Mitochondrial permeability transition pore: sensitivity to opening and mechanistic dependence on substrate availability

Abstract

Mitochondrial Ca²⁺ uptake has a key role in cellular Ca²⁺ homeostasis. Excessive matrix Ca²⁺ concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH₂-linked substrates. The functional consequences of Ca²⁺-induced mPTP opening were assessed by Ca²⁺ retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca²⁺ handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca²⁺ overload.

Figure 1

Mitochondrial metabolic substrate determines sensitivity to Ca²⁺-induced mPTP opening. Mitochondria (1 mg protein ml⁻¹) were incubated with Fluo-4FF (0.35 μM) or TMRM (2 μM) in the presence of either glutamate/malate (10 mM/2 mM), succinate (10 mM) or succinate/rotenone (10 mM/1 μM). Pulses of CaCl₂ (10 μM) were added sequentially and fluorescence measured. (a) Trace of extracellular Ca²⁺ fluorescence (using Fluo-4FF) and Ca²⁺ uptake in the presence different metabolic substrates prior to mPTP opening. Data was normalised using baseline and maximal Fluo-4FF fluorescence. (b) Trace of TMRM fluorescence and time-dependent transmembrane depolarisation after Ca²⁺ overloading and mPTP opening using different metabolic substrates. Data was normalised to baseline and maximal TMRM fluorescence (maximal depolarisation/maximal TMRM de-quench). (c) Area under the curve using Fluo-4FF calculated between CaCl₂ injection numbers 4 and 5. Data were analysed using one-way ANOVA corrected for multiple comparisons using Holm-Sidak method. (d) Area under the curve using TMRM, calculated between CaCl₂ injection numbers 4 and 5. Data were analysed using one-way ANOVA, corrected for multiple comparisons using Holm-Sidak method. Data are expressed as means with error bars indicating standard deviation of 6 independent experiments. (e) Fluo-4FF trace from (a), showing deviation of response as a function of glutamate/malate and (f) TMRM trace from (b) showing deviation of response as a function of succinate. ns; P > 0.05, *P = 0.01–0.05, **P = 0.001–0.01, ***P < 0.001. Abbreviations: ns; not significant, CsA; cyclosporin A, TMRM; tetramethylrhodamine methylester, a.u; arbitrary unit, AUC; area under the curve.

Figure 2

Mitochondrial metabolic substrate determines bioenergetic response to Ca²⁺-induced mPTP opening. Representative oxygen flux recording using closed-chamber, high-resolution respirometry. Mitochondria (1 mg protein ml⁻¹) were maintained in the presence of either (a,b) glutamate/malate (10 mM/2 mM), (c,d) succinate (10 mM) or (e,f) succinate/rotenone (10 mM/1 μM) under constant stirring. Sequential additions of CaCl₂ (2.5 μM) were added as indicated to induce mPTP opening. Oxygen consumption (blue trace) and extra-mitochondrial Ca²⁺ 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). Antimycin A (2.5 μM) was used to completely inhibit respiration. Traces are representative of at least 3 independent experiments. Abbreviations: TMRM; tetramethylrhodamine methylester, AA; antimycin A.

Figure 3

Respiratory consequences of mPTP opening are sensitive to mPTP inhibitors. Representative oxygen flux recording using closed-chamber, high-resolution respirometry. (a) Mitochondria (1 mg protein ml⁻¹) were maintained in the presence of glutamate/malate (10 mM/2 mM) under constant stirring. CsA (0.5 μM) and RuR (0.5 μM) were added after baseline stabilisation. Percentage change in respiration was calculated after 5 minutes compound incubation. Data are expressed as means with error bars indicating standard deviation of at least three independent experiments. Data was analysed using one-way ANOVA, corrected for multiple comparisons using Holm-Sidak method. (b–e) Mitochondria were incubated as above and parallel measurements of oxygen consumption (blue trace) and either extra-mitochondrial Ca²⁺ (Fluo-4FF; green trace) or membrane potential (TMRM; red trace)were measured using the Oxygraph 2 K equipped with fluorimeter and fluorescent control unit (Oroboros Instruments, Innsbruck, Austria). Mitochondria were pre-treated with (b,c) CsA (0.5 μM) or (d,e) RuR (0.5 μM) prior to sequential additions of CaCl₂ (2.5 μM). Abbreviations: Mito; mitochondria, TMRM; tetramethylrhodamine methylester, NADH; nicotinamide adenine dinucleotide, Succ; succinate, AA; antimycin A.

Figure 4

Respiratory consequences of mPTP opening are rescued by exogenous NADH or succinate. (a) Mitochondria (1 mg protein ml⁻¹) were maintained in the presence of glutamate/malate (10 mM/2 mM) under constant stirring and subject to a Ca²⁺ retention challenge. Following pore opening, NADH (3 mM) or (b) succinate (10 mM) were added as indicated and respiration (blue trace), Ca²⁺ uptake (solid red trace) and ΔΨ_m (dashed red trace) measured simultaneously. Antimycin A (2.5 μM) was used to inhibit all respiration. Traces are representative of at least 3 independent experiments. Abbreviations: TMRM; tetramethylrhodamine methylester, NADH; nicotinamide adenine dinucleotide, Succ; succinate, AA; antimycin A.

Figure 5

Metabolic substrate determines mitochondrial Ca²⁺-induced H₂O₂ 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 CaCl₂ (10 μM) were added sequentially and extra-mitochondria Ca²⁺ (Fluo-4FF; solid trace) and H₂O₂ (AmpR; dashed trace) recorded in parallel. Area under the curve (Fluo-4FF) and slope (AmpR) were calculated between each CaCl₂ injection in mitochondrial energised using defined substrates. (c) H₂O₂ production calculated between CaCl₂ injections 5–6 in the presence and absence of CsA (1 μM) or RuR (1 μM) to inhibit mPTP opening and Ca²⁺ 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 CaCl₂ (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. H₂O₂ production was measured following CaCl₂ 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 CaCl₂ (2.5 μM) were added as indicated to induce mPTP opening. Oxygen consumption (blue trace) and H₂O₂ 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 Ca²⁺ 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.

Figure 6

Mitochondrial metabolic substrate determines H₂O₂ production in response to mitochondrially-active compounds. Mitochondria (1 mg protein ml⁻¹) were incubated with AmpR/HRP (10 μM/1 U ml⁻¹) in the presence of either (A) glutamate/malate (10 mM/2 mM), (B) succinate (10 mM) or (C) succinate/rotenone (10 mM/1 μM). Mitochondria were subject to Ca²⁺ retention assay and 10 pulses of CaCl₂ (10 μM) were added sequentially over time. H₂O₂ production was measured following the addition of mitochondrially-active compounds. Mitochondrially-active compounds were incubated for 10 minutes and fluorescence measured. CsA (1 μM) and RuR (1 μM) were included to inhibit mPTP opening and Ca²⁺ uptake respectively. Data are expressed as means with error bars indicating standard deviation of at least three independent experiments, normalised to vehicle (DMSO). Data was analysed using two-way ANOVA, corrected for multiple comparisons using Holm-Sidak method. No symbol P > 0.05, ⁺ P = 0.01–0.05, ^$ P = 0.001–0.01, ^{^} P = 0.0001–0.001, *P < 0.0001. Abbreviations: CsA; cyclosporin A, RuR; ruthenium red, Glu; glutamate, Mal; malate, Succ; succinate, Rot; rotenone, AmpR; Amplex Red.