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. 2019 Jan 8;29(1):78-90.e5.
doi: 10.1016/j.cmet.2018.08.002. Epub 2018 Aug 30.

Mitochondrial DNA Variation Dictates Expressivity and Progression of Nuclear DNA Mutations Causing Cardiomyopathy

Meagan J McManus  1 , Martin Picard  2 , Hsiao-Wen Chen  1 , Hans J De Haas  3 , Prasanth Potluri  1 , Jeremy Leipzig  1 , Atif Towheed  1 , Alessia Angelin  1 , Partho Sengupta  3 , Ryan M Morrow  1 , Brett A Kauffman  4 , Marc Vermulst  1 , Jagat Narula  3 , Douglas C Wallace  5
Affiliations

Mitochondrial DNA Variation Dictates Expressivity and Progression of Nuclear DNA Mutations Causing Cardiomyopathy

Meagan J McManus et al. Cell Metab. .

Abstract

Nuclear-encoded mutations causing metabolic and degenerative diseases have highly variable expressivity. Patients sharing the homozygous mutation (c.523delC) in the adenine nucleotide translocator 1 gene (SLC25A4, ANT1) develop cardiomyopathy that varies from slowly progressive to fulminant. This variability correlates with the mitochondrial DNA (mtDNA) lineage. To confirm that mtDNA variants can modulate the expressivity of nuclear DNA (nDNA)-encoded diseases, we combined in mice the nDNA Slc25a4-/- null mutation with a homoplasmic mtDNA ND6P25L or COIV421A variant. The ND6P25L variant significantly increased the severity of cardiomyopathy while the COIV421A variant was phenotypically neutral. The adverse Slc25a4-/- and ND6P25L combination was associated with impaired mitochondrial complex I activity, increased oxidative damage, decreased l-Opa1, altered mitochondrial morphology, sensitization of the mitochondrial permeability transition pore, augmented somatic mtDNA mutation levels, and shortened lifespan. The strikingly different phenotypic effects of these mild mtDNA variants demonstrate that mtDNA can be an important modulator of autosomal disease.

Keywords: F(1)F(o)-ATPase; OPA1; adenine nucleotide translocator; aging; cardiomyopathy; complex I; complex IV; mitochondrial DNA; mitochondrial-nuclear interaction; mtDNA instability.

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Figures

Figure 1.
Figure 1.. ANT1-Deficiency Induces Transcriptional Changes Associated with Pathological Remodeling of the Heart
(A and B) Twenty-five most significantly downregulated (>2-fold, total = 459; A) and upregulated (<0.4-fold, total = 363; B) functionally annotated gene categories in Ant1-null myocardium compared with WT. Searched categories include Gene Ontology, Protein Information Resource, Sequence (Seq) Features, Kyoto Encyclopedia of Genes and Genomes, InterPro protein sequence, and analysis classification. Analysis performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.7). (C and D) (C) Fold change in mRNA transcripts from nDNA and (D) mtDNA OXPHOS genes by complex relative to WT (*p < 0.05). (E) Portion of differentially expressed RNA transcripts from mtDNA. n = 4. See also Figure S1 and Table S1.
Figure 2.
Figure 2.. Effects of Six nDNA-mtDNA Anti and COIV421A and ND6P25L Combination Strains on Longevity, Activity, and Thermal Tolerance
(A) Kaplan-Meier analysis of six nDNA-mtDNA combinations. Median lifespan and n are depicted in the chart below. (B) Progeroid morphology evident in ANT1 and ANTI/ND6 mice as early kyphosis at 6 months, which progresses to gray hair, alopecia, and advanced kyphosis at 15 months compared with WT and ANTI/COI. (C) Indirect calorimetry recordings of activity counts during the dark cycle of all strains at 6 months. (D) Core body temperature for 6-month-old mice measured at 22°C (filled symbols; red box) showing reduction of all Ant1-null strains. Core body temperature after 4° Ccold stress (open symbols; blue box) showing that the ANT1 and ANTI/ND6 strains were unable to maintain normal body temperature while the ANTI/COI mice were unaffected by cold stress. *p < 0.02 versus WT; n = 6. See also Figure S2.
Figure 3.
Figure 3.. Progression of Cardiomyopathy and Left Ventricular Mechanics in the nDNA-mtDNA Combination Strains
(A) Correlation between the relative rate of cardiac enlargement (heart weight/body weight) over the lifespan for each strain (p < 0.0001; n = 65–111). (B) Gross morphology of hearts by H&E stain at 12 months of age. (C) Cardiac contractility measured by two-dimensional speckle-tracking echocardiography (2D-STE). Representative strain curves obtained from the B-mode long-axis view of the left ventricle over the cardiac cycle (x axis) showing longitudinal strain (% deformation; y axis) and region of the left ventricle (z axis). Each panel shows 49 regional strain curves topographically extending from infero-lateral base (BaseLat.) toward LV apex and back toward antero-septal base (BaseSep ). Note the progressive variations in magnitude and timing of the strain curves between different nDNA-mtDNA combinations. n = 10–34. See also Table S2.
Figure 4.
Figure 4.. Opposing Effects of mtDNA Variation in Complexes I and IV on Mitochondrial Morphology and Cristae Architecture
(A-F) Representative electron micrographs of (A-F) ventricular cardiomyocytes and (A’-F’) mitochondria from each nDNA-mtDNA combination (n = 3). Scale bars, 2 μm (A-F); 500 nm (A’-F’). Sarcomeric and mitochondrial alignment in (A) WT and (B) COI versus structural disarray in all other nDNA-mtDNA combinations (C-F). (B and B’) Mitochondrial enlargement in COI myocardium, suggestive of hyperfusion or impaired fission (green arrow; B’). Mitochondrial fragmentation, autophagic vesicles, and lipofuscin accumulation (blue and purple arrows) in (C and C’) ND6 and (F and F’) ANTI/ND6 myocardium. (D-F) Mitochondrial proliferation and (D’-F’) cristae abnormalities, paracrystalline inclusions (yellow arrow; E, ANTI/COI), cristolysis (red asterisk; D’, ANT1), and reticular morphology (yellow arrowhead; E’, ANTI/COI), present in all Ant1-null strains. (G-I) (G) Quantification of age-related lipofuscin deposits, normalized to WT. Ultrastructural quantification of (H) mitochondrial content and (I) average size per strain (n = 496–1,541). (J) Percentage distribution of mitochondrial size showing the shift in mitochondrial morphology by the ANTI/ND6 compared with WT. (K) Percent of abnormal mitochondria counted in each strain. (L-O) Average number of mitochondria per mm2 with the following most common defects in cristae: (L) cristolysis, and (M) reticular, (N) partitioning, and (O) circular morphologies. *p < 0.05 versus WT; ^p < 0.001 versus ANT1. Data are represented as means ± SE. See also Figures S3 and S4.
Figure 5.
Figure 5.. Consequences of Mitochondrial-Nuclear Interaction on OPA1 Processing and Mitochondrial OXPHOS Complexes
(A-D)(A) Representative western blot of OPA1 and VDAC from isolated heart mitochondria. Densitometric analysis of (B) total OPA1, (C) long Opal (I-OPA1), and (D) short OPA1 (s-OPA1) isoforms, normalized to VDAC and shown as fold change from WT. (E and F) (E) Complex I (C-1) NADH:quinone oxidoreductase (NQR) activity determined by rotenone-sensitive NADH oxidation in the presence of coenzyme Q1 using isolated heart mitochondria (25 μg). (F) Age-dependent decline in C-I diaphorase activity (dOD/min) determined by C-I immunocaptured from 10 μg of isolated heart mitochondria (**p < 0.0001 versus WT at 6 months; &p < 0.05 versus WT at 18 months). (G) C-I assembly measured by blue native electrophoresis of heart mitochondria (20 μg) and immunodetection with anti-NDUFA1. (H) Citrate synthase (CS) activity per mg myocardial tissue as a marker of mitochondrial content at 6 months (**p < 0.01 versus ANT1). (I) Cytochrome c oxidase (COX) activity from the same samples normalize to CS activity. Data are represented as means ± SE. (J) Resolution of oligomeric states of complex V (C-V) F1F0-ATPase by clear native PAGE. Oligomers were undetectable in Ant1−/− mitochondria solubilized in digitonin (3% w/v). V0, oligomers; Vd, dimers; Vm, monomers; V*a and V*b, partial C-V components. Each well was loaded with 30 μg of mitochondrial protein, evident by the anti-VDAC loading control from a duplicate gel. *p ≤ 0.05, **p < 0.001 versus age-matched WT, except as noted in (F) and (H); n = 3–7. See also Figure S5.
Figure 6.
Figure 6.. Mitochondrial Bioenergetics, ROS Production, and mtPTP Stability in Isolated Cardiac Mitochondria from Six nDNA-mtDNA Genetic Combinations
(A) Mitochondrial membrane potential determined bytetramethylrhodamine methyl ester fluorescence respiring on glutamate and malate (GM), graphed relative to WT. (B) Mitochondrial oxygen consumption rate metabolizing glutamate and malate (GM) in the absence of ADP or uncoupler (state II or LEAK rate). (C) Mitochondrial oxygen consumption rate metabolizing GM in the presence of ADP (state III or P rate). (D) Relative inhibition of respiration by oligomycin in mitochondria during state III. (E) Hydrogen peroxide (H2O2) production detected by Amplex red in isolated mitochondria incubated with GM, rotenone (R), or succinate (Succ) and oligomycin (Oligo). (F) Nitroxidative damage determined by 3-nitrotyrosine (3NT) protein adducts in heart tissue from 12-month-old mice. (G) Ca2+ levels required to activate the mtPTP and collapse the mitochondrial membrane potential. (H) Representative traces of extramitochondrial Ca2+ following 20 μM Ca2+ pulses delivered every 2 min until the spontaneous release of mitochondrial Ca2+, marking the onset of mtPTP opening in WT and ANTI/ND6 mice. (I) Activation of intrinsic apoptosis determined by effector caspase-3 and −7 activities. Data are represented as means ± SE. *p ≤ 0.01 versus WT; ^p ≤ 0.01 versus ANT1; n = 3–5. See also Figure S6.
Figure 7.
Figure 7.. Effect of Mitochondrial-Nuclear Interaction on Somatic mtDNA Mutation Accumulation
(A and B) Long-range amplification of mtDNA(12.7 kb) from each nDNA-mtDNAcombination at 12–15monthsindicating multiple, large-scaledeletions (*p = 0.01; n = 4–6). (C) Linear regression analysis of the frequency of mtDNA deletions per genome detected by qPCR of the most common deletion hotspot flanking two 15-bp repeats in mtDNA of mice from each strain over 4–25 months of age (p < 0.001; n = 8–14). (D and E) Correlation between (D) median lifespan or (E) cardiac function and the mtDNA somatic mutation rate, as determined by random mutation capture assay. See also Figure S7.
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