Research ArticleGENETICS

Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics

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Science Advances  29 Jul 2020:
Vol. 6, no. 31, eaba5345
DOI: 10.1126/sciadv.aba5345
  • Fig. 1 Nonrandom segregation of wild-type mtDNA heteroplasmy.

    Estimation of heteroplasmy shift for the indicated tissue from birth to more than 600 days old. (A) Tissues that shift toward C57 mtDNA. (B) Tissues that shift toward NZB mtDNA. (C) Tissues that do not resolve heteroplasmy or display very slow shifts. Red lines give inferred mean segregation behavior with 95% confidence intervals (n = 119 BL/6C57-NZB mice). Black dots show heteroplasmy data in the given tissue plotted relative to eye (see Materials and Methods), which is inferred to have a low segregation rate and is hence used as an approximate control tissue. Absolute values for each tissue are also shown in fig. S1.

  • Fig. 2 Absence of mtDNA-driven cell competition in chimeric mice.

    Estimation of mtDNA proportion shift using eye as the reference tissue for the indicated tissue from birth to 279-day-old chimeric mice (n = 54 mice; red dots, newborn pups; black dots, 29-49 days; green dots, 65-97 days old; blue dots, 279 day-old mice) generated by morula aggregation of homoplasmic C57 and homoplasmic NZB embryos. Black lines give inferred mean segregation behavior with 95% confidence intervals (shadowed areas). No statistically significant P values were observed after correction for multiple testing.

  • Fig. 3 Analysis of heteroplasmy in brain and cardiac cell populations.

    (A and B) Transformed heteroplasmy shift in isolated astrocytes (A) and neurons (B) using tail as reference tissue. In (A) and (B), red lines correspond to the mean and discontinuous lines show 95% confidence intervals of inferred segregation trajectories. Each dot corresponds to a different animal at the indicated age. (C) Bright-field microscopy of single isolated adult cardiomyocytes. (D) Evaluation of intracellular mtDNA heteroplasmy in single cardiomyocytes from 20-week-old heteroplasmic mice (n = 4) Each dot corresponds to a different cardiomyocyte. Dotted line: average of entire tissue. (E to H) Evaluation of transformed heteroplasmy shift using tail as the reference tissue in isolated cardiac cell populations from heteroplasmic hearts at different ages. (E) Total cardiac tissue, (F) myeloid cells, (G) endothelial cells, and (H) lymphoid cells. Each dot corresponds to a different animal. *P < 0.05 and ****P < 0.0001, linear regression coefficient. (A, B, E, F, G, and H): positive toward mtDNA NZB and negative toward mtDNA C57.

  • Fig. 4 mtDNA segregation is sensitive to modulation of organismal metabolism.

    (A) Tissue-specific segregation patterns in basal and intervention conditions. Triangles indicate magnitude (size), s.e.m. (thickness of lighter border), and direction of the segregation toward NZB mtDNA (red, upward) or C57 mtDNA (blue, downward). Control: Segregation with no intervention. Experimental: Segregation resulting from chemical (A) and genetic (F) perturbations. DCA, n = 22 (age 57 to 159); NAC, n = 16 (age 96 to 273); HFD, n = 25 (age 59 to 188). (B) Rate of segregation of mtDNA in control (red line) and hepatectomized (black line) mice. (C and D) Heteroplasmy in different T cell subsets in 15-week-old (C) naïve and (D) immunized mice. Circles indicate CD8+ (filled) or CD4+ (open) T cells, and the colors indicate the different T cell subsets [naïve (TN), memory (TM), or effector (TE) T cells]. FAO, fatty acid oxidation; M, mouse. Data are representative of two independent experiments. (E) Oxygen consumption rate (OCR) Seahorse profiles for indicated cells (n = 5 BL/6C57-NZB). Oligo, oligomycin; FCCP, carbonilcyanide p-triflouromethoxyphenylhydrazone; ROT, rotenone; AA, antimycin. (F) Tissue-specific segregation patterns in heteroplasmic mouse with the indicated nuclear genetic variations. ND, not described. BL/6C57-NZB:NNTKO: n = 27 ovaries, n = 16 testes, and n = 25 rest of the tissues. BL/6C57-NZB:SCAF1113: n = 11 ovaries, n = 10 testes, and n = 20 to 21 for the rest of the tissues. BL/6C57-NZB:OMA1KO: n = 11 ovaries, n = 11 testes, and n = 14 to 16 for the rest. In (A) and (F), P < 0.05 and ††P < 0.01, at the side for significance affecting the overall pattern of segregation and under triangles for significance at individual tissues. Asterisks: Significant differences from zero segregation (bootstrap percentile method with Bonferroni correction; *P < 0.05, **P < 0.01, and ***P < 0.001).

  • Fig. 5 Impact of heteroplasmy on MAF metabolic performance.

    Estimation of the respiratory performance of homoplasmic and heteroplasmic MAFs at low (A) or high (B) glucose concentration. Left panels show representative profiles of OCRs determined by Seahorse analysis, and right panels show the summary of five to six independent assays per clone. Data are means ± SEM. (C) Assessment of the mitochondrial membrane potential (MMP) of the indicated cell clones. (D) ROS production normalized by the maximum respiration rate (MRR) for the indicated clone. (E) Extracellular acidification rate (ECAR) per oxygen consumption rate (OCR) as an index of glycolytic versus OXPHOS metabolism of the indicated cell clones. Data are means ± SD. Significant differences were assessed by ordinary two-way ANOVA and for multiple comparisons of the means. (F) Dynamics of mtDNA segregation in heteroplasmic MAFs under different nutritional conditions: 25 mM glucose displays linear segregation toward C57 mtDNA, and 5 mM glucose displays linear segregation toward NZB mtDNA. Data are means ± SD. (G) Evaluation of the respiratory performance of control homoplasmic and heteroplasmic MAFs under different carbon source media conditions: 5 mM glucose, 5 mM galactose, and Albumax lipid-rich BSA [fatty acids (FA), 1 mg/ml]. Data are means ± SEM. (H) Representative BNE showing the assembly and superassembly status of respiratory complexes in BL/6C57 and heteroplasmic MAFs under different nutritional conditions (left) and quantification of the proportion of free versus super-assembled complex I (right) (n = 5 in all cases except BL/6C57 (n = 4); significance assessed by two-way ANOVA with Fisher’s least significant difference post hoc test for multiple comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

  • Fig. 6 Evaluation of mtDNA dynamics using in vivo modulation of autophagy.

    The graphic represents the heteroplasmy shift calculated using eye as reference tissue (positive toward mtDNA NZB and negative toward mtDNA C57) after 30 days of treatment with PERK activator (in red) or rapamycin (in green). Blue dots represent control heteroplasmic mice (injected with the vehicle) (n = 9 to 10 12-week-old mice per treatment). Data expressed as means ± SEM. Differences between cases were analyzed by fitting a mixed model with correction for multiple comparisons by controlling false discovery rate.

Supplementary Materials

  • Supplementary Materials

    Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics

    Ana Victoria Lechuga-Vieco, Ana Latorre-Pellicer, Iain G. Johnston, Gennaro Prota, Uzi Gileadi, Raquel Justo-Méndez, Rebeca Acín-Pérez, Raquel Martínez-de-Mena, Jose María Fernández-Toro, Daniel Jimenez-Blasco, Alfonso Mora, Jose A. Nicolás-Ávila, Demetrio J. Santiago, Silvia G. Priori, Juan Pedro Bolaños, Guadalupe Sabio, Luis Miguel Criado, Jesús Ruíz-Cabello, Vincenzo Cerundolo, Nick S. Jones, José Antonio Enríquez

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