Noninvasive diagnostics of mitochondrial disorders in isolated lymphocytes with high resolution respirometry

Abstract

Background: Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts.

Methods: To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitonin-permeabilized cells from 48 children were analyzed by high resolution respirometry, cytofluorometric detection of mitochondrial membrane potential and immunodetection of respiratory chain proteins with SDS and Blue Native electrophoreses.

Results: Evaluation of individual respiratory complex activities, ATP synthesis, kinetic parameters of mitochondrial respiratory chain and the content and subunit composition of respiratory chain complexes enabled detection of inborn defects of respiratory complexes I, IV and V within 2 days. Low respiration with NADH-dependent substrates and increased respiration with glycerol-3-phosphate revealed complex I defects; changes in p 50 for oxygen and elevated uncoupling control ratio pointed to complex IV deficiency due to SURF1 or SCO2 mutation; high oligomycin sensitivity of state 3-ADP respiration, upregulated mitochondrial membrane potential and low content of complex V were found in lymphocytes with ATP synthase deficiency due to TMEM70 mutations.

Conclusion: Based on our results, we propose the best biochemical parameters predictive for defects of respiratory complexes I, IV and V manifesting in peripheral blood lymphocytes.

General significance: The noninvasiveness, reliability and speed of an approach utilizing novel biochemical criteria demonstrate the high potential of isolated lymphocytes for diagnostics of oxidative phosphorylation disorders in pediatric patients.

Keywords: AA, antimycin A; BNE, Blue Native PAGE; COX, cytochrome c oxidase; Diagnostics; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GP, glycerol-3-phosphate; GPDH, mitochondrial FAD-dependent glycerophosphate dehydrogenase; Lymphocytes; Mitochondrial diseases; OXPHOS, oxidative phosphorylation; Oxidative phosphorylation; PAGE, polyacrylamide gel electrophoresis; Respirometry; TMPD, tetramethylphenylenediamine; TMRM, tetramethylrhodamine methyl ester; cI–cV, respiratory chain complexes I–V; s3, state 3-ADP; s3u, state 3-uncoupled; s4o, state 4-oligomycin; ΔΨm, mitochondrial membrane potential.

Fig. 1

Flow chart of mitochondrial oxidative phosphorylation system (OXPHOS) analysis in isolated lymphocytes.

Fig. 2

Substrate-inhibitor respiratory measurements using isolated lymphocytes. Complex analysis of respiratory chain functions includes the following steps: 1 — Cells at 0.3 mg protein.ml^− 1 + permeabilizing agent digitonin at 0.05 mg per mg of protein; 2 — Malate 3 mM + pyruvate 10 mM, substrates generating NADH oxidized by complex I; 3 — ADP 1 mM, phosphorylation substrate for ATP synthase, activates respiration by utilizing ΔΨ_m; 4 — Glutamate 10 mM, additional substrate generating NADH; 5 — Succinate 10 mM, oxidized by complex II, additional electron input into respiratory chain; 6 — Glycerol 3-phosphate 10 mM, oxidized by GPDH, additional electron input into respiratory chain; Maximal rate at respiratory state 3 (s3);; 7 — Oligomycin, ATP synthase inhibitor, sensitive assessment of enzyme capacity by step titration in 5, 10, 20, 40, 60, 80, 120, 160 and 200 nM final concentration (red ellipse), respiratory state 4 oligomycin (s4o); 8 — FCCP, uncoupler, achievement of maximal respiratory rate at state 3 uncoupled (s3u) by step titration, usually at 150–200 nM (yellow ellipse); 9 — Full depletion of oxygen — calculation of oxygen kinetics (p₅₀), followed by reoxygenation of medium (blue ellipse); 10 — Antimycin A 0.25 μM, complex III inhibition; 11 — Ascorbate 2 mM + TMPD 0.6 mM, artificial substrates feeding electrons directly to cytochrome c/complex IV; 12 — KCN 0.5 mM — inhibition of complex IV, KCN insensitive oxygen consumption due to ascorbate + TMPD autooxidation.

Fig. 3

Respiratory rates and respiratory control indexes in isolated lymphocytes. Statistical analysis of data from lymphocytes of 48 children includes controls as well as patients with mitochondrial disorders. (A) Absolute respiratory rates. The following abbreviations are used — cI, cII, cIV, respiratory complexes I, II, IV; GPDH, mitochondrial glycerophosphate dehydrogenase; s3 respiration at state 3-ADP; s3u, respiration at state 3-uncoupled; s4o, respiration at state 4 oligomycin. (B) Respiratory control ratios (RCR). Box plots represent Q1, median, Q3 (box), and minimum and maximum (whiskers), asterisks denote means.

Fig. 4

Complex I deficient lymphocytes. (A) Decreased function of complex I in patient P3 lymphocytes detected as low respiration with NADH-dependent substrates. The figures above the experimental traces represent the relative portion (%) of maximal state 3 respiration contributed by individual dehydrogenases. (B) Statistical presentation of decreased relative contribution of complex I to the maximum rate of state 3-ADP respiration. Box plots represent Q1, median, Q3 (box), and minimum and maximum (whiskers), asterisks denote means, n = 60. (C) Scatter plot showing negative correlation between cI-specific and GPDH-specific respiration (n = 60), highest ratio of these parameters indicates to the suspected cI disorders. Values of patients P1–P3 are marked by red circle.

Fig. 5

Detection of complex IV defects in lymphocytes. (A) Oxygen p₅₀ value (n = 47) and (B) the uncoupling control ratio (n = 60) are increased in lymphocytes with (C) isolated decrease of cIV (subunit cox4) in comparison to normal content of cI (NDUFA9 subunit), cII (SDHA subunit), cIII (core2 subunit) and cV (α subunit) detected by SDS-PAGE/WB from patients P4 and P5 harboring SURF1 mutations (red arrows) and P6 patient harboring SCO2 mutation (blue arrows), respectively; in (D) the elevated ratio of oxygen p₅₀ relative to the content of cIV (n = 28) is shown. Box plots represent Q1, median, Q3 (box), and minimum and maximum (whiskers), asterisks denote means.

Fig. 6

Detection of ATP synthase deficiency in lymphocytes. (A) Sensitivity of state 3-ADP respiration (with glutamate + malate + succinate + glycerol-3-phosphate) to oligomycin is increased (n = 50, box plots represent Q1, median, Q3 (box), minimum and maximum (whiskers), asterisks denote means) and (B) uncoupling control ratio (n = 60) is elevated in lymphocytes from patients (P7–P9) with ATP synthase deficiency due to TMEM70 mutation (data points of TMEM70 patients are marked by red circles). (C) High levels of mitochondrial membrane potential ΔΨ_m at state 3-ADP detected by TMRM (20 nM) cytofluorometry (analysis was performed using 10 mM succinate (state 4o), after addition of 1 mM ADP (state 3) or 1 μM FCCP (state 3u), decrease in ΔΨ_m is expressed as % change from state 4 value). (D) diminished content of ATP synthase complex (cV) in comparison to the content of other respiratory chain complexes detected by BNE/WB and by (E) SDS-PAGE/WB using subunit specific antibodies to cV (α subunit), cI (NDUFA9 subunit), cII (SDHA subunit), cIII (core2 subunit) and cIV (cox4 subunit), P — P7, C — control.