Journal: Scientific Reports
Article Title: Mitochondrial permeability transition pore: sensitivity to opening and mechanistic dependence on substrate availability
doi: 10.1038/s41598-017-10673-8
Figure Lengend Snippet: Metabolic substrate determines mitochondrial Ca 2+ -induced H 2 O 2 production. Mitochondria (1 mg protein ml −1 ) were incubated with AmpR/HRP (10 μM/1 U ml −1 ) or Fluo-4FF (0.35 μM) in the presence of either glutamate/malate (10 mM/2 mM), succinate (10 mM) or succinate/rotenone (10 mM/1 μM). ( a ) Baseline rates of mtROS production under distinct metabolic conditions were measured using the FLIPR TETRA . Data was analysed using one-way ANOVA, corrected for multiple comparisons using Holm-Sidak method. ( b ) Pulses of CaCl 2 (10 μM) were added sequentially and extra-mitochondria Ca 2+ (Fluo-4FF; solid trace) and H 2 O 2 (AmpR; dashed trace) recorded in parallel. Area under the curve (Fluo-4FF) and slope (AmpR) were calculated between each CaCl 2 injection in mitochondrial energised using defined substrates. ( c ) H 2 O 2 production calculated between CaCl 2 injections 5–6 in the presence and absence of CsA (1 μM) or RuR (1 μM) to inhibit mPTP opening and Ca 2+ uptake respectively. Data are normalised to baseline and are expressed as means with error bars indicating standard deviation of at least three independent experiments. Data was analysed by two-way ANOVA, corrected for multiple comparisons using Holm-Sidak method. Comparisons between groups: ns; P > 0.05, * P = 0.01–0.05, ** P = 0.001–0.01 *** P < 0.001. Comparisons within groups: No symbol P > 0.05, + P = 0.01–0.05, $ P = 0.001–0.01 ^ P = 0.0001–0.001, * P < 0.0001. ( d , e ) Mitochondria (1 mg protein ml −1 ) were incubated as above in the presence of either glutamate/malate (10 mM/2 mM), succinate (10 mM) or succinate/rotenone (10 mM/1 μM). Sequential additions of CaCl 2 (10 μM) were added as indicated. Mitochondria were incubated in the presence of ( d ) CsA and ( e ) RuR in a 2-fold dilution series under distinct metabolic conditions. H 2 O 2 production was measured following CaCl 2 injection 10. Data are presented as change in AmpR fluorescence over time (slope). ( f ) Mitochondria (1 mg protein ml −1 ) were incubated with AmpR/HRP (10 μM/1 U ml −1 ) in the presence of either glutamate/malate (10 mM/2 mM), succinate (10 mM) or succinate/rotenone (10 mM/1 μM). FCCP was added for 10 minutes and fluorescence measured. Data are presented as change in AmpR fluorescence over time (slope) normalised to data in the presence of DMSO alone. ( g , h ) Representative oxygen flux recording using closed-chamber, high-resolution respirometry. Mitochondria (1 mg protein ml −1 ) were maintained in the presence of glutamate/malate (10 mM/2 mM) under constant stirring. Sequential additions of CaCl 2 (2.5 μM) were added as indicated to induce mPTP opening. Oxygen consumption (blue trace) and H 2 O 2 production (AmpR; red trace) were recorded in parallel using the Oxygraph 2 K equipped with fluorimeter and fluorescent control unit (Oroboros Instruments, Innsbruck, Austria), in the presence of ( g ) CsA (0.5 μM) and ( h ) RuR (0.5 μM; dashed traces) to inhibit mPTP opening and Ca 2+ uptake respectively. Abbreviations: Mito; mitochondria, CsA; cyclosporin A, RuR; ruthenium red, AmpR; Amplex Red, AA; antimycin A, Succ; succinate, Rot; rotenone, a.u; arbitrary unit, AUC; area under the curve, Glu; glutamate, Mal; malate.
Article Snippet: Oxygen consumption (blue trace) and extra-mitochondrial Ca 2+ fluorescence ( a , c , e ) Fluo-4FF; green trace) or ΔΨ m ( b , d , f ): TMRM; red trace) were measured in parallel using the Oxygraph 2 K equipped with fluorimeter and fluorescent control unit (Oroboros Instruments, Innsbruck, Austria).
Techniques: Incubation, Injection, Standard Deviation, Fluorescence, Control
