Peroxynitrite nitrates adenine nucleotide translocase and voltage-dependent anion channel 1 and alters their interactions and association with hexokinase II in mitochondria

Abstract

Cardiac ischemia and reperfusion (IR) injury induces excessive emission of deleterious reactive O₂ and N₂ species (ROS/RNS), including the non-radical oxidant peroxynitrite (ONOO^-) that can cause mitochondria dysfunction and cell death. In this study, we explored whether IR injury in isolated hearts induces tyrosine nitration of adenine nucleotide translocase (ANT) and alters its interaction with the voltage-dependent anion channel 1 (VDAC1). We found that IR injury induced tyrosine nitration of ANT and that exposure of isolated cardiac mitochondria to ONOO^- induced ANT tyrosine, Y⁸¹, nitration. The exposure of isolated cardiac mitochondria to ONOO^- also led ANT to form high molecular weight proteins and dissociation of ANT from VDAC1. We found that IR injury in isolated hearts, hypoxic injury in H9c2 cells, and ONOO^- treatment of H9c2 cells and isolated mitochondria, each decreased mitochondrial bound-hexokinase II (HK II), which suggests that ONOO^- caused HK II to dissociate from mitochondria. Moreover, we found that mitochondria exposed to ONOO^- induced VDAC1 oligomerization which may decrease its binding with HK II. We have reported that ONOO^- produced during cardiac IR injury induced tyrosine nitration of VDAC1, which resulted in conformational changes of the protein and increased channel conductance associated with compromised cardiac function on reperfusion. Thus, our results imply that ONOO^- produced during IR injury and hypoxic stress impeded HK II association with VDAC1. ONOO^- exposure nitrated mitochondrial proteins and also led to cytochrome c (cyt c) release from mitochondria. In addition, in isolated mitochondria exposed to ONOO^- or obtained after IR, there was significant compromise in mitochondrial respiration and delayed repolarization of membrane potential during oxidative (ADP) phosphorylation. Taken together, ONOO^- produced during cardiac IR injury can nitrate tyrosine residues of two key mitochondrial membrane proteins involved in bioenergetics and energy transfer to contribute to mitochondrial and cellular dysfunction.

Keywords: Adenine nucleotide translocase; Cardiac ischemia reperfusion injury; Hexokinase II; Mitochondria; Peroxynitrite; Tyrosine nitration; Voltage-dependent anion channel 1.

Figure 1:

Tyrosine nitration of mitochondrial ANT induced by ONOO⁻. A: Immunostaining of H9c2 cells treated with 200 µM ONOO⁻ using anti-3-NT and mitochondrial marker MitoTracker™ Red CMXRos. The histogram panel below represents the mean fluorescence intensity from 15 cells in each group. *: p < 0.05 vs. control. B: Western blot analyses of H9c2 cells treated with 0 to 500 µM ONOO⁻ using anti-3-NT and β-tubulin (loading control) antibodies. The histogram panel below represents the mean relative 3-NT band intensity (normalized to β-tubulin) from three independent experiments. *: p 0.05 vs. control. C: Western blot analyses of protein tyrosine nitration in isolated mitochondria treated with 0 to 500 µM ONOO⁻ using anti-3-NT and COX IV (loading control) antibodies. Histogram panel represents the mean relative 3-NT band intensity (normalized to COX IV) from three time experiments. *: p < 0.05 vs. control. D: Coverage of ANT residues detected by mass spectrometric assay. Highlighted yellow are detected peptides; highlighted green are detected nitrated tyrosine Y⁸¹. E: Spectrum of peptide displaying the nitration of Y⁸¹. F: Western blot analyses of tyrosine nitration of enriched ANT and mitochondrial lysate isolated from mitochondria treated with 200 µM ONOO⁻ in the absence or presence of MCGR using anti-3-NT antibody (3-NT panel); ANT band confirmed the loading of similar amount of enriched ANT and mitochondrial lysate (ANT panel). The histogram panel below represents the mean relative 3-NT band intensity (normalized to ANT) from three independent experiments. *: p < 0.05 vs. presence of MCGR for mitochondrial lysate; #: p < 0.05 vs. presence of MCGR for enriched ANT.

Figure 2:

Tyrosine nitration of mitochondrial ANT induced by IR injury. A: Coomassie blue stain of enriched ANT from mitochondria isolated from hearts subjected to time control (TC) or 35 min ischemia and 20 min reperfusion (I35R20). B: Western blot of enriched ANT with 3-NT antibody; ANT was used as loading control. C: Data summary of 3-NT band intensity to ANT band ratio for TC and I35R20; *p < 0.05 vs. TC. D: Time course of changes in diastolic LVP (left panel) and coronary flow rate (right panel) during 35 min ischemia and 20 min reperfusion alone (IR) or in the presence of MCGR (IR+MCGR). *: p < 0.05 vs. IR.

Figure 3:

Dissociation of VDAC1 and ANT after I35R20 or ONOO⁻ treatment. A: Immunoprecipitation of mitochondrial proteins with anti-ANT antibody (goat) followed by western blot with VDAC1 (upper panel) and ANT (rabbit (R), low panel) antibodies; ONOO⁻, mitochondria treated with 200 μM ONOO⁻; TC, time control; I35R20, 35 min ischemia and 20 min reperfusion; IgG, negative control. B: Summary of VDAC1/ANT ratio from 3 experiments. *p < 0.05 vs. TC. C: Evaluation of VDAC1 oligomerization based on EGS cross-linking and western blot using anti-VDAC1 and COX IV (loading control) antibodies in isolated mitochondria treated with 0 to 500 µM ONOO⁻. D: Evaluation of ANT disulfide- linked protein complexes in isolated mitochondria treated with 0 to 500 µM ONOO⁻ using SDS-PAGE under non-reducing conditions followed by immunoblotting with anti-ANT antibody.

Figure 4:

Dissociation of HK II from mitochondria. A: Western blot of mitochondrial and cytosolic proteins from TC, I35R20 (IR) and I35R20+MCGR (IR+MCGR) with anti-HK II antibody. The histogram panel below shows summary of HK II/VDAC1 ratio (for mitochondrial fraction) and HK II/β-tubulin ratio (for cytosolic fraction) from 3 experiments. *: p < 0.05 vs. TC. B: Immunoprecipitation of mitochondrial proteins with HK II antibody followed by western blot with VDAC1 and HK II antibod-ies. Mitochondria were treated with or without ONOO⁻. C: Immunostaining of H9c2 cells treated with 200 µM ONOO⁻ or exposed to 4 h hypoxia with HK II and COX IV antibodies. COX IV was used as mitochondrial marker. The intensity profile panels, alongside the immunostaining shows the degree of overlapping of HK II with COX IV. The histogram represents the mean Mander’s split co-localization coefficients for HK II to COX IV and COX IV to HK II in control, ONOO⁻ exposed, and hypoxia treated cells, in that order. The mean Mander’s split co-localization coefficients was calculated from n=10 cells. *: p < 0.05 HK II/COX IV in ONOO⁻ or hypoxia group vs. control group.

Figure 5:

Cytochrome c release from isolated mitochondria. A: Western blot of mitochondrial proteins treated without (CON) or with ONOO⁻, MCGR, DIDS or BKA using anti-cyt c antibody; *: p < 0.05 vs. CON. NDUFS2, complex I subunit 2, was used as loading control. B: Western blot of mitochondrial protein treated with ONOO⁻ (CON), or incubated with MCGR, DIDS or BKA before treatment with ONOO⁻; *: p < 0.05 vs. ONOO⁻ alone. VDAC1 was used as loading control.

Figure 6:

Lower respiration control index and delayed ΔΨ_m repolarization (state 3 respiration) after ONOO− treatment or 35 min of ischemia and 20 min of reperfusion (I35R20). A: Representative traces of respiration in mitochondria subjected to treatment with NaOH (control), 100 or 200 µM ONOO−, or I35R20. B: Summary of the RCIs for control, I35R20 and ONOO⁻-treated mitochondria; *:p < 0.05 vs. control. C: Time course of ΔΨ_m with NaOH treatment (control) or treatment with 100 or 200 µM ONOO⁻. D: Summary of effects of ONOO⁻ compared to control on duration of recovery from ADP- induced state 3 ΔΨ_m (S3 time, sec) depolarization; *: p < 0.05 vs. control. E: Time course of ΔΨ_m from mitochondria isolated from ex vivo hearts subjected to either time control (TC) or I35R20. F: Summary of effects of TC or I35R20 on duration of recovery from ADP-induced state 3 ΔΨ_m (S3 time, sec) depolarization; *: p < 0.05 vs. control.