, Mitochondrial function Quantifications of mitoCa 2+ accumulation (n=5). Data are expressed as means ± sem, p.677
, 001 vs WT or Veh, ###p<0.001 vs Tyr)
, MAO-A/4-HNE axis regulates mitoCa 2+ -induced mitochondrial dysfunction through MCU-680 binding and higher order complex formation. (A) Quantifications of mitoCa 2+ accumulation in 681, vol.6
, AMCMs isolated from WT mice treated with 4-HNE (5 µM, 15 min) in the presence of RU360
, C) Representative immunoblots showing the interaction of 4-HNE with MCU (B) in AMCMs of 683
, Immunoprecipitation (IP) 684 experiments were performed with 4-HNE or MCU antibodies, NRVMs stimulated with Tyr (500 µM, 1h)
, Input is a control of cell lysates. n=4. (D) Representative elution profile and immunoblots showing MCU 686 expression in different protein complexes obtained after size exclusion chromatography (SEC) of non-687
, denatured NRVMs lysates using HPLC (AKTA purifier 10, vol.6
, The elution profile of molecular complexes 689 are shown in four different fractions (F1-F4) with estimated sizes: F1 at ? 400 kDa, F2 at ? 100 kDa, after stimulation with Tyr (500µM, 4h), p.3
, The annotated molecular weights were estimated from the elution profiles 691 of standard markers injected onto the SEC column. (E, F) Representative non-reducing immunoblots 692 showing MCU higher order complex formation (E) in AMCMs of MAO-A Tg mice and (F) in NRVMs 693 stimulated with Tyr in the presence of Alda-1, at ? 75 kDa and F4 at ? 35 kDa, p.694
, AMCMs of WT mice treated with Tyr, p.695
, ). (H) Oxygen 696 consumption rate (OCR) associated with maximal respiratory capacity (n=5-6). (I) TMRE fluorescence 697 normalized to fluorescence in the presence of FCCP (F/F FCCP ) in AMCMs of WT mice stimulated with 698, pp.5-6
, µM) (n=6). (J) ATP content in AMCMs of WT mice 699 stimulated with Tyr (50 µM, 3 h) in the presence of RU360 (10 µM) (n=6). Data are expressed as means 700 ± sem, Tyr (50 µM, 3 h) in the presence of RU360
, A-B) Immunoblots and quantifications of ALDH2, MAO-A and 704 4-HNE protein adducts in (A) cardiac homogenates of SHAM or MI mice (n=4) and (B) left ventricular 705 myocardium of CTL (n=4) or human ischemic cardiomyopathy patients (hICM) (n=5). (C) Model of MI 706 experiments with moclobemide treatment (20 mg/kg/day) or in mice with deletion of MAO-A, Effect of Moclobemide or MAO-A deficiency on myocardial infarction (MI)-induced 4-HNE 703 accumulation and cardiac remodeling
, MAO cKO: n=4 sham, n=8 MI; Moclobemide: n=5 sham, n=5 MI). (E) Echocardiographic parameters of 709
, Ejection Fraction (EF, %). (F) Representative pictures of 3D-reconstructed hearts by LSFM with scar 710 zone in blue in upper panel (Scale Bar = 2 mm) or Masson's Trichrome staining in lower panel, p.711
, Scale Bar = 50 ?m) immunofluorescence staining with quantifications of mean 712 cardiomyocyte area. (MAO-A cKO: n=4 sham, n=8 MI). (H) Immunoprecipitation experiments with 713 MCU or 4-HNE in heart homogenates of mice subjected to 4 weeks ischemia (MI) in the presence of 714 moclobemide (20 mg/kg/day). (n=3 per group). (I) Representative non-reducing immunoblots showing 715 MCU higher order complex formation in heart homogenates of mice, p.716
, 20 mg/kg/day). (n=3 per group). (J) Quantification of Ca 2+ accumulation 717 in isolated mitochondria of WT and MAO-A cKO mice subjected to 1 week ischemia, pp.5-7
, Fig 8. Schema illustrating the mechanisms associated with MAO-A/4-HNE signaling during chronic 720 ischemia (right panel) compared to normal conditions