MTO1-deficient mouse model mirrors the human phenotype showing complex I defect and cardiomyopathy

Abstract

Recently, mutations in the mitochondrial translation optimization factor 1 gene (MTO1) were identified as causative in children with hypertrophic cardiomyopathy, lactic acidosis and respiratory chain defect. Here, we describe an MTO1-deficient mouse model generated by gene trap mutagenesis that mirrors the human phenotype remarkably well. As in patients, the most prominent signs and symptoms were cardiovascular and included bradycardia and cardiomyopathy. In addition, the mutant mice showed a marked worsening of arrhythmias during induction and reversal of anaesthesia. The detailed morphological and biochemical workup of murine hearts indicated that the myocardial damage was due to complex I deficiency and mitochondrial dysfunction. In contrast, neurological examination was largely normal in Mto1-deficient mice. A translational consequence of this mouse model may be to caution against anaesthesia-related cardiac arrhythmias which may be fatal in patients.

Figure 1. Generation of Mto1 knockdown mice by gene trap mutagenesis.

(A) Integration of gene trap vector U3CEO in intron 6 of Mto1. Arrows indicate amplificons for RT-PCR. (B) RT-PCR of heart tissue shows reduced levels of Mto1 transcripts using primers covering sequences 5′ or 3′ to the integration site, ***p<0.001. (C) Western blot showing a clear reduction of MTO1 protein in Mto1−/− mouse embryonic fibroblasts as compared to Mto1+/+ controls.

Figure 2. Behavioral analysis of Mto1 mutant mice using a modified holeboard.

Mto1−/−mice show increased horizontal locomotor activity (A) as well as increased mean velocity (B) as compared to controls. Anxiety-related behavior measured via the time on board (C) and group contact (D) was also significantly changed; object exploration was increased (E) and acoustic startle response decreased (F), *p<0.05; **p<0.01.

Figure 3. Cardiovascular analysis.

(A) Heart rate was reduced in Mto1−/− mutants (white bars) as compared to controls (black bars) at different ages and under different conditions in three cohorts of mice (4, 10 and 15 months old). (B). Electrocardiography in anaesthetized mice showing increased QRS complex duration in Mto1−/− mutant mice as compared to controls (C−F) Echocardiography in awake mice showing increased left ventricular internal diameter in systole and diastole in Mto1−/− mutant mice (C) but no significant differences in fractional shortening (D), left ventricular posterior wall thickness (E) and intraventricular septum thickness (F), *p<0.05; ***p<0.001.

Figure 4. Heart weight related to body weight was significantly increased in Mto1−/− mutant mice as compared to controls, both in 5 months old mice (n = 15, *p<0.05) and as a trend in 10 months old mice (n = 8, n.s.).

Figure 5. Morphological analysis of heart.

(A, C) Focal myocard degeneration in the left ventricle with myofiber atrophy(arrows) and fibrosis(asterisk) in a 303 day old Mto1−/− mouse (A) as compared to control (C) (Masson stain, scale bar 200 µm). (B, D) Focal vacuolar degeneration (arrowheads) of myofibers in the heart of a 303 day old MTO1−/− mouse (B) as compared to control (D) (H&E stain, scale bar 200 µm). (E, F) Transmission electron microscopy showing degeneration of heart muscle in Mto1−/−mice (upper panel E) as compared to wild-type (lower panel F) at different magnifications (as indicated).

Figure 6. Molecular analysis of mouse tissue.

(A) mtDNA copy number in hearts of Mto1−/− mutant mice was significantly reduced as compared to controls (*p<0.05) (B) Respiratory chain complex I was visibly reduced in a Western Blot using a cocktail of OXPHOS antibodies (Mitosciences.com) in Mto1−/− mutant mice as compared to controls while respiratory chain complexes II, II, IV and V were unchanged. (C) Enzymatic activity of complex I in heart tissue lysates was only mildly reduced in Mto1−/− mutant mice but (D) maximal respiratory capacity was significantly reduced in freshly isolated mitochondria (**p<0.01).

Figure 7. Blue Native Gel electrophoresis of heart tissue.

Mitochondrial complexes from wild-type and Mto1−/− heart homogenates were solubilized with dodecyl-β-d-maltoside and separated by BNE. (A) Coomassie stained 1-D BNE gels, (B) 1-D BNE/complex I in gel activity stain were used for (C) densitometric quantification of mitochondrial complexes. Complex values from 3 wild-type and 3 Mto1−/− mice are expressed as percent of the wild-type mean. The Complex I defect is confirmed. Assignment of Coomassie stained complexes: I, complex I or NADH dehydrogenase, V, monomeric complex V or ATP synthase; III, complex III or cytochrome c reductase; IV, complex IV or cytochrome c oxidase; Ia, in-gel activity stain; *, significant differences (p<0.05); **, significant differences (p<0.01) by Students t test; error bars indicate standard deviation (SD).

Figure 8. Mouse embryonic fibroblasts experiments.

In vivo pulse labeling of mitochondrial translation products in WT and Mto1−/− showing a clear loss of signal intensity for complex I subunits ND5, ND6 and ND3, while all other mtDNA-encoded proteins showed a trend to increased levels.