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. 2020 Jun;27(6):1907-1923.
doi: 10.1038/s41418-019-0470-y. Epub 2019 Dec 9.

Mitochondrial 4-HNE derived from MAO-A promotes mitoCa2+ overload in chronic postischemic cardiac remodeling

Yohan Santin  1 , Loubina Fazal  1 , Yannis Sainte-Marie  1 , Pierre Sicard  1   2 , Damien Maggiorani  1 , Florence Tortosa  1 , Yasemin Yücel Yücel  3 , Lise Teyssedre  4 , Jacques Rouquette  4 , Marlene Marcellin  5 , Cécile Vindis  1 , Jean C Shih  6 , Olivier Lairez  1 , Odile Burlet-Schiltz  5 , Angelo Parini  7 , Frank Lezoualc'h  1 , Jeanne Mialet-Perez  8
Affiliations

Mitochondrial 4-HNE derived from MAO-A promotes mitoCa2+ overload in chronic postischemic cardiac remodeling

Yohan Santin et al. Cell Death Differ. 2020 Jun.

Abstract

Chronic remodeling postmyocardial infarction consists in various maladaptive changes including interstitial fibrosis, cardiomyocyte death and mitochondrial dysfunction that lead to heart failure (HF). Reactive aldehydes such as 4-hydroxynonenal (4-HNE) are critical mediators of mitochondrial dysfunction but the sources of mitochondrial 4-HNE in cardiac diseases together with its mechanisms of action remain poorly understood. Here, we evaluated whether the mitochondrial enzyme monoamine oxidase-A (MAO-A), which generates H2O2 as a by-product of catecholamine metabolism, is a source of deleterious 4-HNE in HF. We found that MAO-A activation increased mitochondrial ROS and promoted local 4-HNE production inside the mitochondria through cardiolipin peroxidation in primary cardiomyocytes. Deleterious effects of MAO-A/4-HNE on cardiac dysfunction were prevented by activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2), the main enzyme for 4-HNE metabolism. Mechanistically, MAO-A-derived 4-HNE bound to newly identified targets VDAC and MCU to promote ER-mitochondria contact sites and MCU higher-order complex formation. The resulting mitochondrial Ca2+ accumulation participated in mitochondrial respiratory dysfunction and loss of membrane potential, as shown with the protective effects of the MCU inhibitor, RU360. Most interestingly, these findings were recapitulated in a chronic model of ischemic remodeling where pharmacological or genetic inhibition of MAO-A protected the mice from 4-HNE accumulation, MCU oligomer formation and Ca2+ overload, thus mitigating ventricular dysfunction. To our knowledge, these are the first evidences linking MAO-A activation to mitoCa2+ mishandling through local 4-HNE production, contributing to energetic failure and postischemic remodeling.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. MAO-A activation leads to intramitochondrial 4-HNE production through H2O2-mediated L4CL peroxidation.
a Representative confocal images of 4-HNE staining (green) on AMCMs isolated from WT mice loaded with Mito-ID red and counterstained with DAPI (blue). Cells were treated with 50 μM Tyr for 1 h. Scale Bar = 10 μm. n = 5. b Immunoblots of MAO-A expression and 4-HNE protein adducts and quantifications in isolated mitochondria of WT and MAO-A Tg hearts at 12 weeks (n = 6 per group). c Representative confocal images of MitoPY1 (green), Mito-ID (red), and Hoechst 33258 staining on AMCMs isolated from WT mice treated with 50 μM Tyr for 30 min. Scale Bar = 10 μm. n = 5. d Amplex red measurements in isolated mitochondria of WT and MAO-A Tg hearts (n = 5) at 12 weeks. Quantifications by LC–MS/MS in isolated mitochondria of WT (n = 6) and MAO-A Tg (n = 4) hearts at 12 weeks showing (e) L4CL content as percent of total cardiolipin and (f) each type of HODEs species as ng/µg prot. g Proposed mechanism for intramitochondrial 4-HNE production through H2O2-mediated L4CL peroxidation downstream MAO-A. Data are expressed as means ± sem (*p < 0.05, **p < 0.01 vs WT mice).
Fig. 2
Fig. 2. 4-HNE-mediated adverse remodeling in MAO-A Tg hearts is prevented by ALDH2 gene transduction.
a Model of AAV9-GFP or AAV9-ALDH2 (3 × 1011 vg/mouse) transduction in WT or MAO-A Tg mice. b Cardiac GSH content and c mitochondrial ALDH2 activity in WT and MAO-A Tg hearts at 12 weeks (n = 4). d Mitochondrial ALDH2 activity on cardiac homogenates of MAO-A Tg mice after AAV9 transduction (n = 7). e 4-HNE (Scale Bar = 100 μm) and Vinculin (Scale Bar = 50 μm) immunofluorescence, and interstitial fibrosis with Sirius Red (Scale Bar = 1 mm) on cardiac cryosections of mice after AAV9 transduction. f Quantification of 4-HNE fluorescence as % of total area (n = 6–7). g Quantification of cardiomyocytes area on at least 100 cells/mouse in three distinct regions of the left ventricle (n = 6–7). h Quantification of fibrosis as % of total area (n = 4–6). Echocardiographic measurements with (i) fractional shortening (%) and (j) systolic LV internal dimension on mice after AAV9 transduction (n = 6–7). Data are expressed as means ± sem (*p < 0.05, **p < 0.01, ***p < 0.001 vs WT GFP mice; #p < 0.05, ##p < 0.01 vs MAO-A Tg GFP mice).
Fig. 3
Fig. 3. Mitochondrial MAO-A/4-HNE axis impairs organelle function.
a Oxygen consumption rate (OCR) measurements in AMCMs of WT mice treated with Tyr (50 µM, 3 h) in the presence of Moclo (15 µM) or Alda-1 (100 µM) at baseline and after addition of Oligomycin, FCCP, and Antimycin A + Rotenone (n = 6). b Maximal respiratory capacity measured from oxygen consumption rate (OCR) (n = 6). c ATP content in AMCMs isolated from WT mice stimulated with Tyr (50 µM, 3 h) in the presence of Moclo (15 µM) or Alda-1 (100 µM) (n = 6). d NAD+/NADH ratio in mitochondria isolated from WT mice and treated with Tyr (50 µM, 3 h) in the presence of Moclo (15 µM) or Alda-1 (100 µM) (n = 5). e TMRE fluorescence normalized to fluorescence in the presence of FCCP (F/FFCCP), in AMCMs isolated from WT mice stimulated with Tyr (50 µM, 3 h) in the presence of Moclo (15 µM) or Alda-1 (100 µM) (n = 6). Data are expressed as means ± sem (**p < 0.01, ***p < 0.001 vs CTL; #p < 0.05, ##p < 0.01, ###p < 0.001 vs Tyr).
Fig. 4
Fig. 4. MAO-A activation induces 4-HNE-dependent increase in mitoCa2+ levels.
a Representative images of mitochondrial Ca2+ (mitoCa2+) accumulation in WT or MAO-A Tg AMCMs. The Ca2+ fluorescent probe Rhod-2-AM overlapped with the mitochondrial marker Mitotracker green. Quantification of mitoCa2+ accumulation in AMCMs (b) and isolated mitochondria (c) of MAO-A Tg mice (n = 5–6). d, e Representative images and quantification of mitoCa2+ accumulation in AMCMs of WT mice treated with Tyr (50 µM, 3 h) in the presence of Moclo (15 µM), Alda-1 (100 µM), or RU360 (10 µM) (n = 5–6). f Quantification of Ca2+ accumulation in isolated mitochondria of WT mice treated with Tyr (50 µM, 30 min) (n = 5–6). Data are expressed as means ± sem (*p < 0.05, ***p < 0.001 vs Veh or WT; ##p < 0.01, ###p < 0.001 vs MI WT or Tyr).
Fig. 5
Fig. 5. Implication of MAMs in MAO-A-induced mitoCa2+ increase.
a Representative immunoblots showing the interaction of 4-HNE with VDAC1 in NRVMs stimulated with Tyr. Immunoprecipitation (IP) experiments were performed with 4-HNE or VDAC1 antibodies. IgG was used as a negative control for IP. Input is a control of cell lysates n = 4. b Representative images of in situ interactions (red fluorescent dots) between IP3R1 and GRP75 or IP3R1 and VDAC1 or GRP75 and VDAC1 in WT or MAO-A Tg cardiomyocytes. Nuclei are stained with DAPI. Scale bar, 20 μm. c–e Quantifications of the proximity ligation assay (n = 3). f Representative images of mitoCa2+ accumulation in AMCMs of WT mice treated with Tyr and challenged with histamine (100 µM) or Xestospongin C (2 µM). g Quantifications of mitoCa2+ accumulation (n = 5). Data are expressed as means ± sem (*p < 0.05, **p < 0.01, ***p < 0.001 vs WT or Veh, ###p < 0.001 vs Tyr).
Fig. 6
Fig. 6. MAO-A/4-HNE axis regulates mitoCa2+-induced mitochondrial dysfunction through MCU-binding and higher-order complex formation.
a Quantifications of mitoCa2+ accumulation in AMCMs isolated from WT mice treated with 4-HNE (5 µM, 15 min) in the presence of RU360 (10 µM) n = 4. Representative immunoblots showing the interaction of 4-HNE with MCU (b) in AMCMs of MAO-A Tg mice, (c) in NRVMs stimulated with Tyr (500 µM, 1 h). Immunoprecipitation (IP) experiments were performed with 4-HNE or MCU antibodies. IgG was used as a negative control for IP. Input is a control of cell lysates n = 4. d Representative elution profile and immunoblots showing MCU expression in different protein complexes obtained after size-exclusion chromatography (SEC) of nondenatured NRVMs lysates using HPLC, after stimulation with Tyr (500 µM, 4 h). The elution profile of molecular complexes is shown in four different fractions (F1–F4) with estimated sizes: F1 at ≈400 kDa, F2 at ≈100 kDa; F3 at ≈75 kDa and F4 at ≈35 kDa. Representative nonreducing immunoblots showing MCU higher-order complex formation (e) in AMCMs of MAO-A Tg mice and (f) in NRVMs stimulated with Tyr in the presence of Alda-1 (100 µM). g Oxygen consumption rate (OCR) measurements in AMCMs of WT mice treated with Tyr (50 µM, 3 h) in the presence of RU360 (10 µM) at baseline and after addition of Oligomycin, FCCP and Antimycin A + Rotenone (n = 5–6). h Oxygen consumption rate (OCR) associated with maximal respiratory capacity (n = 5–6). i TMRE fluorescence normalized to fluorescence in the presence of FCCP (F/FFCCP) in AMCMs of WT mice stimulated with Tyr (50 µM, 3 h) in the presence of RU360 (10 µM) (n = 6). j ATP content in AMCMs of WT mice stimulated with Tyr (50 µM, 3 h) in the presence of RU360 (10 µM) (n = 6). Data are expressed as means ± sem (*p < 0.05, **p < 0.01, ***p < 0.001 vs CTL; #p < 0.05, vs 4-HNE or Tyr).
Fig. 7
Fig. 7. Effect of Moclobemide or MAO-A deficiency on myocardial infarction (MI)-induced 4-HNE accumulation and cardiac remodeling.
Immunoblots and quantifications of ALDH2, MAO-A, and 4-HNE protein adducts in (a) cardiac homogenates of SHAM or MI mice (n = 4) and (b) left ventricular myocardium of CTL (n = 4) or human ischemic cardiomyopathy patients (hICM) (n = 5). c Model of MI experiments with moclobemide treatment (20 mg/kg/day) or in mice with deletion of MAO-A in cardiomyocytes (MAO-A cKO). d Quantifications of 4-HNE protein adducts in mouse hearts after MI. (MAO cKO: n = 4 sham, n = 8 MI; Moclobemide: n = 5 sham, n = 5 MI). e Echocardiographic parameters of Ejection Fraction (EF, %). f Representative pictures of 3D-reconstructed hearts by LSFM with scar zone in blue in upper panel (Scale Bar = 2 mm) or Masson’s Trichrome staining in lower panel (Scale Bar = 1 mm). g Vinculin (Scale Bar = 50 μm) immunofluorescence staining with quantifications of mean cardiomyocyte area. (MAO-A cKO: n = 4 sham, n = 8 MI). h Immunoprecipitation experiments with MCU or 4-HNE in heart homogenates of mice subjected to 4 weeks ischemia (MI) in the presence of moclobemide (20 mg/kg/day) (n = 3 per group). i Representative nonreducing immunoblots showing MCU higher-order complex formation in heart homogenates of mice subjected to 4 weeks ischemia (MI) in the presence of moclobemide (20 mg/kg/day) (n = 3 per group). j Quantification of Ca2+ accumulation in isolated mitochondria of WT and MAO-A cKO mice subjected to 1 week ischemia (n = 5–7).
Fig. 8
Fig. 8
Schema illustrating the mechanisms associated with MAO-A/4-HNE signaling during chronic ischemia (right panel) compared with normal conditions (left panel).
See this image and copyright information in PMC

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