Deficiencies in tRNA synthetase editing activity cause cardioproteinopathy

Abstract

Misfolded proteins are an emerging hallmark of cardiac diseases. Although some misfolded proteins, such as desmin, are associated with mutations in the genes encoding these disease-associated proteins, little is known regarding more general mechanisms that contribute to the generation of misfolded proteins in the heart. Reduced translational fidelity, caused by a hypomorphic mutation in the editing domain of alanyl-tRNA synthetase (AlaRS), resulted in accumulation of misfolded proteins in specific mouse neurons. By further genetic modulation of the editing activity of AlaRS, we generated mouse models with broader phenotypes, the severity of which was directly related to the degree of compromised editing. Severe disruption of the editing activity of AlaRS caused embryonic lethality, whereas an intermediate reduction in AlaRS editing efficacy resulted in ubiquitinated protein aggregates and mitochondrial defects in cardiomyocytes that were accompanied by progressive cardiac fibrosis and dysfunction. In addition, autophagic vacuoles accumulated in mutant cardiomyocytes, suggesting that autophagy is insufficient to eliminate misfolded proteins. These findings demonstrate that the pathological consequences of diminished tRNA synthetase editing activity, and thus translational infidelity, are dependent on the cell type and the extent of editing disruption, and provide a previously unidentified mechanism underlying cardiac proteinopathy.

Keywords: cardiomyopathy; mistranslation; protein misfolding; protein quality control; translational fidelity.

Fig. 1.

Generation of editing-defective AlaRS mouse models. (A, Left) Misacylation of tRNA^Ala with serine by mouse wild-type (WT) or C723A AlaRS. (Right) Relative level of mischarged Ser-tRNA^Ala in the presence of WT, C723A, or equal molar ratio of WT and C723A AlaRS at 15 min. Assays were run in duplicate, and points (mean ± SD) were averaged from two experiments. (B) To generate the conditional knock-in allele of AlaRS^C723A, a loxP-flanked transcriptional stop cassette was inserted in intron 1, and the TGT codon encoding cysteine at position 723 in exon 15 was replaced with a GCG alanine codon. Aars transcription remains off until the stop cassette is deleted via Cre expression. The Southern blot probe (5′ probe), the size of the left and right “arm” of the targeting vector, and the distance between the neo cassette and exon 15 are indicated. (C) Body weight of WT (n = 13), Aars^sti/sti (sti/sti; n = 8), and Aars^stop/sti (stop/sti; n = 11) mice at postnatal ages (P) 14 and P21. Values represent mean ± SD; ***P < 0.0001 (one-way ANOVA). (D) Photograph of 2-mo-old WT, Aars^sti/sti, and Aars^stop/sti mice. Note the dorsal alopecia characteristic of the Aars^stop/sti mouse.

Fig. 2.

Cardiac abnormalites in Aars^stop/sti mice. (A) Ratio of the heart weight to femur length (HW/FL) from 10-mo-old WT (n = 11), Aars^sti/sti (sti/sti; n = 6), and Aars^stop/sti (stop/sti; n = 8) mice. Values represent mean ± SD; *P < 0.01 (one-way ANOVA). (B) Hematoxylin and eosin staining of longitudinal sections of hearts from 10-mo-old mice with the indicated genotypes. (Scale bar, 2 mm.) (C–F) Masson’s trichrome stain of heart sections from 4- (C) and 10-mo-old (D) Aars^stop/sti and 10-mo-old Aars^sti/sti (E) and WT (F) mice. Higher magnification photos of a representative area of each image are shown in the Lower panel. Note the high levels of interstitial fibrosis (blue; white arrows) and perivascular fibrosis (blue; black arrows) in Aars^stop/sti but not Aars^sti/sti or WT hearts. (Scale bars, 200 μm and 60 μm for the Upper and Lower panel, respectively.) (G) Quantification of fibrotic areas (as a percentage of total area). Values are mean ± SD; *P < 0.01 (unpaired t test) and **P < 0.001 (one-way ANOVA); n = 4 mice for each data point. (H) Ejection fraction (EF%; Left graph) and fractional shortening (FS%; Right graph) of 10-mo-old WT (n = 15), Aars^sti/sti (n = 6), and Aars^stop/sti (n = 9) mice. Values are mean ± SD; ***P < 0.0001 (one-way ANOVA).

Fig. 3.

Formation of misfolded protein aggregates in Aars^stop/sti mutant cardiomyocytes. (A) Immunofluorescence with antibodies to ubiquitin on heart sections from 10-mo-old WT and Aars^stop/sti mice. Nuclei are counterstained with Hoechst dye (blue). Higher magnification photos of Aars^stop/sti cardiomyocytes are shown in the Lower panel. Arrowheads indicate myofibers with intense ubiquitin signals and reduced fiber diameter. An arrow indicates a cardiomyocyte with elevated ubiquitin and vacuoles. (B–D) High-magnification confocal microscopic images showing colocalization of p62 with ubiquitin (B), LC3 (C), and Hsp70 (D) within the cytoplasm of representative cardiomyocytes of 2-mo-old Aars^stop/sti mice. [Scale bars, (A, Upper and Middle panels) 100 μm, (A, Lower panel) 20 μm, and (B–D) 10 μm.]

Fig. 4.

Ultrastructural changes in Aars^stop/sti cardiomyocytes. (A–I) Representative transmission electron microscope images of hearts from 2-mo- (B–E, G, and H) and 5-mo-old (A and F) Aars^stop/sti mice and 2-mo-old WT (I) mice. (A) Formation of large protein aggregates (arrowheads) that disrupt myofibril (Mf) structures. (B and C) Accumulation of autophagic vacuoles (white arrow, early autophagosome; black arrow, autolysosome) accompanied by loss of Mf structures. (D–F) Multilamellar bodies (MLBs, arrows), products of active autophagy and a hallmark of insufficient lysosomal activity, are also present. (G) Defective mitochondria (Mt) with disrupted cristae (arrowheads). (H) Disrupted sarcoplasmic reticulum (arrowheads) in Aars^stop/sti cardiomyocytes. [Scale bars, (A, B, and D–I) 1 μm and (C) 1 μm.]