were designated Tars2^Flox/Flox-Cre (mutant) mice. Litter-mate mice expressing wild-type mmtThrRS in cardiomyocytes were designated Tars2^Flox/Flox (control, wild-type) mice. (B) The genotyping results after crossing Myhc6 promoter-Cre mice with Tars2^Flox/Flox mice; the outcome followed Mendelian laws. (C) Survival curves for the wild-type (n = 13) and the mutant mice (n = 16). (D and E) LVEF (Tars2^Flox/Flox, n = 7; Tars2^Flox/Flox-Cre, n = 6) and LVFS (Tars2^Flox/Flox, n = 7; Tars2^Flox/Flox-Cre, n = 4) measurements of the wild-type and the mutant mice over time. (F) Representative hematoxylin and eosin (H&E) staining of the wild-type and the mutant mouse hearts. (G) Representative image showing Masson’s trichrome staining of the wild-type and the mutant mouse hearts. The data are presented as the mean ± SEM (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; NS, not significant). Student’s t test was performed.

Fig. 6.

Increased ROS levels and inhibited proliferation of cardiomyocytes from the mutant mice. (A) Mitochondria (indicated by a black arrow) were examined by TEM (×30,000). Representative images (Left) showing disorganized mitochondrial cluttering (indicated by a red arrow) in the mutant mice hearts (scale bar: 0.2 µm.) The quantification (Right) of the surface area of mitochondria from the wild-type (n = 128) and the mutant (n = 73) mice. (B) Representative images showing DHR 123 staining of heart tissue sections from the wild-type and the mutant mice. (C) Representative images showing WGA staining (Left) and quantification (Right) of parameters measured in the heart tissue sections prepared from the wild-type (n = 3) and the mutant (n = 3) mice. (D) Bright-field images (Upper) and number (Lower) of isolated mouse cardiomyocytes (the wild-type and the mutant mice, n = 3). (E) The types (Left) and quantification (Right) of the nuclei in the isolated mouse cardiomyocytes from the wild-type (n = 3) and the mutant (n = 4) mice. The data are presented as the mean ± SEM (^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001). Student’s t test was performed.

Fig. 7.

Proposed model showing how mmtThrRS editing deficiency leads to cellular and heart dysfunctions. Mutations in His¹³⁸ and His¹⁴² abolished the posttransfer editing function of mmtThrRS, leading to gain-of-function effects that increased the Ser-mtRNA^Thr mischarging, in addition to Thr-mtRNA^Thr charging, by mmtThrRS-H138A/H142A. The accumulation of Ser-mtRNA^Thr led to abundant Thr-to-Ser misincorporation during mitochondrial mRNA translation. This disruption to translation impaired the assembly and function of the mitochondrial respiratory chain complexes and caused oxidative stress, which subsequently led to cell cycle arrest in the G0/G1 phase. This comprehensive mitochondrial and cellular dysfunction likely affected cardiomyocytes after mutations of His¹³⁸ and His¹⁴², which were associated with dilated cardiomyopathy in heart-specific mmtThrRS editing–defective mice. This figure was created with BioRender.