PhD Yohan Santin

PhD Loubina Fazal

PhD Yannis Sainte-Marie

PhD Pierre Sicard

PhD Damien Maggiorani

Florence Tortosa

PhD Yasemin Yücel Yücel

Lise Teyssedre

PhD Jacques Rouquette

Marlène Marcellin

Marlene Marcellin

PhD Cécile Vindis

PhD Jean C Shih

MD, PhD Olivier Lairez

PhD Odile Burlet-Schiltz

MD, PhD Angelo Parini
email: angelo.parini@inserm.fr

PhD Frank Lezoualc'h

PhD Jeanne Mialet-Perez

Mitochondrial 4-Hne Santin

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

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Post-myocardial infarction (MI) remodeling is a detrimental mechanism leading to Heart Failure (HF) and poor patient outcome. Although it is known that chronic remodeling consists in various maladaptive changes such as cardiomyocyte death, interstitial fibrosis, inflammation and contractile dysfunction, there is still a strong need to better understand underlying pathological processes in order to develop novel therapeutic strategies. Mitochondrial dysfunction has emerged as a key player in post-MI ventricular failure and new advances put forward that mitochondria-targeted therapeutics may lead to improved energy production in the heart and better contractile function [START_REF] Brown | Mitochondrial function as a therapeutic target in heart failure[END_REF]. Mitochondria control key processes of cardiac remodeling such as ATP production, lipid metabolism, cell survival/death and calcium (Ca 2+ ) buffering [START_REF] Rizzuto | Mitochondria as sensors and regulators of calcium signalling[END_REF]. Uptake of cytosolic Ca 2+ into mitochondrial matrix regulates important enzymes of the Krebs cycle and electron transport chain and is necessary for matching energy supply to the demand (3).

Hence, deregulation of mitochondrial Ca 2+ (mitoCa 2+ ) levels contribute to energetic failure in rodent models of HF, implying that its concentration needs to be fine-tuned thanks to the activity of the mitoCa 2+ uniporter (MCU) and the Na + /Ca 2+ exchanger (NCLX) [START_REF] Luongo | The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability[END_REF][START_REF] Santulli | Mitochondrial calcium overload is a key determinant in heart failure[END_REF]. In the particular situation of chronic ischemia, it is still unclear how mitoCa 2+ is regulated and contributes to cardiac remodeling, especially when it is accompanied by another stressor, oxidative stress [START_REF] Bertero | Calcium Signaling and Reactive Oxygen Species in Mitochondria[END_REF].

Mitochondria are the main source of cellular reactive oxygen species (ROS) and, when exceeding antioxidant capacities, ROS can lead to the oxidation of fatty acids in a process termed lipid peroxidation [START_REF] Kornfeld | Mitochondrial reactive oxygen species at the heart of the matter: new therapeutic approaches for cardiovascular diseases[END_REF]. Mounting evidence has linked lipid peroxidation with various human pathological states, such as neurodegenerative diseases, cancer, diabetes and HF. 4-hydroxy-trans-2-nonenal (4-HNE) is the most abundant lipid peroxidation product and forms adducts with proteins, thereby affecting their biological function to impair cellular homeostasis [START_REF] Gueraud | 4-Hydroxynonenal metabolites and adducts in pre-carcinogenic conditions and cancer[END_REF]. Interestingly, 4-HNE levels are elevated in plasma of HF patients and inversely correlate with cardiac function [START_REF] Mak | Unsaturated aldehydes including 4-OH-nonenal are elevated in patients with congestive heart failure[END_REF]. Furthermore, enhancing the degradation of mitochondrial 4-HNE is beneficial in rodent models of acute and chronic heart diseases [START_REF] Chen | Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart[END_REF][START_REF] Gomes | Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling[END_REF]. At present, there is a strong need to identify the source of mitochondrial 4-HNE in pathological states and to understand the underlying mechanisms of its toxicity on mitochondrial function.

The monoamine oxidase-A (MAO-A) is an enzyme located at the outer mitochondrial membrane which represents the main degradative pathway for catecholamines and serotonin. MAO-A generates H 2 O 2 as a by-product of substrate metabolism and has been recognized as a significant source of mitochondrial ROS in the heart [START_REF] Kaludercic | Monoamine oxidases as sources of oxidants in the heart[END_REF][START_REF] Villeneuve | p53-PGC-1alpha pathway mediates oxidative mitochondrial damage and cardiomyocyte necrosis induced by monoamine oxidase-A upregulation: role in chronic left ventricular dysfunction in mice[END_REF]. During post-MI remodeling, the sustained sympathetic drive and chronic elevation of circulating catecholamines could fuel MAO-A activity and ROS generation leading to 4-HNE production.

To date, this putative role of MAO-A in post-MI remodeling has never been investigated.

In the present study, we found that MAO-A was responsible for an intra-mitochondrial generation of 4-HNE that, in turn, bound to voltage-dependent anion channel (VDAC) and MCU proteins, leading to mitoCa 2+ overload and impaired mitochondrial function. In ischemic failing human heart samples, there was a concomitant increase in MAO-A levels and 4-HNE-bound proteins and selective inactivation of MAO-A in mice with chronic MI prevented cardiac remodeling, 4-HNE accumulation and mitoCa 2+ overload.

METHODS

Human heart tissues

All studies are conformed to the Declaration of Helsinki and institutional ethical regulations. Informed consent was obtained from all subjects. Human failing hearts samples were a kind gift from Dr J.L Samuel (Lariboisière Hospital, France). Explanted failing hearts were obtained from patients undergoing cardiac transplantation for end-stage cardiac HF secondary to ischemic cardiomyopathy. All patients had New York Heart Association (NYHA) Class IV HF, with a mean pretransplant left ventricular EF of 22±4 %.

None had received chronic intravenous inotropic support over at least 7 days immediately prior to transplantation. HF therapy consisted of angiotensin converting enzyme inhibitors and diuretics in all patients. Non-failing hearts were obtained from prospective multiorgan donors who had died from head trauma or intracranial bleeds; these hearts were unsuitable for transplantation for technical reasons. All tissues were stored at -80 °C until further analyses.

Animal models

All animal procedures were performed in accordance with International Guidelines on Animal Experimentation and with a French Ministry of Agriculture license. Moreover, this investigation conformed to the guide for Care and Use of Laboratory Animals published by the Directive 2010/63/EU of the European Parliament. All mice were housed in temperature-controlled cages with a 12-h light-dark cycle and given free access to water and food. MAO-A cKO mice: maoa gene on Chromosome X was floxed with two LoxP sites around Ex12 in the laboratory of Jean C. Shih (University of Southern California, CA, USA) (13) . The Myh6-Cre mice expressing Cre recombinase in cardiomyocytes were purchased from Janvier labs. The males with cardiomyocyte-specific deletion of Maoa (Maoa fl/Y xCre + , referred as MAO-A cKO) and their control littermates (Maoa WT/Y xCre + , referred as WT) on the C57Bl6J background were obtained by crossing Maoa WT/Y xCre + males with Maoa WT/fl females. MAO-A Tg mice: MAO-A transgenic mice (MAO-A Tg) on the C57Bl6J background were maintained by breeding of MAO-A Tg males (Myh6-maoa) with C57BL6/J females [START_REF] Villeneuve | p53-PGC-1alpha pathway mediates oxidative mitochondrial damage and cardiomyocyte necrosis induced by monoamine oxidase-A upregulation: role in chronic left ventricular dysfunction in mice[END_REF]. MAO-A Tg males and their WT littermates were used for the experiments. AAV9 model: AAV9-cTnT-GFP and AAV9-cTnT-ALDH2 were purchased from Penn Vector Core (Pennsylvania, USA). AAV9-cTnT-ALDH2 was made from the cDNA of mouse ALDH2 cloned into pCMV6-Entry vector (OriGene, Rockville, USA). The AAV9 vectors were injected by i.v.in the jugular vein of 3 week-old mice under anesthesia at a dose of 3x10 11 vg/mouse. Mice were kept for 4 months to assess cardiac remodeling.

Myocardial ischemia.

Mice were anaesthetized by 2% isofluorane inhalation. After orotracheal intubation, mice were mechanically ventilated (minivent type845, harvard apparatus) with 100% oxygen. Analgesia was provided by buprenorphine injection (0.02 mg/kg s.c.) and local lidocaïne injection (50 µL of 10 mg/mL solution). Electrocardiogram (ADinstrument) was used to monitor the mice during the procedure. A left thoracotomy was performed at the 4 th intercostal space to expose the heart. After opening of the pericardium using an 8-0 pvdf suture, the left anterior descending (LAD) coronary artery was tied 1 mm below the tip of the left auricle. Ischemia was verified by the sudden regional paleness of the myocardium and ST elevation. The chest cavity was closed by bringing the ribs together with 6-0 suture while pressure was applied at the level of the xyphoïd process. Muscle and skin layers were closed and the anesthesia was stopped. Mice were extubated at first sign of waking up and allowed to recover in a heating box. Only LAD ligation was omitted in the SHAM procedure. Moclobemide treatment: moclobemide was given 15 min after coronary artery ligation by IP injection at a dose of 20 mg/kg. In the following day, moclobemide was given in the drinking water at a concentration of 20/mg/kg/day and kept during the 4 remaining weeks. The number of animals required for the study has been evaluated from the first validation experiment performed on a small cohort of mice (n=12). Power analysis was conducted to estimate the appropriate sample size by setting the probability of a Type I error (α) at 0.05, power at 0.95, and effect size at 1,5.

Echocardiography

Mice were anesthetized with 2% isoflurane and examined with noninvasive echocardiography (Vivid 7 ultrasound, GE; vevo2100 Visual Sonics). Cardiac ventricular dimensions were measured in a blinded fashion on M-MODE/2D images for the number of animals indicated.

Whole heart imaging

Heart optical clearing was performed as follow. Formalin-fixed hearts were dehydrated with three successive immersions in 100% methanol solutions. Samples were transferred into methanol/BABB solution (one part benzyl alcohol, two parts benzyl benzoate and three parts of methanol) then in BABB solution (one part benzyl alcohol and two parts benzyl benzoate), and incubated for 2 days. Cleared hearts were imaged using Light-Sheet Fluorescence Microscopy (LSFM) as described in Abadie et al. [START_REF] Abadie | 3D imaging of cleared human skin biopsies using light-sheet microscopy: A new way to visualize in-depth skin structure[END_REF].

Images were processed with the open-source image-processing Fiji. 3D volume rendering, segmentation and measurements were performed with Amira ® software (FEI Visualization Sciences Group, Hillsborough, OR, USA).

Primary cultures of cardiomyocytes

Neonatal rat ventricular myocytes (NRVM) isolation: Neonatal rats of 1-2 days old were euthanized by decapitation. The heart was excised and the atria were removed. Primary culture of NRVMs was subsequently performed as previously described [START_REF] Morel | cAMP-binding protein Epac induces cardiomyocyte hypertrophy[END_REF]. NRVMs were transduced with an adenovirus expressing rat MAO-A under the control of the CMV promoter to drive expression of MAO-A. 24h later, the medium was replaced with Ham-F12 medium supplemented with 3% dialyzed FBS, and pharmacological treatments were performed.

Adult Mice Cardiomyocytes (AMCMs):

The heart was quickly excised, and the aorta was cannulated for retrograde perfusion in a Langendorff apparatus at a constant flow rate of 3 mL/min at 37°C as previously described [START_REF] Fazal | Multifunctional Mitochondrial Epac1 Controls Myocardial Cell Death[END_REF]. Freshly isolated cardiomyocytes were plated on laminin-coated culture dishes in M199 complete medium (M199 medium with added 100 IU/mL penicillin, 2 mmol/L L-carnitine, 5 mmol/L creatine and 5 mmol/L taurine) at 37 °C in a humidified atmosphere with 5 % CO 2 . The culture protocol yielded an average of 80 % rod-shaped myocytes at a plating density of 50 cells/mm 2 that were viable at pH 7.2 for 48 h.

Mitochondria isolation

Hearts were excised, washed in phosphate-buffered saline (PBS), and crushed using a tissue grinder in mitochondrial isolation buffer (0,3 M sucrose, 5 mM TES, 200 μM EGTA) supplemented with Proteinase bacterial (Sigma-Aldrich). Mitochondrial isolation buffer supplemented with BSA was then added to the homogenates before centrifugation at 500g for 10 min at 4°C. Supernatant was collected and centrifuged at 3000g for 10 min at 4°C and pellet corresponding to mitochondria was either immediately used (for mitoH 2 O 2 , mitoNAD + /NADH, mitoCa 2+ measurements) or kept at -80°C (for western blots, LC-MS/MS for cardiolipin and HODEs).

Immunofluorescence and histological studies

Heart tissues were embedded in optimal cutting temperature compound (OCT) (Sigma-Aldrich) under icecold 2-methylbutane. For immunofluorescence studies, frozen sections (5 µm) were fixed in 4% paraformaldehyde, followed by permeabilization and blocking in PBS with 0.02% FBS, 1% bovine serum albumine and 0.3% Triton X-100 at RT. Sections were immunostained overnight with the following antibodies: anti-4-HNE (Abcam 46545), anti-Vinculin (Sigma-Aldrich V9131) followed by secondary Alexafluor antibodies (Molecular Probes). Nuclei were visualized with DAPI. Images were acquired by confocal Microscope Zeiss LSM 780 and ZEN image analysis software (Zeiss) or digitized with a Hamamatsu NanoZoomer and fluorescence intensity was quantified by ImageJ software. Average cardiomyocyte diameter and the number of cardiomyocytes per total myocardial area were measured manually after vinculin staining (500-600 cells counted per heart) on 6 different regions of the peri-infarct zone. For fibrosis, hearts were transversely sectioned at 10 μm thickness, fixed with 4% paraformaldehyde and stained with Masson's Trichrome. Fibrosis was measured as positively stained area with Masson's Trichrome and expressed as percent of total area, using ImageJ software (RSB). For NRVMs, cells stained with MitoTracker™ Red (Thermo Fischer Scientific M22425) were fixed with 4% PFA and incubated in TBS-0,2% triton O/N at 4°C with 4-HNE antibody. After secondary antibody (anti-rabbit IgG alexa-fluor 488, Invitrogen), slides were observed under confocal microscopy (LSM780, Carl Zeiss).

Western blot

Total extracts of ventricle or NRVMs were lysed in RIPA buffer (50mM Tris pH 7.2, 500mM NaCl, 1% Triton X-100, 1mM EDTA, 100mM sodium fluoride, 5mM sodium metavanadate, 10mM sodium pyrophosphate) and equal protein amounts were subjected to SDS-PAGE. After electrophoresis, proteins were transferred to PVDF membranes and immunoblotted with the following antibodies: anti-4-HNE (Abcam 46545), anti-MAO-A (Abcam 126751), anti-ALDH2 (Abcam 108306), anti-GAPDH (CST5174), anti-Vinculin (Sigma V9131), anti-COX-IV (CST4844), anti-CBARA1/MICU1 (CST12524), anti-EFHA1/MICU2 (Abcam 101465), anti-MCU (CST14997), anti-VDAC1 (CST4866). Proteins were detected by chemiluminescence with a Bio-Rad ChemiDoc. Relative densities were quantified using the ImageLab 5.2.1 software (Bio-Rad). All data were normalized by internal controls.

Immunoprecipitation

NRVMs, AMCMs or heart tissues were lysed in buffer containing 20 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X100, protease and phosphatase cocktail inhibitors (Roche). Proteins were put in agitation O/N at 4°C with 5-10 μg of appropriated antibodies. Purification steps were performed with protein A/G agarose according to the manufacturer's instructions (SantaCruz Biotechnologies). For improved Western blotting detection to avoid heavy and light chains signals, blots were incubated 2 h at RT with a Protein G-HRP (dilution 1:5000, Biorad), prepared in blocking buffer.

After washing with TBST, blots were developed with ECL Plus (Amersham Biosciences).

ALDH2 activity, GSH measurements and ATP content

ALDH2 activity was determined in ventricular homogenates or NRVMs using ALDH2 assay kit (Abcam).

GSH content was evaluated in ventricle samples using "Total Glutathione detection kit" from Assay Designs (Enzo life sciences). Intracellular ATP content was determined by measuring the conversion of D-luciferin to oxyluciferin using ATP bioluminescence assay kit II according to the manufacturer's instructions (Roche).

Mitochondrial H 2 O 2

Cells were incubated with mitoPY1 probe (Mitochondria Peroxy Yellow-1, Sigma) at a final concentration of 10 μM at 37°C for 60 min. Mitochondrial specificity was confirmed by co-staining with MITO-ID Red (Enzo life sciences). Representative pictures were taken with a confocal microscope (LSM780, Carl Zeiss). Mitochondrial H 2 O 2 production in mouse hearts was measured using fluorescent probe Amplex Red (10 µmol/L) in the presence of 0.6 U/ml horseradish peroxidase (excitation and emission wavelengths set to 530 and 590 nm, respectively) on isolated mitochondria.

Mitochondrial membrane potential (ΔΨm)

TMRE (Tetramethylrhodamine, ethyl ester) probe was added to each well at a final concentration of 500 nM at 37°C for 30 min. Then, medium was replaced by HBSS and the fluorescence was recorded in the presence or not of FCCP at Ex/Em = 549/575 nm, using a fluorimeter TECAN infinite pro F200.

Mitochondrial respiration

Oxygen consumption rate (OCR) was measured in AMCMs with a Seahorse XFe24 Analyzer (Agilent).

Cells were plated in laminin-coated Seahorse 24-well assay plates (2500 cells per well) in M199 complete medium. After 2 h of cell attachment, cell medium was replaced with XF base medium supplemented with 10 mM glucose, 4 mM L-glutamine and 1 mM sodium pyruvate (pH 7,4) and maintained at 37°C for 1h.

The wells of a hydrated sensor cartridge were then loaded with 1 µM oligomycin (port A), 1 µM FCCP (port B) and 1 µM antimycin A + 1 µM rotenone (port C). Data were analyzed with Seahorse Wave software. Maximal respiratory capacity was calculated by subtracting the non-mitochondrial OCR (minimum rate measurement after antimycin A/rotenone injection) from the OCR after FCCP injection.

Spare respiratory capacity was calculated by subtracting the baseline OCR from the OCR after FCCP injection.

Measurement of mitochondrial Ca 2+ uptake

Intact cardiomyocytes or freshly isolated mitochondria were suspended in Tyrode buffer and incubated with 10 μmol/L Rhod-2-acetoxymethyl ester (Rhod-2-AM; Life Technologies, France) for 30 min at 4°C, and then for 30 min at 37°C [START_REF] Fazal | Multifunctional Mitochondrial Epac1 Controls Myocardial Cell Death[END_REF]. With this cold loading/warm incubation procedure, Rhod-2-AM localized to mitochondria. Typically, successive 1ml aliquots of cells/mitochondria suspension were loaded and experimented on for the same amount of time. Fluorescence (λ excitation: 540 nm, λ emission: 605nm) was acquired from cells/mitochondria treated or not with Rhod-2-AM on a Varioskan Flash Multimode Microplate Reader for 270 sec. In each experiment, 20 μmol/L CaCl 2 was added. Experiments were performed at least with 5-6 mice/group.

Duolink Proximity Ligation In Situ Assay

Briefly adult mouse cardiomyocytes were fixed with 4 % paraformaldehyde and permeabilized with 0.1 % Triton X-100. Subsequent blocking, antibody hybridizations, proximity ligations, and detections were performed according to recommendations from manufacturers (OLINK Bioscience, Sigma-Aldrich, France). The cells were incubated with the primary antibodies overnight at 4°C and then washed 3 times with TBS-0.05 % Tween 20. Briefly, after incubation with primary antibodies, we applied combinations of corresponding in situ proximity ligation assay (PLA) probes for 1 h at 37°C. The cells were washed with TBS-0.05 % Tween 20, incubated for 30 min with ligase, and finally washed with TBS-0.05% Tween 20. Then cells were incubated with polymerase for 100 min. Fluorescence was analyzed with a laser confocal Zeiss LSM780 microscope. Quantification of signals was performed with BlobFinder software (Center for Image Analysis, Uppsala University) and expressed as interactions per cell relative to the non-treated group. Experiments were performed at least 4 times, with a minimum of 6-8 fields taken per condition.

Size-exclusion Chromatography

Cells were washed in PBS and lysed in IP buffer (20 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 % Triton X100, protease and phosphatase inhibitors cocktail (Roche). Using a AKTA purifier 10 (GE Life Science), lysates were injected (200 μL per injection) onto a Superose 6 10/300GL column (GE Life Sciences) equilibrated with IP Buffer (pH 7.5). The flow rate was 0.2 mL.min-1 and 40x400 μL fractions were collected using a low protein binding 96-deep-well plate Eppendorf (Hamburg, Germany) and tested on western blots. The system was previously calibrated with High molecular weight markers (GE Life Science).

Statistical analysis

Statistical analysis was carried out using Student's t-test or 2-way ANOVA with the Tukey post hoc test, when appropriate. Lung edema was analyzed statistically using chi square test. The results are shown as the mean ± SEM. Values of p<0.05 were considered to be significant.

RESULTS

MAO-A activation promotes intra-mitochondrial 4-HNE formation through cardiolipin peroxidation

We first sought to determine whether monoamine oxidase-A (MAO-A) influenced mitochondrial 4-HNE.

Application of tyramine (Tyr), a MAO substrate, led to 4-HNE mitochondrial accumulation in adult mice cardiomyocytes (AMCMs), as revealed by co-staining of 4-HNE with the mitochondrial marker Mito-ID (Fig. 1A, Supp Fig. 1A). In transgenic mice with cardiac overexpression of MAO-A, a model of oxidative stress and HF (MAO-A Tg) [START_REF] Villeneuve | p53-PGC-1alpha pathway mediates oxidative mitochondrial damage and cardiomyocyte necrosis induced by monoamine oxidase-A upregulation: role in chronic left ventricular dysfunction in mice[END_REF], isolated mitochondria showed accumulation of 4-HNE-bound proteins compared to WT mice (Fig. 1B). 4-HNE can be formed upon ROS-dependent peroxidation of lipids, in particular cardiolipin (CL) the signature phospholipids of mitochondrial inner membrane, via the synthesis of hydroxyoctadecadienoic acids (HODEs) [START_REF] Liu | Formation of 4-hydroxynonenal from cardiolipin oxidation: Intramolecular peroxyl radical addition and decomposition[END_REF]. We thus investigated whether MAO-A favored intramitochondrial 4-HNE accumulation through ROS-dependent CL peroxidation. In AMCMs, application of Tyr for 30 min increased mitochondrial H 2 O 2 , as shown by overlapped staining of the H 2 O 2 -sensitive probe MitoPY1 with Mito-ID red (Fig. 1C, Supp Fig. 1B). Consistently, mitochondria of MAO-A Tg hearts also displayed elevated levels of H 2 O 2 compared to WT (Fig. 1D). In neonatal rat ventricular myocytes (NRVMs) transduced with MAO-A adenovirus, Tyr-induced mitoH 2 O 2 accumulation preceded the accumulation of 4-HNE (at 1 h and later) (Supp Fig. 1C-E). We next performed LC-MS/MS to analyze mitochondrial content in cardiolipins and HODEs. The major species tetralinoleoylcardiolipin (L 4 CL or 72:8) was significantly decreased in MAO-A Tg mitochondria compared to WT (Fig. 1E, Supp Fig. 1F). This is particularly interesting since L 4 CL is the most abundant CL in mammalian tissues, and is the only form made of four linoleic moieties, the main precursor of 4-HNE. Consequently, we observed an increase in 12(S), 13(S) and 9(S) species of HODEs, which are different oxidation products of linoleic acid (Fig. 1F). Our results indicate that MAO-A-dependent ROS generation promotes intra-mitochondrial formation of 4-HNE through L 4 CL peroxidation and HODEs intermediate formation (Fig. 1G).

MitoALDH2 mitigates MAO-A-induced cardiac dysfunction

In order to evaluate if 4-HNE could mediate the deleterious effects of MAO-A, we overexpressed mitochondrial ALDH2, the main enzyme for 4-HNE detoxification, in hearts of MAO-A Tg mice using a cardiotropic AAV9-ALDH2 (Fig. 2A). Major 4-HNE detoxification systems were inhibited in Tg hearts, as shown by a decrease in cardiac glutathione content (GSH) and mitochondrial ALDH2 activity (Fig. 2B-C). As expected, AAV9-ALDH2 restored basal level of ALDH2 activity and reduced 4-HNE accumulation in MAO-A Tg hearts (Fig. 2D-F). Importantly, this effect of ALDH2 was associated with a reduction in reactive hypertrophy and interstitial fibrosis (Fig. 2E-H). An improved cardiac function was also observed in AAV9-ALDH2 Tg mice compared to AAV9-GFP Tg mice, as demonstrated by the echocardiographic data (Fig. 2I-J, Table 1). These results show that mitochondrial ALDH2 protects from MAO-A-induced 4-HNE accumulation and cardiac dysfunction in MAO-A Tg.

MAO-A-induced 4-HNE accumulation alters mitochondrial function

As we demonstrated that mitochondrial 4-HNE mediated the deleterious effects of MAO-A, we next sought to investigate by which downstream mechanisms this occurred. Quantitative mass spectrometrybased (nanoLC-MS/MS) proteomic study after immunoprecipitation with an anti-4-HNE antibody identified 53 proteins enriched at least 2-fold in MAO-A Tg vs WT cardiomyocytes, with a cutoff p-value <0.05 (Table 2). The majority of proteins were mitochondrial, confirming the existence of an organellespecific regulation of 4-HNE downstream MAO-A. Analysis of over-represented networks of 4-HNEbound proteins with Reactome (https://reactome.org/) identified five major pathways that belong to mitochondrial metabolism (respiratory electron transport, citric acid cycle), mitoCa 2+ transport, glucose metabolism and muscle contraction (Supp Fig. 2). We thus assessed the functional relevance of these pathways in response to MAO-A/4-HNE axis in AMCMs of WT mice. Activation of MAO-A with Tyr altered mitochondrial respiration, as shown by the impairment in oxygen consumption rate (OCR), an effect prevented with the selective MAO-A inhibitor moclobemide and with Alda-1, an allosteric activator of ALDH2 (Fig. 3A-B, Supp Fig. 3A-D). Interestingly, direct application of 4-HNE on AMCMs was sufficient to impair mitochondrial respiration (Supp Fig. 4). A decrease in ATP concentration was observed after Tyr stimulation, and this effect was prevented by Moclobemide and Alda-1 (Fig. 3C).

MAO-A activation also decreased NAD+/NADH ratio and altered mitochondrial membrane potential but not in the presence of Moclobemide and Alda-1 (Fig. 3D-E). Altogether, our data show that mitochondrial 4-HNE mediates the deleterious effects of MAO-A on mitochondrial function.

MAO-A/4-HNE axis induces mitochondrial Ca 2+ accumulation

Because mitoCa 2+ handling proteins were identified as potential 4-HNE targets (Supp Fig. 2), we measured the effects of MAO-A/4-HNE on mitoCa 2+ . Cardiomyocytes or mitochondria of MAO-A Tg mice showed elevated levels of mitoCa 2+ compared to WT mice, as shown by the colocalization of the fluorescent probe Rhod-2-AM with MitoTracker (Fig. 4A,4B,4C). Interestingly, activation of MAO-A with Tyr in WT AMCMs or mitochondria induced mitoCa 2+ accumulation, an effect prevented with moclobemide or Alda-1 (Fig. 4D,4E,4F). MitoCa 2+ uptake is regulated at different levels. It is driven by close interactions between the endoplasmic reticulum (ER) and the mitochondria called MAMs, resulting in the transfer of Ca 2+ through the outer membrane by VDACs, followed by the accumulation Ca 2+ into the matrix via the MCU on the inner membrane [START_REF] Giorgi | Mitochondriaassociated membranes: composition, molecular mechanisms, and physiopathological implications[END_REF]. As observed in the proteomic study, an increased association between VDAC1 and 4-HNE was shown by co-immunoprecipitation experiments in AMCMs stimulated with Tyr (Fig. 5A). We evaluated the interactions between ER and mitochondria, which involve associations of VDAC1, IP3R1 and GRP75. Proximity Ligation Assay showed that AMCMs of MAO-A Tg mice displayed increased interactions between VDAC1/IP3R1, VDAC1/GRP75 and IP3R1/GRP75 compared to WT mice, potentially favoring Ca 2+ transfer (Fig. 5B-E). Interestingly, in WT AMCMs, histamine, a known stimulator of IP3R-mediated Ca 2+ transfer from ER to mitochondria, enhanced Tyr-induced mitoCa 2+ accumulation (Fig. 5F,5G). On the other hand, Xestospongin C, a specific inhibitor of IP3R, reduced Tyrmediated mitoCa 2+ transfer (Fig. 5F,5G). Thus, MAO-A/4-HNE axis may potentiate Ca 2+ transfer through increased interactions between ER and mitochondria and enhanced Ca 2+ outer membrane transfer through VDAC1.

4-HNE binding to MCU induces MitoCa 2+ overload and mitochondrial dysfunction

Ca 2+ transport through the inner membrane occurs via the MCU, which is regulated by its two main partners MICU1 and MICU2 [START_REF] Patron | MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity[END_REF]. We investigated whether MCU also participated in mitoCa 2+ accumulation in response to MAO-A/4-HNE. Tyr-mediated mitoCa 2+ accumulation was significantly reduced by the MCU inhibitor Ru360, in both isolated WT AMCMs and purified mitochondria (Fig. 4E,4F). In line with these findings, direct application of 4-HNE on AMCMs promoted mitoCa 2+ accumulation, an effect inhibited by RU360 (Fig. 6A). This finding prompted us to determine whether MCU, MICU1 or MICU2 were potential targets of 4-HNE covalent binding. In AMCMs of MAO-A Tg mice, immunoprecipitation assays showed that 4-HNE levels increased on MCU, but not on MICU1 and MICU2 (Fig. 6B, Supp Fig. 5A). In NRVMs treated with Tyr, we also found increased association of MCU and 4-HNE, but not MICU1 or MICU2 (Fig. 6C, Supp Fig. 5B). In order to better understand how 4-HNE adducts could modify MCU-mediated Ca 2+ uptake, we evaluated MCU complex formation, the active form of the channel. Indeed, the MCU per se is not able to form the channel but should organize in oligomers allowing efficient mitoCa 2+ uptake [START_REF] Tomar | MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics[END_REF][START_REF] Wang | Structural Mechanism of EMRE-Dependent Gating of the Human Mitochondrial Calcium Uniporter[END_REF]. Size exclusion chromatography followed by western blot on NRVMs showed that MCU was eluted and detected in a high molecular weight fraction (F1 ≈ 400 kDa) only in the Tyr-stimulated condition, which corresponded to higher-order oligomers (Fig. 6D). Furthermore, in non-reducing western blots conditions, MAO-A Tg cardiomyocytes displayed larger MCU complexes compared to WT (Fig. 6E). Similar findings were observed in NRVMs treated with Tyr or 4-HNE (Fig. 6F, Supp. Fig 5C-D). Importantly, Alda-1 prevented MCU oligomerization, confirming the role of 4-HNE in MAO-A-induced MCU conformational modifications (Fig 6F). In order to assess if MCU functionally regulated mitochondrial function in response to MAO-A activation, we incubated WT AMCMs with Tyr in the presence of the MCU inhibitor RU360. Seahorse measurements indicated that respiration impairment induced by MAO-A could be prevented with RU360 (Fig. 6G, 6H and Supp Fig. 6). Loss of ATP content and mitochondrial membrane depolarization induced by MAO-A were also partially reduced in the presence of RU360 in WT AMCMs (Fig. 6I,6J). Altogether, these results identify for the first time MAO-A as a regulator of MCU channel through 4-HNE-mediated conformational changes and show that MCU activation participates in mitochondrial dysfunction.

MAO-A inactivation protects from 4-HNE accumulation and cardiac remodeling during chronic ischemia

In order to evaluate the pathophysiological relevance of our findings, we developed a model of myocardial infarction (MI) induced by permanent coronary artery ligation in mice. Four weeks post-MI, while ALDH2 expression remained unchanged, both MAO-A levels and 4-HNE-bound proteins were elevated compared to sham-operated mice (Fig. 7A). The same observation was made in ventricular samples of human ischemic cardiomyopathy (Fig. 7B). In order to test if MAO-A/4-HNE axis could be associated with chronic cardiac remodeling, mice were treated with the selective and reversible MAO-A inhibitor, moclobemide (20 mg/kg/day), after the surgery and the treatment was sustained during 4 weeks (Fig. 7C).

Moclobemide efficiently inhibited MAO-A activity and reduced 4-HNE accumulation post-MI (Supp Fig. 7A; Fig. 7D). This was accompanied by reduced cardiac remodeling, as shown by improved ejection fraction, reduced ventricular dilatation, decreased heart-to-body weight ratios and lung edema (Fig. 7E, Table 3, Supp Fig. 7D-E). In order to test if cardiomyocytes were the primary cells responsible for these effects, we used mice with cardio-selective deletion of MAO-A (Maoa fl crossed with αMHC-Cre + mice) (Supp Fig. 7B-C). Cardiomyocyte-specific deletion of MAO-A reduced 4-HNE accumulation and mitigated contractility impairment and dilatation at 4 weeks post-MI (Fig. 7D-E, Supp Fig. 7G-H, Table 3). Heart-to-body weight ratios and lung congestions were also reduced in MAO-A cKO mice compared to WT mice (Supp Fig. 7D-E). To evaluate the volume of the scar zone, we performed 3D-whole heart imaging using high-resolution LSFM. As shown by 3D reconstructions in Fig. 7F, the scar zone (blue) represented 14 % of total heart in WT mice while it was reduced to 5% in MAO-A cKO mice, suggesting that MAO-A deletion limited expansion of the infarct (Supp Fig. 7F). These results were confirmed with Masson's Trichrome showing a marked decrease in the scar area of MAO-A cKO hearts compared to WT (Fig. 7F, Supp Fig. 7F). Histologically, MAO-A cKO hearts had lessened reactive hypertrophy in the remote zone of the infarct compared to WT mice (Fig. 7G) while interstitial fibrosis was slightly decreased (Supp Fig. 7G-I). Next, we evaluated if the newly identified mechanism of action of 4-HNE on MCU/mitoCa 2+ was dysregulated in ischemic remodeling. Most interestingly, we observed a coimmunoprecipitation of 4-HNE with MCU in MI hearts, associated with MCU oligomer formation, and this was inhibited by moclobemide (Fig. 7H-I). Moreover, cardiac mitochondria of ischemic hearts displayed higher mitoCa 2+ levels compared to SHAM mice, and this effect was attenuated in MAO-A cKO mice (Fig. 7J). Altogether, these results strongly support a role for cardiac MAO-A in the generation of deleterious 4-HNE and MCU-mediated Ca 2+ overload during chronic remodeling.

Discussion

In this study, we show for the first time that overactivation of MAO-A leads to intra-mitochondrial generation of 4-HNE with functional consequences on mitoCa 2+ transport, mitochondrial dysfunction and chronic ventricular remodeling post-MI (Fig. 8). To our knowledge, this constitutes the first evidence of a link between MAO-A activation during cardiac injury and the previously recognized deleterious effects of

4-HNE. Recently, it has been shown that human endstage ischemic hearts displayed an increase in MAO-

A and MAO-B activities, but the causative role of these enzymes in the pathogenesis of chronic ischemic remodeling and the associated mechanisms were not investigated [START_REF] Manni | Monoamine Oxidase Is Overactivated in Left and Right Ventricles from Ischemic Hearts: An Intriguing Therapeutic Target[END_REF]. Here, we show that pharmacological or genetic inhibition of MAO-A reduces deleterious remodeling in mice with coronary artery ligation. The role of MAO-A has been previously demonstrated in acute ischemia/reperfusion injury [START_REF] Pchejetski | Oxidative stressdependent sphingosine kinase-1 inhibition mediates monoamine oxidase A-associated cardiac cell apoptosis[END_REF] and in pressure overload-induced cardiac remodeling [START_REF] Kaludercic | Monoamine oxidase Amediated enhanced catabolism of norepinephrine contributes to adverse remodeling and pump failure in hearts with pressure overload[END_REF]. Its mechanisms of action have been delineated in a mouse model of MAO-A overexpression that, despite high MAO-A expression levels, is useful in deciphering specific pathways downstream MAO-A [START_REF] Santin | Oxidative Stress by Monoamine Oxidase-A Impairs Transcription Factor EB Activation and Autophagosome Clearance, Leading to Cardiomyocyte Necrosis and Heart Failure[END_REF]. However, previous studies mainly focused on the mechanisms of action of H 2 O 2 . Recently, Kaludercic et al. suggested a role for aldehydes in MAO-B-induced deleterious effects, showing a worsening effect of in vitro ALDH2 inhibition after dopamine treatment [START_REF] Kaludercic | Monoamine oxidase B prompts mitochondrial and cardiac dysfunction in pressure overloaded hearts[END_REF]. Here, we unravel for the first time that lipid-derived aldehydes, in particular the most abundant 4-HNE, act downstream MAO-A to mediate mitochondria dysfunction. Mounting evidence has linked lipid peroxidation aldehydes such as 4-HNE with various human pathological states [START_REF] Mak | Unsaturated aldehydes including 4-OH-nonenal are elevated in patients with congestive heart failure[END_REF] and with mice models of HF [START_REF] Gomes | Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling[END_REF]. However, the mechanisms of 4-HNE mitochondrial accumulation as well as the identification of its mitochondrial targets remained poorly understood [START_REF] Xiao | Pathophysiology of mitochondrial lipid oxidation: Role of 4-hydroxynonenal (4-HNE) and other bioactive lipids in mitochondria[END_REF]. Here, we show that MAO-A is one of the factors driving 4-HNE mitochondrial accumulation through L 4 CL peroxidation. The loss of L 4 CL in mitochondrial membrane constitutes a hallmark of cardiomyopathy, with consequences on the enzymatic activity of cytochrome oxidase and respiratory capacity [START_REF] Sparagna | Loss of cardiac tetralinoleoyl cardiolipin in human and experimental heart failure[END_REF]. However, this alteration of L 4 CL is not sufficient to explain the mitochondrial dysfunction observed with MAO-A, as ALDH2 activation conferred significant protection. This indicates that 4-HNE mitochondrial accumulation plays a major role in MAO-dependent mitochondrial damage.

Our results also put forward that 4-HNE acts at its site of formation since: i) 4-HNE accumulates within the mitochondria after MAO activation, ii) overexpression of ALDH2 activity, a mitochondrial enzyme, rescues MAO-A-mediated HF ; iii) Mass spectrometry of 4-HNE-bound proteins shows overrepresentation of mitochondrial proteins. The main pathways identified by proteomic study were "mitochondrial metabolism, glucose metabolism, mitoCa 2+ transport and muscle contraction". Interestingly, two targets of 4-HNE (NDUFS2, SDHA) were also found in large-scale analysis of 4-HNEbound proteins in doxorubicin-induced cardiomyopathy, which suggests the existence of common targets in stressed cardiomyocytes [START_REF] Zhao | Redox proteomic identification of HNE-bound mitochondrial proteins in cardiac tissues reveals a systemic effect on energy metabolism after doxorubicin treatment[END_REF].

Due to its key role in bioenergetics, redox homeostasis and cell death, mitochondrial dysfunction is a critical factor in the development and progression of HF [START_REF] Brown | Mitochondrial function as a therapeutic target in heart failure[END_REF]. Of particular interest, mitoCa 2+ overload seems to be an important determinant of ischemic ventricular remodeling. A recent study showed that blockade of mitoCa 2+ extrusion by NCX channel promoted infarct expansion and death of border cardiomyocytes (4). Santelli et al. also demonstrated that leaky Ryr2 caused mitoCa 2+ overload and dysfunction in HF [START_REF] Santulli | Mitochondrial calcium overload is a key determinant in heart failure[END_REF]. Interestingly, mitoCa 2+ amounts were lessened in MAO-A cKO mice compared to WT after ischemia, which was associated with a better cardiac function, identifying MAO-A as an activator of mitoCa 2+ entry. In addition, direct 4-HNE treatment led to mitoCa 2+ overload and mitochondrial dysfunction in both isolated cardiomyocytes and mitochondria. The mechanism by which Ca 2+ overload induces mitochondrial dysfunction remains incompletely understood. One hypothesis could be that mitoCa 2+ exacerbates ROS generation, further impairing mitochondrial function through the generation of superoxide due to mild uncoupling of mitochondria (4).

Of particular importance, we identified VDAC and MCU as relevant targets of 4-HNE adduct formation following MAO-A activation. The entry of Ca 2+ inside the mitochondria is highly regulated by the communication between the ER and the mitochondria called MAMs [START_REF] Giorgi | Mitochondriaassociated membranes: composition, molecular mechanisms, and physiopathological implications[END_REF] and the association of the VDAC1/GRP75/IP3R complex [START_REF] Paillard | Depressing Mitochondria-Reticulum Interactions Protects Cardiomyocytes From Lethal Hypoxia-Reoxygenation Injury[END_REF]. MAO-A Tg mice exhibited higher levels of MAMs compared to WT mice, which could favor mitoCa 2+ entry from the ER. As we and others [START_REF] Dodson | Regulation of autophagy, mitochondrial dynamics, and cellular bioenergetics by 4-hydroxynonenal in primary neurons[END_REF] demonstrated that 4-HNE covalently bound to VDAC1, it is possible that the carbonylated form of VDAC1 has better interactions with its partners GRP75 and IP3R1, strengthening mitoCa 2+ transfer during MAO-A activation. We also observed that MAO-A-dependant 4-HNE formation led to the formation of higher-order MCU oligomers.

MCU oligomer formation is sensitive to post-translational modifications, among which oxidation of Cys-97, resulting in persistent MCU channel activity and higher mitoCa 2+ uptake [START_REF] Dong | Mitochondrial Ca(2+) Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity[END_REF]. As 4-HNE exhibits the strongest reactivity for Cys residues, we can hypothesize that increased 4-HNE binding could modify MCU activity through similar conformational changes. These results put forward MAO-A as a regulator of MCU through 4-HNE-mediated post-translational modification and subsequent conformational changes. Finally, we provide evidences that MCU-mediated mitoCa 2+ overload following MAO-A/4-HNE activation is deleterious, as mitochondrial dysfunction is prevented with the MCU inhibitor RU360. While it has been clearly demonstrated that increasing the levels of mitoCa 2+ was deleterious in pathological cardiac remodeling [START_REF] Luongo | The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability[END_REF][START_REF] Santulli | Mitochondrial calcium overload is a key determinant in heart failure[END_REF], the selective inhibition of MCU has given conflicting results. Global MCU-/had normal response to isoproterenol or pressure overload stress [START_REF] Holmstrom | Assessment of cardiac function in mice lacking the mitochondrial calcium uniporter[END_REF][START_REF] Pan | The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter[END_REF], suggesting the existence of compensatory mechanisms. On the other hand, cardiac-specific ablation of MCU in adult mice protected mice from I/R damage [START_REF] Luongo | The Mitochondrial Calcium Uniporter Matches Energetic Supply with Cardiac Workload during Stress and Modulates Permeability Transition[END_REF] and pharmacological inhibition of MCU was beneficial in pressure-overloaded hearts [START_REF] Yu | Mitochondrial calcium uniporter inhibition provides cardioprotection in pressure overload-induced heart failure through autophagy enhancement[END_REF]. In the present study, our results highlight for the first time a deleterious role for MCUdependent Ca 2+ overload in chronic ischemic remodeling. In conclusion, we propose a model whereby excessive activation of MAO-A in cardiac stress conditions promotes mitochondrial 4-HNE accumulation and binding on VDAC1 and MCU, both acting in a synergistic way to promote excessive mitoCa 2+ entry and mitochondrial dysfunction (Fig. 8).

Finally, our identification of MAO-A as a major source of mitochondrial 4-HNE during post-MI remodelling paves the way toward targeted therapies aimed at decreasing aldehyde load, mitoCa 2+ dysregulation and energetic depletion in HF. As the MAO-A inhibitor moclobemide is approved in Europe and used as an antidepressant, it would be interesting to consider the possibility of repurposing this drug for HF therapy. IVSs (cm) 0,18 ± 0,01 0,17 ± 0,004 0,16 ± 0,01*** 0,15 ± 0,003**

LVIDs (cm)

0,19 ± 0,02 0,17 ± 0,01 0,27 ± 0,01*** 0,23 ± 0,01 # LVIDd (cm) 0,34 ± 0,02 0,31 ± 0,01 0,38 ± 0,01 0,36 ± 0,01

LVPWs (cm)

0,16 ± 0,01 0,19 ± 0,01 0,15 ± 0,01 0,17 ± 0,01

EDV (ml)

0,10 ± 0,01 0,08 ± 0,01 0,14 ± 0,01 0,12 ± 0,01

ESV (ml)

0,020 ± 0,004 0,013 ± 0,002 0,053 ± 0,006*** 0,036 ± 0,003 # EF (%)

Figure legends Fig 1 .

 legends1 Figure legends

Fig 2 . 4 -

 24 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 (3x10 11 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 = 100μm) 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 3 distinct regions of the left ventricle (n=6-7). (H) Quantification of fibrosis as % of total area (n= 4-6). (I, J) 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 .

 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, 3h) 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/F FCCP ), 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 .

 4 Fig 4. MAO-A activation induces 4-HNE-dependent increase in mitoCa 2+ levels. (A) Representative images of mitochondrial Ca 2+ (mitoCa 2+ ) accumulation in WT or MAO-A Tg AMCMs. The Ca 2+ fluorescent probe Rhod-2-AM overlapped with the mitochondrial marker Mitotracker green. Quantification of mitoCa 2+ accumulation in AMCMs (B) and isolated mitochondria (C) of MAO-A Tg mice (n=5-6). (D, E) Representative images and quantification of mitoCa 2+ 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 Ca 2+ 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 .

 5 Fig 5. Implication of MAMs in MAO-A-induced mitoCa 2+ 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 mitoCa 2+ accumulation in AMCMs of WT mice treated with Tyr and challenged with histamine (100 µM) or Xestospongin C (2 µM). (G) Quantifications of mitoCa 2+ 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 .

 6 Fig 6. MAO-A/4-HNE axis regulates mitoCa 2+ -induced mitochondrial dysfunction through MCUbinding and higher order complex formation. (A) Quantifications of mitoCa 2+ accumulation in AMCMs isolated from WT mice treated with 4-HNE (5 µM, 15 min) in the presence of RU360 (10 µM). n=4. (B, C) 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, 1h). 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 (AKTA purifier 10, GE Life Science) coupled to a Superose 6 10/300GL column, after stimulation with Tyr (500µM, 4h). The elution profile of molecular complexes are 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. The annotated molecular weights were estimated from the elution profiles of standard markers injected onto the SEC column. (E, F) Representative non-reducing 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/F FCCP ) 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 .

 7 Fig 7. Effect of Moclobemide or MAO-A deficiency on myocardial infarction (MI)-induced 4-HNE accumulation and cardiac remodeling. (A-B) 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 non-reducing 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 Ca 2+ accumulation in isolated mitochondria of WT and MAO-A cKO mice subjected to 1 week ischemia (n=5-7).

Fig 8 .

 8 Fig 8. Schema illustrating the mechanisms associated with MAO-A/4-HNE signaling during chronic ischemia (right panel) compared to normal conditions (left panel).

TABLE 3 .

 3 Echocardiographic parameters of mice treated with Moclobemide (20 mg/kg/d) or MAO-A cKO mice 4 weeks after myocardial ischemia (2D imaging) Ad, end-diastolic area ; EDV, end-diastolic volume ; As, end-systolic area ; ESV, end-systolic volume ; EF, ejection fraction. **p<0.01, ***p<0.001 vs. corresponding SHAM mice; #p<0.05, ##p<0.01 vs. mice treated with Vehicle or WT mice for MI condition.

		SHAM		MI
		Vehicle	Moclobemide	Vehicle	Moclobemide
	N	6	5	13	11
	Ad, mm 2	24,3 ± 1,2	26,6 ± 0,6	43,7 ± 1,6***	38,1 ± 2,3** ,#
	EDV, ml	0,068 ± 0,005	0,077 ± 0,002	0,199 ± 0,093***	0,15 ± 0,01*** ,##
	As, mm 2	15,7 ± 0,7	19,0 ± 0,6	40,4 ± 1,7***	32,95 ± 2,25*** ,##
	ESV, ml	0,033 ± 0,002	0,045 ± 0,002	0,170 ± 0,012***	0,125 ± 0,014*** ,#
	EF (%)	50,7 ± 0,7	43,9 ± 1,6	11,7 ± 1,7***	20,0 ± 2,5*** ,##
	HR, bpm	479,38 ± 12,61	398,49 ± 6,88*	503,79 ± 18,65	470,66 ± 15,91
		SHAM		MI
		WT	MAO-A cKO	WT	MAO-A cKO
	N	4	4	8	8
	Ad, mm 2	26,10 ± 0,97	29,45 ± 0,98	49,94 ± 3,15***	37,82 ± 1,69* ,##
	EDV, ml	0,078 ± 0,005	0,073 ± 0,018	0,234 ± 0,025***	0,151 ± 0,012* ,#
	As, mm 2	12,5 ± 2,5	17,5 ±2,5	42,9 ± 3,6***	32,5 ± 1,6*** ,#
	ESV, ml	0,030 ± 0,004	0,035 ± 0,006	0,188 ± 0,023***	0,106 ± 0,010* ,##
	EF (%)	47,5 ± 1,2	48,3 ± 0,6	21,2 ± 2,2***	29,5 ± 1,8*** ,#
	HR, bpm	500,25 ± 22,93	460,75 ± 14,59	540,13 ± 21,17	466,78 ± 21,52

TABLE 2. List of 53 4-HNE-bound proteins in cardiomyocytes of MAO-A Tg mice compared to WT

mice (cut-off ratio>2 and p-value<0.05)

Acknowledgements

We thank X. Sudre (Zootechnie UMS006), R. D'angelo (Imaging Platform I2MC) and A. Lucas (We-Met Platform I2MC) for technical support, L. Pieruccioni for whole heart imaging (TRI-ITAV, USR3505, Toulouse), Julien Colombelli for LSFM development (Advanced Digital Microscopy Core Facility, IRB, Barcelona, Spain), JP Pais-De-Barros (Plateforme de Lipidomique-uBourgogne, INSERM UMR1231, Dijon, France) for cardiolipin measurements. This work was supported by grants from Agence Nationale pour la Recherche referenced as "ANRJCJC CARDIOMAO", "ProFI-ANR-10-INBS-08", "ANR-17-CE14-0014-01", grants from European funds (FEDER), Fondazione Cariplo (2014-0672), Fondation pour la Recherche Médicale (équipe FRM2016, DEQ20160334892) and Région Occitanie.


Tiago Silva
email: tiago.silva@eui.eu

'Trendy' Cities: Exploring the adoption of different types of social media by Portuguese municipalities

Keywords: Local Government, Social Media, Transparency, E-Participation

What are the determinants of social media adoption by local government? This ongoing research provides a tentative answer to this question by analysing the 308 municipalities in Portugal. Extending previous analyses of Facebook and/or Twitter usage levels, we examine why local governments adopt a particular social media platform. More concretely, we explore, with statistical analyses, the determinants of the adoption of different types of social media. We investigate the adoption of three extremely popular social media (i.e. Facebook, Twitter and YouTube) as well as possible alternatives to those, more popular, applications. Since these platforms have distinct natures and can serve diverse purposes, we examine to what extent aspects such as local government's commitment to transparency and participation, administrative capacity, media landscape, and socio-demographic and economic factors can explain the adoption of social media platforms. The results show that, indeed, demographic characteristics and administrative capacity are important factors for the adoption of less popular social media. Surprisingly, we also observe a geographical difference in municipalities' social media adoption, with the south, in this regard, being 'trendier', or more innovative, than the north.

Introduction and short literature review

Recently, much has been written in academia about social media. Undoubtedly, these inexpensive and user-friendly online platforms became extremely popular and widely used around the world. With billions of users worldwide, we live in a post mass media society where a large part of the population relies on those online applications to get information [START_REF] Gottfried | News Use Across social medial Platforms 2016[END_REF]. Naturally, the popularity of social media has also captured the attention of public administration and different levels of government. By allowing their users to generate and share original content within very large, and relatively diverse, networks, social media have opened the door to real-time interaction between public sector officials and citizens [START_REF] Rybalko | Dialogic communication in 140 characters or less: How Fortune 500 companies engage stakeholders using Twitter[END_REF]. Understandably, this has brought some expectations to improve public administration, different levels and sectors of government and, more broadly, democracy as well. Amid reports of a steady increase, in the last decades, of citizens' discontent with democratic institutions [START_REF] Dalton | Citizen Politics: Public Opinion and Political Parties in Advanced Industrial Democracies[END_REF][4], it seems that social media can play an important role in the future of our societies by promoting, together with other ICTs, a culture of receptiveness and transparency in public organizations and political institutions [START_REF] Bertot | Using ICTs to create a culture of transparency: E-government and social media as openness and anti-corruption tools for societies[END_REF]. Indeed, under certain conditions, the Internet and social media can contribute positively to political participation [START_REF] Bakker | Good news for the future? Young people, Internet use, and political participation[END_REF], even though, despite this recognized capacity, they can as well lead to the exact opposite effect [START_REF] Kent | Using social media dialogically: Public relations role in reviving democracy[END_REF]. In the end, this perceived potential of social media to mitigate important challenges in established democracies make the study of the adoption and usage of those platforms by political and public organizations extremely relevant. In this paper we focus on social media use by local government.

Regardless of the reasons behind their adoption and use (i.e. either simply to inform or to promote citizens' interaction, participation and collaboration), a large number of local governments are nowadays incorporating social media in their communication repertoire [START_REF] Bonsón | Local e-government 2.0: social media and corporate transparency in municipalities[END_REF]. The study of local government use of social media has been mainly focusing on frequency of use (e.g. post count) and levels of citizen's engagement with the messages [START_REF] Lameiras | An Empirical Analysis of social media Usage by Local Governments in Portugal[END_REF]. Much less attention has been paid to a) what are the types of social media adopted by the municipalities and b) the communication strategies of the municipalities for those platforms. This paper addresses precisely those two untapped aspects of social media use by local governments, offering an empirical contribution to the former and providing an initial discussion and hypotheses for the latter.

There are two fundamental assumptions, or social media aspects, guiding this study. The first one is that there is a great amount of uncertainty in both using and adopting those platforms. Regarding their use, differently from mainstream media, the reach and impact of the content generated on social media is extremely uncertain. As Castells [START_REF] Castells | Communication power[END_REF] nicely puts it, communication on social media is like casting a message in a bottle into the ocean. Any message can either go viral or unnoticed in the massive ocean of information called cyberspace. In other words, institutions cannot fully control the visibility and impact of their online communication. The uncertainty in the adoption of social media concerns the fact that institutions have an extremely vast and growing catalogue to 'choose' from. It is difficult to foresee which applications will become popular and persevere and which ones will abruptly end. For this reason, institutions must choose carefully those applications, especially since "being active" is a key requirement for their success [START_REF] Kaplan | Users of the world, unite! The challenges and opportunities of social media[END_REF].

The second important aspect of social media is that those online applications can be quite distinct. In fact, the only common aspect shared by all of them is the fact that, differently from Internet forums, they are structured or built around people rather than topics [START_REF] Boyd | social network sites: Definition, history, and scholarship[END_REF]. However, they can be public, semi-private or private, and they differ regarding the type of content that they allow its users to generate and share. Depending on the type of content allowed on those platforms, the costs of adopting a particular social media will vary as well (i.e. sharing original text/photo is easier/cheaper than sharing original video). As a consequence, not only the users of those platforms vary considerably, in terms of volume and characteristics, but also, partially as a consequence of that, social media can be used in different ways and for different purposes. It is normal for institutional actors to give different uses to different social media [START_REF] Morini | Twitter for Politics and Facebook for Leisure? The social media Behaviour of Italian Politicians[END_REF].

Overall, besides focusing on usage levels, it is important to take a step back and understand why municipalities adopt a particular type of social media. While there are relevant benefits from using them, the risks associated with their use are equally important. The goal of this paper is precisely to explore, what factors explain the adoption of a particular social media by the Portuguese local governments.

Research question, hypotheses and methodology

This paper's research question is: What are the determinants of social media adoption by the Portuguese local governments? Even though our main research hypotheses derive from the literature, this study, at this stage, has a strong exploratory component.

We investigate this research question by looking at three of the most internationally popular social media: Facebook, Twitter and YouTube. Our three dependent variables are dichotomous measures for whether a municipality has a Facebook, Twitter, and YouTube account (1=Yes). Additionally, a fourth dependent variable is included to assess the presence of municipalities in alternative social media platforms besides those three main ones (e.g Flickr, Google+, etc.) (1=Yes). All variables refer to 2016. Concerning our research hypotheses and independent variables, the use of social media by different organizations is often associated with efforts to increase both transparency and citizens' participation and collaboration with those organizations, since it allows dialogic communication [START_REF] Linders | From e-government to we-government: Defining a typology for citizen coproduction in the age of social media[END_REF]. Indeed, studies have found a positive relationship between social media usage levels and the municipalities' commitment to transparency [8][9]. We therefore expect those two motivations to be associated with the adoption of different social media. More concretely, we expect that: H1) Municipalities with higher commitment to participation are more likely to be present on social media. H2) Municipalities with higher commitment to transparency are more likely to be present on social media.

In this study we have two variables measuring the municipalities' commitment to participation and transparency. The former is measured by a dummy variable called "Participation" coded "1" if the municipality has been conducting participatory budgeting initiatives and "0" otherwise. Commitment to local government transparency is measured with an index, ranging from 0 to 100, based on an assessment of the information provided in municipalities' websites [START_REF] Da Cruz | Measuring local government transparency[END_REF].

Even though creating a social media account is an easy and, usually, costless process, being active (and original) on them, which is a crucial aspect for the success of the online communication of any organization, is not as easy or straightforward. An effective use of social media requires the allocation of important resources. In fact, lack of resources can be a factor hindering local governments' use of those platforms [16] [17]. This factor is particularly important in the case of less popular social media, with higher cost/benefit, and the ones that only allow more elaborated/costly content (e.g. YouTube). Therefore, we expect that: H3) Municipalities with higher administrative/economic capacity are more likely to adopt social media, particularly the less popular or costlier ones.

Administrative capacity is measured with two different variables. The first one is the number of local government employees. The second is the proportion of own revenues raised by the municipalities.

Two other aspects examined in this study are the political setting and the media landscape of the Portuguese municipalities. Concerning the first one, we expect political participation and political competition to have an effect on municipalities' adoption of social media. Citizens' use of social media and political behavior should be positively correlated [START_REF] Gil De Zúñiga | social media Use for News and Individuals' social Capital, Civic Engagement and Political Participation[END_REF] and political competition should also affect positively governments' commitment to transparency and disclosure of information [START_REF] Tavares | Explaining the transparency of local government websites through a political market framework[END_REF] [20] [START_REF] García | Determinants of online reporting of accounting information by Spanish local government authorities[END_REF]. For this reason, we expect that:

H4) The higher the level of political participation and political competitiveness, the more likely municipalities will be to adopt different social media platforms.

Political participation is measured by the average turnout in the last three local elections. Similarly, political competitiveness is the average margin of victory (i.e. the difference, in percentage points, between the two first candidates/parties) in the last three mayoral elections. We use an average of three elections in order to capture long-lasting trends of these two variables.

Regarding media landscape, we employ the number of local radios and newspapers in the municipality. The goal is to explore a possible relationship between the local media environment and the use of social media. We expect that local governments might rely more on different social media when the number of local media outlets is lower.

Finally, we include a set of control variables in the estimations: population size, level of education, number of parishes, proportion of foreign population, purchase power and latitude.

Results

Concerning the adoption of different social media by the Portuguese municipalities, our analysis shows that Facebook is, by far, the most popular social media platform. As displayed in Table 1, 85% of all Portuguese municipalities had, in 2016, an official Facebook account. The second most popular Social Networking Site (SNS) was YouTube. More than half of the Portuguese municipalities had an account in this platform. With respect to Twitter, only 36% of the municipalities used this platform. Finally, around 33% of the 308 Portuguese municipalities had accounts in some other social media applications that were not one of the three majors (e.g. Google+, Flickr). The results of the logistic regression analyses are presented in Table 2. Odds ratios (OR) and robust standard errors (in parentheses) are reported. OR higher than one indicate a positive relationship between the variables. Contrarily, OR lower than 1 imply a negative relationship between variables. The only difference between the four models is the dependent variable.

The first model investigates the adoption of Facebook. Since a large number of municipalities have adopted this platform, nothing very relevant can be taken or interpreted from these results. The only variable that is statistically significant is Latitude (with an OR lower than 1), which measures the geographical location of the municipality in the country (more north or south). The results suggest that municipalities from the north are less likely than the ones in the south to have a Facebook account. Overall, we can just say that Facebook is widely popular among Portuguese municipalities.

The results become far more interesting when we look at the other, less popular, social media Sites. In the case of Twitter, five variables display statistical significance. Again, municipalities from the south are more likely to use Twitter. Indeed, this was a consistent finding across all dependent variables, suggesting that municipalities from the north of Portugal are less likely to adopt social media compared to their southern counterparts. In addition to that, the results show that municipalities more committed to participation initiatives (i.e. with participatory budgeting) are almost twice as likely as the others to have a Twitter account. Besides that, municipalities with a higher number of parishes (sub-municipal governments/districts), with more economic resources and more employees are also more likely to be present on Twitter. Overall, our analysis shows that Twitter is not a popular platform among Portuguese local governments and that its adoption is positively associated with the municipalities' resources and commitment to engage with the citizens. However, it is also possible that the use of Twitter is not a direct consequence of municipalities wanting to engage citizens but rather a consequence of their efforts to get more visibility to specific e-participation initiatives.

The results for YouTube are similar to the ones we found for Twitter. There are only two exceptions. The first one is that the difference in "Participation" is not statistically significant. The second one is that population size becomes statistically significant with a negative coefficient. This means that municipalities with larger populations are less likely to adopt YouTube. Overall, these results further confirm that, when it comes to the adoption of social media, human and financial resources are important for municipalities to venture on alternatives to Facebook. Indeed, we can say that having less resources hinders local governments from being present on less popular social media.

Finally, when it comes to alternative social media, we also get very interesting results. The first one is that "Participation" is significant. The municipalities with Participatory Budgeting initiatives are about 70% more likely to have an account in one alternative (less popular) social media. Again, it might be the case that rather than using them to interact with citizens, municipalities are simply trying to expand their online communication channels to give more visibility to those initiatives. The municipalities' resources are an important factor to explain presence in alternative SNS. The number of parishes, population size, the percentage of foreigners and latitude are also statistically significant. In the case of population, foreign population and latitude, their OR is lower than 1, meaning that the relationship between the variables is negative. However, what is perhaps more interesting in this last model is the statistically significant and negative relationship between local media and the adoption of alternative social media. To put it in other words, municipalities with a higher number of local radio stations and newspapers are less likely to adopt alternative (less popular) social media. This suggests that perhaps Portuguese local governments rely on social media to circumvent some limitations (or lack of traditional alternatives) to inform their citizens.

Conclusion

There are important limitations in this paper due to the fact that it is an ongoing research. In specific, the nature of our dependent variables does not allow us to explore short-term factors such as the characteristics of mayors and executives and the institutional configuration of communication departments inside municipalities. These are factors that indubitably might play an important role in the adoption of different social media and that allow an in-depth study of the determinants of social media adoption by Portuguese local governments. Therefore, we exclusively focus on long-term, less mutable, variables. Nevertheless, our study revealed important aspects regarding the adoption of those platforms by local government and provides grounded clues for further research and methodology. Perhaps the most important, or at least consistent, finding of this study was that resources and local government capacity matter. And they seem to matter a lot. That was the only hypothesis (H3) that the quantitative analysis fully confirmed. We knew already, from previous studies, that resources matter for usage levels (i.e. municipalities with more resources are more active on social media [START_REF] Lameiras | An Empirical Analysis of social media Usage by Local Governments in Portugal[END_REF]). Now we also know that these are important factors for the adoption of less popular social media. Contrarily, political aspects (turnout and competition) (H4) do not seem to affect the adoption of those platforms. Overall, this study suggests that Portuguese municipalities are indeed aware of the risks involved in adopting social media. They are more likely to adopt more alternative platforms when they have resources to guarantee their successful use (i.e. being active on them).

We also found that while commitment to transparency does not have any impact on social media adoption, municipalities that have participatory budgeting initiatives are more likely to have accounts on alternative, less popular platforms. We can, however, interpret this result in two different ways. It can indeed be that these alternative SNS are more suitable to engage citizens, since there is an understanding that Twitter is more suitable for sharing ideas while Facebook is used for leisure or networking. However, it can also be that these municipalities are simply using a larger variety of online applications to promote their participatory budgeting initiatives. Further analyses must be conducted to better investigate these two hypotheses. This is, indeed, the next step of this project that aims, with a multi-method approach, to better understand the use of social media by local government.

One final aspect worth mentioning is that we found that municipalities from the north of Portugal tend to be more conservative when it comes to social media adoption, a difference observed even for the most popular application -Facebook. This is interesting since the north is also more conservative when it comes to local politics and the importance of religion. When it comes to the adoption of new communication channels, we can say that southern municipalities seem to be trendier than their counterparts in the north.

5

Table 1 .

 1 Descriptive statistics of the variables.

		mean	sd	min	max
	Facebook	0.851	0.357	0	1
	Twitter	0.357	0.480	0	1
	YouTube	0.523	0.500	0	1
	Other Social Networking	0.331	0.471	0	1
	Sites (SNS)
	Participation	0.338	0.474	0	1
	Transparency	44.30	17.21	0.82	94.23
	#Employees	385.0	580.7	27	7417
	Own revenues	39.01	18.33	2.31	90.21
	Political participation	62.86	6.39	44.86	79.13
	Political competition	27.85	7.77	10.99	49.16
	Latitude	39.60	1.86	32.65	42.11
	#Parishes	10.04	8.51	1	61
	Population (log)	9.70	1.14	6.11	13.21
	Education	9.944	4.554	2.87	33.55
	Foreign Pop.	2.677	2.829	0.31	21.63
	Purchase Power	80.55	18.15	56.54	207.9
	Local Media	3.640	5.019	0	49

Table 2 .

 2 Logistic regression analysis of the determinants of social media adoption by the 308 Portuguese Municipalities.

		(1)	(2)	(3)	(4)
		Facebook	Twitter	YouTube	Alternative SNS
	Participation 1.000	1.970**	1.087	1.719*
		(0.381)	(0.549)	(0.301)	(0.491)
	Transp.	1.016	1.010	1.006	1.000
		(0.0106)	(0.00808)	(0.00785)	(0.00855)
	Parishes	1.031	1.062***	1.071**	1.043*
		(0.0267)	(0.0244)	(0.0306)	(0.0248)
	Pop. (log)	1.168	0.639	0.340***	0.556*
		(0.448)	(0.206)	(0.111)	(0.178)
	Education	1.000	0.950	0.979	0.969
		(0.0835)	(0.0625)	(0.0668)	(0.0625)
	Foreign Pop. 0.908	0.961	0.975	0.849**
		(0.0716)	(0.0653)	(0.0715)	(0.0574)
	Purchase P.	1.013	0.991	1.004	1.004
		(0.0227)	(0.0173)	(0.0175)	(0.0166)
	Local Media 0.965	0.972	0.981	0.900**
		(0.0484)	(0.0720)	(0.0453)	(0.0470)

Acknowledgments

This paper is a result of the project "SmartEGOV: Harnessing EGOV for Smart Governance (Foundations, methods, Tools) / NORTE-01-0145-FEDER-000037", supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (EFDR). António Tavares acknowledges the financial support of the Portuguese Foundation for Science and Technology and the Portuguese Ministry of Education and Science through national funds [Grant No. UID/CPO/0758/2019].