Audrey Joannes
email: audrey.joannes@univ-rennes1.fr

Claudie Morzadec

Maëla Duclos

Francisco Llamas Gutierrez

Dan Cristian Chiforeanu

Cécile Le Naoures

Bertrand De Latour

Simon Rouzé

Lutz Wollin

Stéphane Jouneau

Laurent Vernhet

Arsenic trioxide inhibits the functions of lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

Keywords: Interstitial lung diseases, primary human lung fibroblasts, metalloid, proliferation, migration, differentiation IPF: idiopathic pulmonary fibrosis, ILD: interstitial lung disease, PDGF-BB: platelet-derived growth factor BB, TGF-β1: transforming growth factor-β1, ATO: arsenic trioxide, HLFs: human lung fibroblasts, α-SMA: α-smooth muscle actin, APL: acute promyelocytic leukemia, COL1: collagen-1, NRF2: nuclear factor erythroid-2-related factor 2, NQO1: NADPH dehydrogenase quinone 1, HO-1: heme-oxygenase-1, MiRNA: microRNA, CDK: cyclin-dependent kinase, PBS: phosphate-buffered saline, ANOVA

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

Background

Patients diagnosed with idiopathic pulmonary fibrosis (IPF), the most frequent idiopathic fibrosing interstitial lung disease (ILD), have an average life expectancy of 3-5 years [START_REF] Lederer | Idiopathic Pulmonary Fibrosis[END_REF][START_REF] Raghu | Diagnosis of Idiopathic Pulmonary Fibrosis. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline[END_REF]. Despite intensive research, the cellular and molecular mechanisms underlying the development of lung fibrosis remain unclear. The disease is probably triggered by repeated injuries to the alveolar epithelium, due to agents such as chronic cigarette smoking, viruses, or air pollutants. The resulting damage disturbes tissue repair and promotes abnormal fibroproliferation, myofibroblast differentiation and excessive collagen deposition eventually leading to chronic respiratory failure and death [START_REF] Lederer | Idiopathic Pulmonary Fibrosis[END_REF]. Profibrotic mediators like transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF), produced by epithelial, endothelial or infiltrating immune cells, play crucial roles in IPF pathogenesis by inducing the formation of fibroblastic foci [START_REF] Budi | TGF-β as a driver of fibrosis: physiological roles and therapeutic opportunities[END_REF][START_REF] Grimminger | The role of tyrosine kinases in the pathogenesis of idiopathic pulmonary fibrosis[END_REF]. PDGF triggers the migration and proliferation of fibroblasts while TGF-β induces the differentiation of lung fibroblasts into myofibroblasts. These activated fibroblasts markedly express the α-smooth muscle actin (α-SMA) and produce abnormal amounts of collagen [START_REF] Grimminger | The role of tyrosine kinases in the pathogenesis of idiopathic pulmonary fibrosis[END_REF][START_REF] Upagupta | Matrix abnormalities in pulmonary fibrosis[END_REF].

Nintedanib and pirfenidone are the only drugs that have been approved worldwide for treating IPF [START_REF] Raghu | Diagnosis of Idiopathic Pulmonary Fibrosis. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline[END_REF]. Low concentrations of these drugs decrease the proliferation and migration of human lung fibroblasts (HLFs) from IPF patients in vitro. Although high concentrations of them also prevent the differentiation of HLF into activated myofibroblasts, such concentrations are seldom reached in the plasma of treated patients [START_REF] Conte | Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts[END_REF][START_REF] Hostettler | Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis[END_REF][START_REF] Rangarajan | Novel Mechanisms for the Antifibrotic Action of Nintedanib[END_REF]. The drugs reduce the progression of IPF by slowing the rate of lung function decline but they do not reverse lung fibrosis [START_REF] King | A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis[END_REF][START_REF] Richeldi | Efficacy and safety of nintedanib in patients with advanced idiopathic pulmonary fibrosis[END_REF][START_REF] Richeldi | Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis[END_REF]. Hence, new antifibrotic drugs are urgently required.

Arsenic trioxide (ATO) has been approved for the treatment of acute promyelocytic leukemia J o u r n a l P r e -p r o o f (APL) that is driven by the t(15:17) translocation [START_REF] Kumana | Systemic availability of arsenic from oral arsenic-trioxide used to treat patients with hematological malignancies[END_REF][START_REF] Soignet | United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia[END_REF].

It is also being assessed for the management of solid tumors [START_REF] Kim | Phase I clinical trial of KML001 monotherapy in patients with advanced solid tumors[END_REF] and immunological disorders such as systemic lupus erythematosus [START_REF] Hamidou | Safety and efficacy of low-dose intravenous arsenic trioxide in systemic lupus erythematosus: an open-label phase IIa trial (Lupsenic)[END_REF]. The concentrations of total arsenic measured in the plasma of patients treated with standard dosing (0.15 mg/kg/d, intravenous), immediately after the completion of administration and during the following hours, were in a range from 5-7 µM and 0.1-0.5 µM, respectively [START_REF] Kumana | Systemic availability of arsenic from oral arsenic-trioxide used to treat patients with hematological malignancies[END_REF][START_REF] Shen | Studies on the clinical efficacy and pharmacokinetics of low-dose arsenic trioxide in the treatment of relapsed acute promyelocytic leukemia: a comparison with conventional dosage[END_REF][START_REF] Shen | Use of Arsenic Trioxide (As2O3 ) in the Treatment of Acute Promyelocytic Leukemia (APL): II. Clinical Efficacy and Pharmacokinetics in Relapsed Patients[END_REF]. ATO administration is generally well tolerated but in few cases the treatment must be temporarily interrupted when adverse effects are observed and judged to be possibly related to ATO. Interestingly, experimental studies have demonstrated that these concentrations of ATO measured in the plasma of patients suffering from APL exhibit, in vitro, strong antifibrotic actions. ATO decreases the myofibroblast differentiation induced by TGF-β1 in both dermal and non-IPF HLFs in vitro [START_REF] Luo | Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo[END_REF][START_REF] Zhong | Arsenic trioxide inhibits the differentiation of fibroblasts to myofibroblasts through nuclear factor erythroid 2-like 2 (NFE2L2) protein and the Smad2/3 pathway[END_REF] and decreased the proliferation of ocular fibroblasts in vitro [START_REF] Su | Arsenic Trioxide Inhibits Proliferation of Rabbit Tenon's Capsule Fibroblasts After Trabeculectomy by Downregulating Expression of Extracellular Matrix Proteins[END_REF]. It attenuates both cisplatin-induced renal fibrosis in rats [START_REF] Maghmomeh | Arsenic trioxide and curcumin attenuate cisplatin-induced renal fibrosis in rats through targeting Hedgehog signaling[END_REF] and lung fibrosis in a murine model of systemic sclerosis in vivo [START_REF] Kavian | Reactive oxygen species-mediated killing of activated fibroblasts by arsenic trioxide ameliorates fibrosis in a murine model of systemic sclerosis[END_REF]. Finally, ATO inhibits bleomycin-induced lung fibrosis in mice and rats by decreasing collagen deposition and inhibiting α-SMA expression [START_REF] Gao | Arsenic trioxide prevents rat pulmonary fibrosis via miR-98 overexpression[END_REF][START_REF] Luo | Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo[END_REF].

ATO is a pro-oxidant metalloid that up-regulates the redox-sensitive expression of the transcription factor, NF-E2-related factor 2 (NRF2), in several cell types. NRF2 mediates the ATO-dependent stress response by stimulating the expression of several antioxidant genes. It also contributes to the arsenic effects on various major cell functions [START_REF] Bourdonnay | Redox-sensitive regulation of gene expression in human primary macrophages exposed to inorganic arsenic[END_REF][START_REF] Morales | Reactive oxygen species are not required for an arsenic trioxide-induced antioxidant response or apoptosis[END_REF][START_REF] Wang | Arsenic modulates heme oxygenase-1, interleukin-6, and vascular endothelial growth factor expression in endothelial cells: roles of ROS, NF-κB, and MAPK pathways[END_REF]. In addition, increased NRF2 expression reduces the development of experimental lung fibrosis [START_REF] Cho | The transcription factor NRF2 protects against pulmonary fibrosis[END_REF] and modulated the differentiation of HLF in vitro [START_REF] Artaud-Macari | Nuclear factor erythroid 2-related factor 2 nuclear translocation induces myofibroblastic dedifferentiation in idiopathic pulmonary fibrosis[END_REF].

This study was designed to determine whether ATO could inhibit the pro-fibrotic functions of HLFs from patients with IPF. We found that nanomolar concentrations of ATO, in the range of plasmatic concentrations achieved in APL patients treated with conventional ATO dosing, inhibited the proliferation and migration of HLFs from patients with IPF. In addition, independently of its ability to induce NRF2 expression ATO reduced the differentiation of fibroblasts to pro-fibrotic myofibroblasts.

Materials and Methods

Primary cultures of HLFs

IPF and control HLFs were isolated from lung tissue samples [START_REF] Marchand-Adam | Defect of hepatocyte growth factor secretion by fibroblasts in idiopathic pulmonary fibrosis[END_REF].

The IPF samples were from patients undergoing open lung biopsy (median age 68 year; range 51-77 years). IPF was diagnosed according to the ATS/ERS/JRS/ALAT criteria, including histopathological features of usual interstitial pneumonia [START_REF] Raghu | Diagnosis of Idiopathic Pulmonary Fibrosis. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline[END_REF]. Control samples were from cancer patients undergoing lobectomy or pneumonectomy, away from the tumor (median age 64.5 year; range 59-74 year). The absence of tumor tissue in control samples was verified histologically. This study was approved by the local ethics committee (Ethics Committee CHU Rennes, no 16123). Written informed consent was obtained from all subjects. All experiments were performed in accordance with relevant guidelines and regulations. Cell morphology was checked by phase contrast microscopy.

Cell culture and treatments

IPF (n=10) and control (n=6) HLFs were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco™, Life Technologies, Courtaboeuf, France) supplemented with 10% fetal calf serum (FCS) (Eurobio scientific, Evry, France), L-glutamine, antibiotic-antimycotic solution (Gibco™, Life Technologies) and used at passages 5 and 6. The HLFs used to analyze mRNA and protein expressions were seeded in 6-well plates for 24 h, and then starved in FCS-free DMEM for 16 h. TGF-β1-induced myofibroblast differentiation was measured by treating serum-starved HLFs with ATO (0.01-1 µM) (Formula: AS 2 O 3, CAS number: 1327-53-3, molecular weight 197,84 g/mol; Sigma-Aldrich, St. Quentin Fallavier, France) for 24 h and then with TGF-β1 (1 ng/ml; Preprotech, Neuilly-sur-Seine, France) for 24 h. The HLFs used for cell growth and cell cycle analysis were serum-starved for 16 h, cultured in DMEM containing 1 % FCS and 0.01-1 μM ATO for 24 h, and finally stimulated with PDGF-BB (50 ng/ml) (R&D Systems, Bio-Techne, Lille, France) for a further 72 h. The HLFs were treated with ATO for 24 h as this incubation time is enough to significantly modulate the phenotype of normal HLFs [START_REF] Luo | Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo[END_REF]. The concentrations of ATO (0.01-1µM) were in a comparable range to the plasma concentrations that can be reached in APL patients treated with standard doses of ATO [START_REF] Kumana | Systemic availability of arsenic from oral arsenic-trioxide used to treat patients with hematological malignancies[END_REF][START_REF] Shen | Studies on the clinical efficacy and pharmacokinetics of low-dose arsenic trioxide in the treatment of relapsed acute promyelocytic leukemia: a comparison with conventional dosage[END_REF][START_REF] Shen | Use of Arsenic Trioxide (As2O3 ) in the Treatment of Acute Promyelocytic Leukemia (APL): II. Clinical Efficacy and Pharmacokinetics in Relapsed Patients[END_REF].

Proliferation and viability

After ATO and PDGF-BB treatments, cells were dissociated with trysin, centrifugated (1500g, 5 min) and resuspended in DMEM containing 10 % FCS. Proliferation and viability were assessed with the acridine orange/propidium iodide staining (Logos Biosystems, Villeneuve d'Ascq, France) and an automated fluorescence cell counter (Luna™, Logos Biosystems). The cell viability dye causes viable nucleated cells to fluoresce green and nonviable nucleated cells to fluoresce red. After cell counting, the relative cell proliferation was calculated by dividing the number of viable cells exposed to ATO +/-PDGF-BB by the number of viable untreated cells arbitrarily set at 1. The cell viability was defined by dividing the number of viable cells by the number of viable and dead cells. It was expressed in percentages (%).

Cell cycle analysis

The HLFs used to analyse the cell cycle distribution were fixed in ice-cold 70 % (v/v) ethanol for 30 min at 4°C, and collected by centrifugation (900g/5 min). The cell pellets were washed, suspended in PBS (pH 7.4) containing propidium iodide (50 μg/mL) and DNase-free RNase A (100 μg/mL) (Sigma-Aldrich, St. Quentin Fallavier, France) and incubated at 4°C for 1 h. DNA was quantified by flow cytometry using an LRSFortessa™X20 flow cytometer (BD Biosciences, Paris, France). Data were analysed with flow cytometry data analysis software FlowLogic (Miltenyi Biotech, Paris, France) using the Watson improved method.

Migration assay

HLF migration was assessed in a modified Boyden chamber assay, using a Transwell permeable support (6.5 mm, 8 μm) (Corning, Tewksbury, MA, USA) coated with fibronectin (0.1 μg/ml) (Sigma-Aldrich, St. Quentin Fallavier, France). The medium in the upper compartment (DMEM-0%FCS) contained 0.01-1µM ATO and that in the lower compartment contained (DMEM-1%FCS) 25 ng/ml PDGF-BB. Cells were incubated for 24 h at 37°C, fixed in 70 % ethanol, and labelled with DAPI. Migration was assessed by counting the number of cells on the lower surface of the filters. The relative cell migration was defined by dividing the number of migrating cells stimulated with PDGF-BB by the number of migrating cells left untreated and arbitrarily set at 1.

siRNA transfection

HLFs were transfected for 8 h with 25 nM Nrf2 siRNA (NM_006164) (Dharmacon SMARTpool siRNA reagents, Fisher Scientific, Illkirch, France), or negative control (scramble) siRNA (D-0018010-10-20, Dharmacon, plus non-targeting pool) using Lipofectamine RNAiMAX Reagent (Invitrogen, Thermo Fisher scientific) according to the manufacturer's instructions. Treated cells were starved, incubated with 1 μM ATO for 24 h and stimulated with 1 ng/ml TGF-β1 for a further 24 h.

Protein analysis

HLFs were lysed in ice-cold RIPA buffer containing cOmplete™ Protease Inhibitor Cocktail (Roche, Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich). Proteins were separated by electrophoresis on 10 % Tris-glycine SDS polyacrylamide gels and transferred to nitrocellulose membranes (pore size: 0.2 μm, Invitrogen). Free binding sites were blocked by incubation in 5 % milk or bovine serum albumin (BSA) (Sigma-Aldrich, St. Quentin

J o u r n a l P r e -p r o o f

Fallavier, France) for 2 h at room temperature. Membranes were then incubated with the appropriate primary human antibody (Ab) overnight at 4°C. The primary Abs were anti-α-SMA (clone 1A4, Sigma-Aldrich), anti-collagen I (Southern Biotechnology Associates, Birmingham, AL), anti-NRF2 (ab62352, abcam, Amsterdam, Netherlands), anti-NADPHquinone-oxydoreductase-1 (NQO1) (sc-271116, Santa Cruz, Santa Cruz, USA), anti-heme oxygenase-1 (HO-1) (ADI-SPA-895, Enzo Life Science, NY, USA), and anti-GAPDH (mab90009-P, Covalab, Bron, France) Abs. Anti-phospho p44/42 MAPK (197G2), anti-p44/42 MAPK (9101), anti-phospho SMAD2 (3104), anti-SMAD2 (D43B4), anti-phospho SMAD3 (C25A9), anti-SMAD3 (C67H9), anti-phospho PDGF receptor (PDGFR) β ( 3161) and anti-PDGFR (28E1) Abs were from Cell Signaling Technology (Massachusetts, USA).

Digital images were acquired on a gel imaging system (Chemi doc, BIO-RAD Laboratories, Marnes-La-Coquette, France) equipped with a CCD camera. Western blots were quantified by densitometry using Image Lab™ software (BIO-RAD) and normalized to GAPDH. The relative protein expression in stimulated cells was determined by arbitrarily setting at 1 the protein expression measured in untreated cells.

RNA isolation and RT-PCR assays

Total RNAs were extracted using the mRNA extraction kit from Macherey Nagel (Hoerdt, France) according to the manufacturer's instructions. RNA concentrations were measured by spectrofluorimetry using a NanoDrop 1000 (Thermo Fisher scientific, Saint-Herblain, France) and were in a range of 23.6 to 183.2 ng/ml. The purity (260/280 ratio) of each RNA sample was between 2 and 2.2. mRNAs were reverse transcribed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific). Quantitative PCR (polymerase chain reaction) was performed using the SYBR Green methodology on a CFX384 Real-Time PCR System (Bio-Rad Laboratories) in duplicates. All the primers were provided by Sigma-Aldrich (KiCqStart™ SYBR® Green Primers, St. Quentin Fallavier, France). The specificity of gene amplification was checked at the end of PCR using the comparative cycle threshold method (CFX Manager Software). The mean Cq values were used to normalize the target mRNA concentrations to those of the 18S ribosomal protein by the 2 (-ΔΔCq) method. The relative mRNA expression was defined by the ratio between the treated conditions compared to the untreated condition which was arbitrarily set at 1.

Statistical analysis

All results are presented as means ± standard errors of mean (SEM) of the indicated numbers of independent biological experiments. All biological experiments were performed with different primary cell cultures. Statistical analyses were performed with Prism 5 (GraphPad Software, La Jolla, CA). Significant differences were determined by Student's t-test ($ p<0.05; $$ p<0.01; $$$ p<0.001) when two groups were compared, or by ANOVA followed by the Dunnett's Multiple comparison t-test (* p<0.05; ** p<0.01; *** p<0.001) for more than 2 groups.

Results

ATO inhibited PDGF-BB-induced proliferation of IPF and control HLFs

Fibroblast proliferation is a major contributor to the formation of fibroblastic foci in the lungs of IPF patients [START_REF] King | Idiopathic pulmonary fibrosis[END_REF]. IPF and control cells were cultured with ATO for 72 h to determine the effects of ATO on basal HLF growth. ATO did not alter the viability of HLF under these conditions (supplemental Fig. 1A and1B) or reduce the proliferation of IPF HLFs (Fig. 1A) although 1 µM ATO significantly decreased the basal growth of control cells (Fig. 1B). Cell proliferation in lung fibrosis is stimulated by several pro-fibrotic cytokines, particularly by PDGF-BB. We therefore investigated the capacity of ATO to prevent the proliferative effects of PDGF-BB. ATO-treated IPF and control HLFs were incubated with PDGF-BB for 72 h. As expected, PDGF-BB significantly increased the proliferation of both IPF and control HLFs (Fig. 1C-1D). ATO did not reduce the viability of these activated HLFs (supplemental Fig. 1C and1D) but attenuated the PDGF-BB-induced proliferation in a concentration-dependent manner (Fig. 1C-1D); the effect was numerically detectable at 0.01 μM and it was maximal at 0.3 µM in IPF HLF and at 1 μM in control HLF.

ATO modified the cell cycle of IPF and control HLFs.

We next determined the influence of ATO on the cell cycle of PDGF-BB-stimulated HLFs.

As shown in the representative pictures displayed in Figure 2A, after 72 h, most IPF HLFs were in the G0/G1 phase whatever their treatment. ATO significantly decreased the percentage of IPF HLFs arrested in G0/G1 while it markedly increased the percentage of cells in G2/M phase after a 72 h-incubation with PDGF-BB (Fig. 2B). It had a similar effect on the cell cycle distribution of activated control HLFs (supplemental Fig. 2A and2B). The third set of experiments examined the capacity of ATO to modulate cell cycle progression by altering the expression of cell cycle regulatory proteins. PDGF-BB increased the expressions of the cyclin D1/CDK4, cyclin A/CDK2 and cyclin B/CDK1 that control progression in the G1, S and G2 phases, respectively (Figure 2C-2D). At 1 µM, ATO had no effect on cyclin D1 concentration while it slightly decreased the expressions of its CDK4 partner. It also significantly reduced the expression of cyclin A/CDK2 and cyclin B (Figure 2C-2D).

ATO prevented the PDGF-BB-induced migration of IPF and control HLFs.

Fibroblast migration is another important step in the formation of fibroblastic foci [START_REF] Chilosi | Migratory marker expression in fibroblast foci of idiopathic pulmonary fibrosis[END_REF][START_REF] King | Idiopathic pulmonary fibrosis[END_REF]. PDGF-BB not only stimulates cell proliferation, its potent chemoattractant properties also promote fibrogenesis in pulmonary tissues. We found that it significantly increased the migration of IPF and control HLFs and that ATO reduced in a concentration-dependent manner, the response of both IPF and control HLFs to PDGF-BB stimulation (Figure 3). Its effect reached significance at 0.1 μM and maximum at 1 μM; 1 μM ATO almost totally blocked the PDGF-BB-induced migration of IPF HLFs.

ATO reduced PDGF-BB-induced phosphorylation of p38-kinase.

We next determined if ATO could prevent the growth and migration of IPF HLFs through inhibition of the PDGF-BB signaling by determining its effects on the phosphorylation of the kinases induced by PDGF receptor (PDGFR) activation. As shown in Figures 4A and4B, PDGF-BB increased the levels of phosphorylated PDGFR, ERK, AKT and p38-kinase. ATO had no effect on the phosphorylation of PDGFR, ERK and AKT but it significantly decreased the phosphorylation of p38-kinase at 1µM.

ATO modulated the TGFβ-1-induced differentiation of HLFs.

Activation of the TGF-β pathway drives the differentiation of fibroblasts to myofibroblasts and is therefore an essential feature of fibrosis [START_REF] Budi | TGF-β as a driver of fibrosis: physiological roles and therapeutic opportunities[END_REF]. We examined the capacity of ATO to prevent the TGF-β1-dependent differentiation of IPF and control HLFs by first determining the effects of ATO on the mRNA levels of two major pro-fibrosis genes, COL1A1 and ACTA2. IPF and control HLFs were incubated with 1 ng/ml TGF-β1 for 24 h, which increased the concentrations of COL1A1 and ACTA2 mRNA levels (Supplemental Figure 3). ATO did not prevent the increase in COL1A1 mRNA in IPF HLFs but it significantly reduced the increase in ACTA2 mRNA in these cells (Supplemental Figure 3A-3B). In contrast, it did not prevent the increase in this profibrotic gene expression in control HLFs (Supplemental Figure 3D). ATO had no effect on the basal expression of COL-1 and -SMA proteins in HLFs (Supplemental Figure 4). However, as shown in the Figures 5A-5D, ATO reduced, in a concentration dependent manner, the expression of COL-1 and α-SMA protein levels in both IPF and control HLFs stimulated with TGF-1.

Because activation of the SMAD2 and SMAD3 pathways plays a key role in the action of TGF-β1, we next examined the capacity of ATO to prevent fibroblast differentiation by inhibiting the phosphorylation of SMAD proteins. TGF-β1 stimulated the phosphorylation of phospho-SMAD2 and phospho-SMAD3 proteins in IPF and control HLFs (Figures 6). ATO significantly reduced the levels of these phosphorylated proteins at its highest concentration (1 µM) in IPF HLFs (Figures 6A-6B) but it decreased only the level of phosphorylated SMAD3 in control cells (Figures 6C-6D).

ATO induced NRF2 and antioxidant protein expression in IPF and control

HLFs.

ATO can modulate cell functions by increasing the expression of the redox-sensitive transcription factor NRF2 [START_REF] Bourdonnay | Redox-sensitive regulation of gene expression in human primary macrophages exposed to inorganic arsenic[END_REF][START_REF] Wang | Arsenic modulates heme oxygenase-1, interleukin-6, and vascular endothelial growth factor expression in endothelial cells: roles of ROS, NF-κB, and MAPK pathways[END_REF]. NRF2 not only controls the cell stress responses but it also limits the development of lung fibrosis by interfering with the TGF-β1 signaling [START_REF] Artaud-Macari | Nuclear factor erythroid 2-related factor 2 nuclear translocation induces myofibroblastic dedifferentiation in idiopathic pulmonary fibrosis[END_REF][START_REF] Gong | Activation of Nrf2/AREs-mediated antioxidant signalling, and suppression of profibrotic TGF-β1/Smad3 pathway: a promising therapeutic strategy for hepatic fibrosis -A review[END_REF]. Thus, we next determined if NRF2 could mediate, at least in part, the antifibrotic effects of ATO on IPF and control HLFs. To this goal, we first studied the impact of ATO on NRF2 expression. Most cell types contain low concentrations of NRF2 because it is bound to Keap1 that promotes its ubiquitination by Cul3 ubiquitin ligase and its rapid degradation by the proteasome. The oxidation of cysteine residues in Keap1 that occurs in cell stress reduces the interaction of NRF2 with Cul3, so preventing its ubiquitination and subsequent degradation [START_REF] He | NRF2 Cysteine Residues Are Critical for Oxidant/Electrophile-Sensing, Kelch-Like ECH-Associated Protein-1-Dependent Ubiquitination-Proteasomal Degradation, and Transcription Activation[END_REF]. Stabilized NRF2 protein moves into the nuclei during cell stress, where it promotes the expression of several antioxidant genes. We measured the influence of ATO on NRF2 protein stabilization and the cell stress response in HLFs. As frequently observed in other cell types [START_REF] Bourdonnay | Redox-sensitive regulation of gene expression in human primary macrophages exposed to inorganic arsenic[END_REF], ATO rapidly increased the concentration of NRF2 protein in IPF HLFs (Fig. 7A); the maximum response was obtained after incubation with 0.5 μM ATO for 1 h (Fig. 7B). We next measured the activities of the antioxidant genes NQO1 and HMOX1, which encode proteins NQO1 and HO-1, as they are induced by NRF2 in many cell types. ATO dose-dependently increased the concentrations of NQO1 and HO-1 in unstimulated and TGF-β1-stimulated IPF HLFs (Figures 7C-7F). Maximal effects were detected in cells treated with 1 μM ATO. TGF-β1 had no significant effect on the expression of these proteins. ATO similarly stabilized NRF2 and stimulated NQO1 and HO-1 synthesis in control HLFs (Supplemental Fig. 5).

NRF2 did not mediate the ATO-dependent inhibition of TGF-β1-induced myofibroblast differentiation in IPF HLF.

In a last of experiments, we examined the contribution of NRF2 to the inhibition of the TGF-β-dependent differentiation of IPF HLFs induced by ATO. We inhibited NRF2 gene expression using SiRNA technology. Both unstimulated and TGF-1-stimulated IPF cells transfected with NRF2 SiRNA had decreased NRF2 mRNA levels whether or not they were cultured in the presence of ATO (Fig. 8A). NRF2 gene silencing also significantly decreased the concentration of basal NQO1 mRNA in unstimulated cell and prevented any increase in stimulated cells that had been treated with ATO (Fig. 8B). However, this treatment neither reduced the induction of ACTA2 mRNA by TGF-β1 nor blocked the inhibition of -SMA protein expression by ATO in TGF-1-stimulated IPF HLFs (Fig. 8C).

Discussion

Our results indicate that ATO inhibits major profibrotic processes in primary HLFs from both IPF and normal lung tissue. It inhibits the proliferation and migration of PDGF-BBstimulated HLFs and reduces the myofibroblast differentiation induced by TGF-1. ATO also triggers a stress response controlled by the NRF2 transcription factor in IPF HLF. However, inhibiting NRF2 synthesis did not block the antifibrotic effects of ATO on TGF-1-stimulated HLFs.

We first showed that ATO had little or no effects on the proliferation of unstimulated IPF and control HLFs but that non-cytotoxic concentrations (0.1 -1 µM) significantly inhibited the proliferation of both IPF and control HLFs stimulated with PDGF-BB. This growth factor plays a major role in lung fibrosis by promoting the formation of fibroblastic foci [START_REF] Bonner | Regulation of PDGF and its receptors in fibrotic diseases[END_REF]. Its concentration is increased in murine models of lung fibrosis [START_REF] Zhuo | Modulation of PDGF-C and PDGF-D expression during bleomycin-induced lung fibrosis[END_REF] and inhibiting PDGF signaling markedly reduces pulmonary fibrosis in mice treated with bleomycin (Aono et al., 2005). We have not identified the molecular mechanism by which ATO reduced HLF proliferation but we have shown that it modulates the cell cycle in PDGF-BB-stimulated HLFs. It significantly reduced the percentages of cells in the G0-G1 phase and greatly increased those arrested in G2/M. These findings suggest that ATO does not alter DNA synthesis but rather prevents the entry of HLFs into mitosis. ATO is known to disturb the cell cycle by modulating the expression of cyclin and CDK [START_REF] Liu | Low-Concentration Arsenic Trioxide Inhibits Skeletal Myoblast Cell Proliferation via a Reactive Oxygen Species-Independent Pathway[END_REF][START_REF] Park | Arsenic trioxide induces G2/M growth arrest and apoptosis after caspase-3 activation and bcl-2 phosphorylation in promonocytic U937 cells[END_REF][START_REF] Su | Arsenic Trioxide Inhibits Proliferation of Rabbit Tenon's Capsule Fibroblasts After Trabeculectomy by Downregulating Expression of Extracellular Matrix Proteins[END_REF][START_REF] Zhao | Effect of As2O3 on cell cycle progression and cyclins D1 and B1 expression in two glioblastoma cell lines differing in p53 status[END_REF]. In HLF ATO did not alter the concentration of cyclin-D1, which controls the G0 to G1 progression, but it decreased the concentrations of cyclin-A, cyclin-B and CDK2, all key regulators of the G2/M phase transition, so retarding mitosis.

We also found that increasing concentrations of ATO greatly reduced the migration of PDGF-BB-stimulated IPF and control HLFs. This is relevant in the pathogenesis of the disease because IPF fibroblasts have increased capacity for migration and invasion, making them more destructive in lung tissue [START_REF] Suganuma | Enhanced migration of fibroblasts derived from lungs with fibrotic lesions[END_REF]. ATO inhibits the migration of several cell types, including vascular endothelial [START_REF] Yang | Antiangiogenic effect of arsenic trioxide in lung cancer via inhibition of endothelial cell migration, proliferation and tube formation[END_REF] , glioblastoma [START_REF] Ghaffari | Arsenic Trioxide Induces Apoptosis and Incapacitates Proliferation and Invasive Properties of U87MG Glioblastoma Cells through a Possible NF-κB-Mediated Mechanism[END_REF], hepatocellular carcinoma [START_REF] Zhang | Combination of canstatin and arsenic trioxide suppresses the development of hepatocellular carcinoma[END_REF], cervical [START_REF] Wei | Arsenic trioxide prevents radiation-enhanced tumor invasiveness and inhibits matrix metalloproteinase-9 through downregulation of nuclear factor kappaB[END_REF], and breast cancer cells [START_REF] Zhang | Arsenic trioxide suppresses cell growth and migration via inhibition of miR-27a in breast cancer cells[END_REF]. However, its effect on the migration of HLFs had never been reported. ATO seems to reduce cell migration by several molecular mechanisms.

It impairs the polymerization of microtubules [START_REF] Gao | Arsenic trioxide disturbs the LIS1/NDEL1/dynein microtubule dynamic complex by disrupting the CLIP170 zinc finger in head and neck cancer[END_REF], decreases the concentrations of the matrix metalloproteinases MMP-2 and MMP-9 [START_REF] Zhang | Arsenic Trioxide Suppressed Migration and Angiogenesis by Targeting FOXO3a in Gastric Cancer Cells[END_REF], modulates the concentrations of certain mi-RNAs [START_REF] Shi | Arsenic trioxide inhibits cell growth and motility via up-regulation of let-7a in breast cancer cells[END_REF][START_REF] Zhang | Arsenic trioxide suppresses cell growth and migration via inhibition of miR-27a in breast cancer cells[END_REF], and interferes with key signalling pathways triggered by vascular endothelial growth factor, basic fibroblast growth factor receptor, and PDGF [START_REF] Tingting | Arsenic trioxide inhibits osteosarcoma cell invasiveness via MAPK signaling pathway[END_REF][START_REF] Yang | Antiangiogenic effect of arsenic trioxide in lung cancer via inhibition of endothelial cell migration, proliferation and tube formation[END_REF]. We found that ATO specifically reduced the phosphorylation of p38-kinase in PDGF-stimulated HLFs. This kinase promotes the cell proliferation and migration induced by PDGF [START_REF] Tangkijvanich | p38 MAP kinase mediates platelet-derived growth factor-stimulated migration of hepatic myofibroblasts[END_REF][START_REF] Yamaguchi | Platelet-derived growth factor BB-induced p38 mitogen-activated protein kinase activation causes cell growth, but not apoptosis, in vascular smooth muscle cells[END_REF] and can contribute to lung fibrogenesis [START_REF] Matsuoka | A p38 MAPK inhibitor, FR-167653, ameliorates murine bleomycin-induced pulmonary fibrosis[END_REF].

It will be interesting to determine whether inhibiting p38-kinase prevents the proliferation and/or migration of PDGF-stimulated IPF HLFs in order to further understand these antifibrotic effects of ATO.

The TGF-β-dependent differentiation of fibroblasts to myofibroblasts is an important pathogenic event in IPF fibrogenesis as myofibroblasts produce excess extracellular matrix leading to lung tissue stiffness [START_REF] Budi | TGF-β as a driver of fibrosis: physiological roles and therapeutic opportunities[END_REF]. We found that ATO significantly decreased the TGF-β1-dependent synthesis of the myofibroblastic markers α-SMA and collagen-1 by HLFs, probably by blocking the activation of SMAD2/3. This agrees well with reports describing its impact on animal models of fibrosis. ATO was shown to decrease the development of pulmonary fibrosis and myofibroblastic marker concentrations in mice and rats treated with bleomycin [START_REF] Gao | Arsenic trioxide prevents rat pulmonary fibrosis via miR-98 overexpression[END_REF][START_REF] Luo | Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo[END_REF]. It also limits dermal thickening and inhibits collagen deposition in a mouse model of systemic sclerosis [START_REF] Kavian | Reactive oxygen species-mediated killing of activated fibroblasts by arsenic trioxide ameliorates fibrosis in a murine model of systemic sclerosis[END_REF]. However, ATO can promote fibrosis in organs such as heart and liver by increasing TGF-1 synthesis or TGF-1 signaling in chronically exposed animals [START_REF] Chu | Arsenic-induced interstitial myocardial fibrosis reveals a new insight into drug-induced long QT syndrome[END_REF][START_REF] Dai | Bi-directional regulation of TGF-β/Smad pathway by arsenic: A systemic review and meta-analysis of in vivo and in vitro studies[END_REF].

In our latest experiments, we found an association between the antifibrotic effects of ATO with a stress response controlled by the transcription factor NRF2. NRF2-dependent expression of the HMOX1 and NQO1 genes was rapidly induced in ATO-treated HLF that had been stimulated with TGF-1 or left unstimulated. We thus hypothesized that NRF2 activation could mediate, at least in part, the ATO-dependent inhibition of TGF-β1 effects in HLF. There is some support for this hypothesis. NRF2-deleted mice develop more severe pulmonary fibrosis in response to bleomycin [START_REF] Cho | The transcription factor NRF2 protects against pulmonary fibrosis[END_REF], whereas NRF2 inducers, such as sulforaphane and esomeprazole, attenuate fibrotic responses in vitro and/or vivo by activating the NRF2 pathway [START_REF] Artaud-Macari | Nuclear factor erythroid 2-related factor 2 nuclear translocation induces myofibroblastic dedifferentiation in idiopathic pulmonary fibrosis[END_REF][START_REF] Ebrahimpour | Esomeprazole attenuates inflammatory and fibrotic response in lung cells through the MAPK/Nrf2/HO1 pathway[END_REF]. NRF2 notably reduces the TGF-β-dependent production of reactive oxygen species by controlling the synthesis of antioxidant proteins; the generation of reactive oxygen species reinforces the TGF- signaling and its subsequent profibrotic effects in several models [START_REF] Guan | NF-E2-Related Factor 2 Suppresses Intestinal Fibrosis by Inhibiting Reactive Oxygen Species-Dependent TGF-β1/SMADs Pathway[END_REF][START_REF] Yang | Trichostatin A inhibits transforming growth factor-β-induced reactive oxygen species accumulation and myofibroblast differentiation via enhanced NF-E2-related factor 2-antioxidant response element signaling[END_REF]. Other studies indicate that NRF2 is a major molecular mediator of the antifibrotic response and that stimulating its synthesis could be a novel strategy for limiting fibrosis [START_REF] Oh | Sulforaphane attenuates hepatic fibrosis via NF-E2-related factor 2-mediated inhibition of transforming growth factor-β/Smad signaling[END_REF][START_REF] Wei | Nrf2 exerts cell-autonomous antifibrotic effects: compromised function in systemic sclerosis and therapeutic rescue with a novel heterocyclic chalcone derivative[END_REF]. Unfortunately, our results do not support this.

Blocking NRF2 synthesis by genetic silencing clearly inhibited NQO1 expression but not that of -SMA in TGF-1-stimulated HLFs cultured with or without ATO. Other molecular mechanisms must therefore drive the ATO-dependent inhibition of TGF-1 affects. For example, the reduction in phosphorylated SMAD3 concentrations in normal HLFs exposed to elemental arsenic is correlated with a concomitant decrease in promyelocytic leukemia (PML) nuclear bodies and PML protein. PML knock-out mouse embryonic fibroblasts produce lower concentrations of fibrotic markers in response to TGF-β1 [START_REF] Luo | Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo[END_REF]. Arsenic inhibits TGF-β1 signaling in human cancer cells by improving the expression of microRNA-155, which could contribute to experimental fibrosis and IPF [START_REF] Ji | Inhibition of transforming growth factor beta/SMAD signal by MiR-155 is involved in arsenic trioxide-induced anti-angiogenesis in prostate cancer[END_REF][START_REF] Kurowska-Stolarska | The role of microRNA-155/liver X receptor pathway in experimental and idiopathic pulmonary fibrosis[END_REF]. Additional experiments are required to determine whether such molecular mechanisms are involved in the antifibrosis effects of ATO on IPF HLFs.

Our results indicate that ATO reduces the proliferation, migration and differentiation of IPF HLF, as reported for nintedanib and pirfenidone, the two drugs approved for the treatment of IPF. At low nanomolar concentrations, nintedanib binds to the ATP binding pocket of various growth factor receptors, including the PDGF-BB receptor, resulting in the inhibition of the receptor autophosphophorylation and the downstream signaling cascades that control proliferation and migration [START_REF] Hostettler | Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis[END_REF][START_REF] Wollin | Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis[END_REF]. At micromolar concentration, nintedanib can also prevent the differentiation of HLFs by inhibiting the activation of the TGF-β receptor [START_REF] Rangarajan | Novel Mechanisms for the Antifibrotic Action of Nintedanib[END_REF]. However, such concentrations are rarely achieved in the plasma of IPF patients treated with standard dosing of nintedanib.

Pirfenidone, a synthetic small-molecule deriving from pyridone, exerts antifibrotic effects by molecular mechanisms that remain still unclear. Although it can interfere with the TGF- and PDGF-BB signaling, its molecular targets have not been identified yet [START_REF] Conte | Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts[END_REF][START_REF] Ruwanpura | Pirfenidone: Molecular Mechanisms and Potential Clinical Applications in Lung Disease[END_REF]. Pirfenidone can also reduce the production of oxygen activated species and limit inflammatory processes [START_REF] Plataki | Mitochondrial Dysfunction in Aged Macrophages and Lung during Primary Streptococcus pneumoniae Infection is Improved with Pirfenidone[END_REF]. The antifibrotic effects of pirfenidone are induced in vitro at concentrations that are generally much higher than those measured in the plasma of IPF patient treated with standard drug dosing [START_REF] Barranco-Garduño | Pharmacokinetic evaluation of two pirfenidone formulations in patients with idiopathic pulmonary fibrosis and chronic hypersensitivity pneumonitis[END_REF]. The present study shows that ATO can block proliferation, migration and differentiation of HLFs at non-cytotoxic concentrations that can be reached in the plasma of ACCEPTED MANUSCRIPT -CLEAN COPY 20 APL patients treated with ATO (0.15 mg/kg) [START_REF] Kumana | Systemic availability of arsenic from oral arsenic-trioxide used to treat patients with hematological malignancies[END_REF][START_REF] Shen | Studies on the clinical efficacy and pharmacokinetics of low-dose arsenic trioxide in the treatment of relapsed acute promyelocytic leukemia: a comparison with conventional dosage[END_REF][START_REF] Soignet | United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia[END_REF]. These antifibrotic effects of ATO might thus occur in patients with IPF treated with a similar dosing. Unexpectedly, the ATO concentrations used in our study were very different from those inhibiting the myofibroblastic differentiation of normal HLFs in a previous study [START_REF] Luo | Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo[END_REF]. These authors showed that nanomolar concentrations (10-20 nM) of ATO inhibited TGF-β1-induced α-SMA and COL-1 synthesis while we found that these low concentrations have no effect on the phenotype of HLFs. The difference may arise from the commercial batches of normal HLFs used by Luo et al. The response of HLFs to ATO may therefore vary from one patient to another.

Conclusion

We demonstrate that non-cytotoxic concentrations of ATO have potent antifibrotic effects in IPF HLFs. This suggests that ATO should be considered for exploration of the therapeutic potential in patients with IPF.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of CHU Rennes, no 16123. Written informed consent was obtained from all subjects.

Availability of data and materials

The datasets supporting the conclusions of this article are included in the article and its additional files.

Competing interests

SJ has received fees, funding or reimbursement for national and international conferences, boards, expert or opinion groups, research projects over the past 5 years from Actelion, AIRB, Astra Zeneca, Bellorophon Therapeutics, Biogen, BMS, Boehringer Ingelheim, Chiesi,

13.

Credit author statement

LV, SJ, AJ conceived and designed the study. AJ, LV, SJ revised the manuscript. AJ, CM, MD performed the experiments. AJ, LV, SJ analyzed and interpreted the data. FLG, DCC, CLN, BDLT, SR provided lung biopsy. AJ, LV, SJ, LW wrote the paper. All authors read and approved the final revised manuscript.

Figure 1 :

 1 Figure 1: ATO reduces HLF proliferation. IPF and control HLFs were treated with 0.01-1

Figure 2 :

 2 Figure 2: ATO alters cell cycle distribution in IPF HLFs. IPF HLFs were treated with 1

Figure 3 :

 3 Figure 3: ATO blocks the PDGF-BB-induced migration of IPF and control HLFs. IPF

Figure 4 :

 4 Figure 4: ATO reduces PDGF-BB-induced p38-kinase phosphorylation. IPF and control

Figure 5 :

 5 Figure 5: ATO decreases TGF-β1-induced cell differentiation in IPF and control HLF.

Figure 6 :

 6 Figure 6: ATO prevents the phosphorylation of SMAD2/3 induced by TGF-β1 in IPF

Figure 7 :

 7 Figure 7: ATO induces the expression of NRF2 and antioxidant proteins in IPF HLFs.

Figure 8 :

 8 Figure 8: NRF2 does not mediate the ATO-dependent inhibition of -SMA expression

ACCEPTED MANUSCRIPT -CLEAN COPY 21 Fibrogen, Galecto Biotech, Genzyme, Gilead, GSK, LVL, Mundipharma, Novartis, Olam Pharm, Pfizer, Pliant Therapeutics, Roche, Sanofi, Savara-Serendex. AJ and LV report grants from Boehringer Ingelheim.

LW is an employee of Boehringer Ingelheim.

The other authors declare that they have no competing interests.

Funding

This work was supported by The Institut National de la Santé et de la Recherche Médicale (INSERM), the Université de Rennes (Univ Rennes) and Boehringer Ingelheim Pharma GmbH.

Authors' contributions

LV, SJ, AJ conceived and designed the study. AJ, CM, MD performed the experiments. AJ, LV, SJ analyzed and interpreted the data. FLG, DCC, CLN, BDLT, SR provided lung biopsy. AJ, LV, SJ, LW wrote the paper. All authors read and approved the final manuscript.

Acknowledgements

We thank Dr Owen Parkes for editing the English text.

Supplementary data

Supplementary material

Aono, Y., Nishioka, Y., Inayama, M., Ugai, M., Kishi, J., Uehara, H., Izumi, K., Sone, S., 2005. Imatinib as a novel antifibrotic agent in bleomycin-induced pulmonary fibrosis in mice. Am. J. Respir. Crit. Care Med. 171, 1279-1285. https://doi.org/10.1164/rccm.200404-531OC

Declaration of interests

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☒ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Laurent Vernhet reports financial support was provided by Boehringer Ingelheim Pharma GmbH and Co KG. Lutz Wollin reports a relationship with Boehringer Ingelheim Pharma GmbH and Co KG Biberach that includes: employment.

Highlights

 ATO counteracted pro-fibrotic activities of primary IPF and control Human Lung Fibroblasts (HLFs).  ATO prevented the PDGF-BB-induced proliferation of IPF and control HLFs  ATO prevented the PDGF-BB-induced migration of IPF and control HLFs.  ATO decreased the TGFβ-1-induced differentiation of HLFs.  ATO induced NRF2 and antioxidant proteins in IPF and control HLFs


MD Sam Sharobeem

MD Dominique Boulmier

MD Guillaume Leurent

MD Marc Bedossa

MD PhD Christophe Leclercq

MD Philippe Mabo

MD PhD Raphael P Martins

MD Jacques Tomasi

MD,PhD Jean-Philippe P Philippe Verhoye

MD, PhD Erwan Donal

MD Gwenaelle Sost

MSc Marielle Le Guellec

MD Hervé Le Breton

MD, PhD Vincent Auffret
email: vincent.auffret@chu-rennes.fr

Prognostic impact of permanent pacemaker implantation after transcatheter aortic valve replacement

Keywords: Permanent pacemaker implantation after TAVR Aortic stenosis, TAVR, Conduction disturbances, Permanent Pacemaker Implantation, Heart Failure

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

INTRODUCTION

In patients with severe, symptomatic, aortic valve stenosis and an intermediate to high surgical risk, transcatheter aortic valve replacement (TAVR) progressively became the standard of care over surgical aortic valve replacement (SAVR) [START_REF] Vahanian | ESC/EACTS Guidelines for the management of valvular heart disease[END_REF][START_REF] Otto | ACC/AHA Guideline for the Management of Patients With Valvular Heart Disease[END_REF] . Following the demonstration of its non-inferiority compared with SAVR among patients at low-surgical risk in recent randomized trials [START_REF] Mack | Transcatheter Aortic-Valve Replacement with a Balloon-Expandable Valve in Low-Risk Patients[END_REF][START_REF] Popma | Transcatheter Aortic-Valve Replacement with a Self-Expanding Valve in Low-Risk Patients[END_REF] , the extension of TAVR indications to younger, less comorbid patients with a longer life expectancy seems inevitable [START_REF] Vahanian | ESC/EACTS Guidelines for the management of valvular heart disease[END_REF][START_REF] Otto | ACC/AHA Guideline for the Management of Patients With Valvular Heart Disease[END_REF] . Therefore, it appears of paramount importance to prevent TAVR-related complications, which may have a deleterious long-term effect. Among these complications, conduction disturbances, i.e. new-onset persistent left bundle branch block (LBBB) and permanent pacemaker implantation (PPI), remain the most common with rates which did not significantly decrease over time despite increased operators experience and refined newer-generation devices [START_REF] Auffret | Conduction Disturbances After Transcatheter Aortic Valve Replacement: Current Status and Future Perspectives[END_REF] . Although several reports have attempted to provide recommendations regarding the in-hospital management of these conduction disturbances [START_REF] Auffret | Conduction Disturbances After Transcatheter Aortic Valve Replacement: Current Status and Future Perspectives[END_REF][START_REF] Lilly | ACC Expert Consensus Decision Pathway on Management of Conduction Disturbances in Patients Undergoing Transcatheter Aortic Valve Replacement[END_REF][START_REF] Rodés-Cabau | Management of Conduction Disturbances Associated With Transcatheter Aortic Valve Replacement: JACC Scientific Expert Panel[END_REF] , guidelines are mainly based upon experts' opinion considering the lack of reliable prospective data to inform their management.

Moreover, long-term data on the prognostic impact of post-TAVR PPI, remain scarce and equivocal [START_REF] Auffret | Conduction Disturbances After Transcatheter Aortic Valve Replacement: Current Status and Future Perspectives[END_REF][START_REF] Chamandi | Long-Term Outcomes in Patients With New Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement[END_REF] even though the deleterious nature of right ventricular cardiac pacing has already been demonstrated in other clinical setting [START_REF] Elder | Pacing-induced heart disease: understanding the pathophysiology and improving outcomes[END_REF][START_REF] Zhang | Mortality risk associated with bundle branch blocks and related repolarization abnormalities (from the Women's Health Initiative [WHI])[END_REF][START_REF] Zannad | Left bundle branch block as a risk factor for progression to heart failure[END_REF][START_REF] Zhou | Different effects of abnormal activation and myocardial disease on left ventricular ejection and filling times[END_REF] . Nonetheless, peri-procedural conduction disturbances usually result from a direct injury to the conduction system, which may have different pathophysiological and prognostic implications than a degenerative impairment. Thus, delineating the long-term impact of PPI, according to its timing relative to TAVR, is crucial to allow an appropriate shared decision-making among patients suitable to TAVR and SAVR.

In this setting, the objectives of this study were to clarify the long-term prognostic impact of PPI, according to its timing relative to TAVR, upon the risk of mortality and hospitalization for heart failure (HHF).

METHODS

A full version of the methods can be found in the appendix.

J o u r n a l P r e -p r o o f

Study population

Patients who underwent TAVR at a single center from February 2009 to June 2018 were studied.

Exclusion criteria were in-hospital death, surgical conversion or unsuccessful TAVR procedure. All patients gave written informed consent for the procedures and anonymous collection of their data, which were prospectively gathered in an electronic database as part of the national registries [START_REF] Auffret | Temporal Trends in Transcatheter Aortic Valve Replacement in France: FRANCE 2 to FRANCE TAVI[END_REF] . The research reported in this paper adhered to the Helsinki Declaration as revised in 2013.

PPI status

Patients were separated in the following four groups according to the occurrence and timing PPI: no PPI, previous PPI (P-PPI), in-hospital PPI (i.e. during the index hospitalization, IH-PPI) and PPI during follow-up (FU-PPI).

Indications for PPI

In accordance with international guidelines [START_REF] Brignole | ESC Guidelines on cardiac pacing and cardiac resynchronization therapy[END_REF][START_REF] Glikson | ESC Guidelines on cardiac pacing and cardiac resynchronization therapy[END_REF] , IH-PPI was indicated for persistent high degree atrioventricular block (HAVB), defined as type 2 second degree AVB or third degree AVB, not expected to resolve or symptomatic sinus node dysfunction. Among patients with milder conduction disturbances such as new-onset LBBB with or without PR interval prolongation, the final indication of PPI was at the discretion of the senior electrophysiologist responsible for the patient, taking into account clinical data and the in-hospital ECG evolution. For such patients with conduction disturbances not representing absolute PPI indications, a local electrophysiological study-guided PPI strategy was implemented in our center in June 2017. Details regarding this strategy have been previously published [START_REF] Bourenane | Electrophysiological Study-Guided Permanent Pacemaker Implantation in Patients With Conduction Disturbances Following Transcatheter Aortic Valve Implantation[END_REF] .

During follow-up, the indication of PPI was at the discretion of the physician responsible for the patient.

Follow-up

Follow-up was performed by on-site clinical visit and/or telephone at 1-month post-TAVR, at 1 year and yearly thereafter.

Outcomes

The primary objective of the present study was to assess the long-term clinical impact of PPI, according to its timing relative to TAVR, on the composite of all-cause death and HHF.

J o u r n a l P r e -p r o o f

Statistical Analysis

Continuous variables were presented as mean ± standard deviation or median (interquartile range) depending on their distribution, which was assessed using the Kolmogorov-Smirnov test, and were compared using the Kruskal-Wallis test. Categorical variables were summarized as numbers (percentages), and compared using chi-square tests or the Fisher exact test as appropriate. HHF was evaluated using competing-risk models with all-cause mortality as a competing event. Competing-risk models were used to determine the predictors of HHF by reporting adjusted sub-hazard ratios (sub HR) with their 95% confidence intervals (CI). All-cause mortality was analyzed using Cox proportional hazard models. Parametric multistate survival models, adjusted for the previously identified variables, were performed to predict the probability of transitioning from one state (post-TAVR status, post-TAVR PPI status, post-TAVR HHF status, death) to another over time.

Generalised linear model for repeated measures with interaction was used to compare the changes in LVEF at different time points between groups. All tests were 2-sided at the 0.05 significance level.

RESULTS

Baseline and in-hospital characteristics

During the study period, 1250 patients underwent a TAVR procedure. Thirty-nine died during the subsequent hospitalization, 9 needed a surgical conversion, whereas the procedure was unsuccessful in 3 patients. A total of 1199 TAVR patients (median age: 83 years old, 45.8% female), discharged alive after a successful TAVR procedure, were included in the present analysis. Among them, 130 had a previous PPI, 116 were implanted during the index hospitalization and 59 during follow-up. Median follow-up was 2.94 years (1.42-4.32

Timing and characteristics of PPI

The clinical indications for PPI, device type and timing of PPI are available in supplementary tables 3 and 4. For IH-PPI, the most common indication was HAVB (86.2%) and most of the devices were dual chamber pacemakers (68.1%). For FU-PPI, only 6 patients (10.2%, 0.6% of patients at risk) underwent PPI within 30 days of TAVR while the median time to implantation was 1.7 (0.5-2.8) years. A significant proportion of patients (16.9%) received cardiac resynchronization therapy (CRT) among this sub-group. The Nelson-Aalen estimates of the rate of PPI during follow-up among the 953 patients discharged without a permanent pacemaker are represented in supplementary Figure 1.

Prognostic impact of permanent pacemaker status

During the study period, 275 patients (22.9%) experienced HHF and 447 (37.3%) died. Annual estimates of HHF within 5 years post-TAVR were: 11.0% (95% CI: 9.3-13.0%), 17.3% (95% CI: 15.3-19.7%), 22.4% (95% CI: 19.8-25.3%), 27.0% (95% CI: 24.0-30.3%), and 33.6% (29.7-37.7%).

In the FU-PPI group, the first HHF occurred before PPI in 22 patients (37.3%) and after in 37 patients (62.7%). In the former subgroup, the first HHF occurred within a week of PPI in 7 patients (31.8%). In the latter subgroup the median delay between PPI and HHF was 1.3 (0.4-2.1) years. Annual survival estimates within 5 years post-TAVR were: 93.5% (95% CI: 91.9-94.8%), 87.3% (95% CI: 85.0-89.1%), 78.4% (95% CI: 75.6-80.9%), 66.2% (95% CI: 62.8-69.4%), and 54.5% (95% CI: 50.7-

58.6%).

Figure 1 depicts the Kaplan-Meier estimates of the rate of the composite primary outcome over time according to PPI status. Variables associated with the composite of all-cause death and HHF in univariate and multivariable regressions are shown in Table 2. FU-PPI was associated with a higher occurrence of the primary outcome (HR: 2.11, 95%CI: 1.39-3.20, p<0.001) whereas P-PPI and IH-PPI J o u r n a l P r e -p r o o f

were not (HR: 0.96, 95%CI: 0.71-1.29, p =0.77 and HR:1.26, 95%CI: 0.88-1.81, p=0.21, respectively).

The Kaplan-Meier estimates of the rate of all-cause mortality according to PPI status are provided in Figure 2. Supplementary Table 5 reports the univariate and multivariable correlates of all-cause death. Only P-PPI demonstrated a protective association with all-cause mortality in multivariable analysis (HR: 0.61, 95%CI: 0.43-0.87, p=0.006) whereas other PPI groups were not significantly associated with survival.

Figure 3 reports the cumulative incidence functions of HHF according to PPI status. FU-PPI associated with a significantly higher risk of HHF (sub HR: 3.21, 95%CI: 2.02-5.11, p<0.001) while this relation was only borderline significant for P-PPI (sub HR: 1.51, 95%CI: 0.99-2.29, p=0.05). In contrast, there was no relationship between IH-PPI and the subsequent risk of HHF (Supplementary Table 6).

The adjusted probabilities of transitioning from post-TAVR status to post-TAVR HHF and death at each time point in the overall population and according to PPI status and timing, estimated by parametric multistate models, are represented in supplementary Figures 2 and3, respectively.

Regarding transition to HHF, there was an increasing probability from the no-PPI to P-PPI to IH-PPI and to FU-PPI within the first three years post-TAVR. Regarding transition to death, the probability steadily increased in all groups with a markedly higher probability at each time point in the FU-PPI group.

Changes in LVEF over time

An echocardiographic LVEF was available at a median follow-up of 408 days (361-961; mean 665 days) in 1089 patients. Changes in LVEF over time according to PPI status are shown in Figure 4.

DISCUSSION

The main findings of this large cohort of patients discharge alive after a TAVR procedure can be summarized as follows: 1-P-PPI patients showed evidence of a more evolved valvular heart disease at baseline. 2-IH-PPI was performed in 10.8% of patients, whereas FU-PPI occurred at a median timing of 1.7 years post-TAVR and involved a significant proportion of CRT (16.9%). 3-No detrimental J o u r n a l P r e -p r o o f association was found between P-PPI or IH-PPI and the composite primary outcome of all-cause death or HHF. On the contrary, FU-PPI was independently associated with the primary outcome; a result driven by a 3-fold higher risk of HHF. 4-Despite an increased probability of transitioning to HHF and death among IH-PPI patients in multistate models, our multivariable regressions results suggest that the pacemaker implantation per se does not play a significant role in the long-term risk of these outcomes.

The rate of IH-PPI in the present study is in the lower range of previously published data [START_REF] Auffret | Conduction Disturbances After Transcatheter Aortic Valve Replacement: Current Status and Future Perspectives[END_REF][START_REF] Chamandi | Reported Versus "Real" Incidence of New Pacemaker Implantation Post-Transcatheter Aortic Valve Replacement[END_REF] , which may be explained by a restrictive implantation strategy. Indeed, the indication for pacing was persistent HAVB in 86.2% and symptomatic sinus node dysfunction in 0.9% of IH-PPI patients, which represent class I indications for pacing in recent guidelines [START_REF] Glikson | ESC Guidelines on cardiac pacing and cardiac resynchronization therapy[END_REF] . In contrast, 71.4% and 5.6% of patients in a recent multicenter series investigating the long-term effect of 30-days PPI following TAVR received a permanent pacemaker for HAVB and sinus node dysfunction, respectively [START_REF] Chamandi | Long-Term Outcomes in Patients With New Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement[END_REF] . The indications for pacing was not reported in another recent single-center report of 816 patients focusing on the longterm impact of post-TAVR conduction disturbances [START_REF] Jørgensen | Mortality and Heart Failure Hospitalization in Patients With Conduction Abnormalities After Transcatheter Aortic Valve Replacement[END_REF] . Furthermore, in the present study, up to 31.3% of other indications were retained after an electrophysiological study-guided implantation strategy, which demonstrated its accuracy in identifying patients who can be safely discharged without PPI, thus avoiding unnecessary PPI [START_REF] Bourenane | Electrophysiological Study-Guided Permanent Pacemaker Implantation in Patients With Conduction Disturbances Following Transcatheter Aortic Valve Implantation[END_REF] . The linear nature of FU-PPI, with only 0.6% of patients at risk undergoing PPI within 30 days of TAVR, also supports the appropriate selection of IH-PPI recipients in the present report.

Previous studies reached conflicting results regarding the impact of early PPI post-TAVRupon the risk of mortality and HHF at follow-up [START_REF] Chamandi | Long-Term Outcomes in Patients With New Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement[END_REF][START_REF] De Carlo | Safety of a conservative strategy of permanent pacemaker implantation after transcatheter aortic CoreValve implantation[END_REF][START_REF] Nazif | Predictors and clinical outcomes of permanent pacemaker implantation after transcatheter aortic valve replacement: the PARTNER trial and registry[END_REF][START_REF] Urena | Permanent pacemaker implantation after transcatheter aortic valve implantation: impact on late clinical outcomes and left ventricular function[END_REF][START_REF] Fadahunsi | Incidence, Predictors, and Outcomes of Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement: Analysis From the U.S. Society of Thoracic Surgeons/American College of Cardiology TVT Registry[END_REF][START_REF] López-Aguilera | Long-term clinical impact of permanent cardiac pacing after transcatheter aortic valve implantation with the CoreValve prosthesis: a single center experience[END_REF] . Most of these studies were of moderate size with a limited follow-up (<2 years). A recent study-level meta-analysis including >20000 patients with post-TAVR PPI suggested its deleterious influence on 1-year all-cause mortality as well as HHF, yet not cardiovascular death [START_REF] Faroux | Clinical impact of conduction disturbances in transcatheter aortic valve replacement recipients: a systematic review and meta-analysis[END_REF] . Chamandi et al. also reported a significant association between PPI within 30 days of TAVR and HHF among 1629 TAVR recipients at a median of 4 years of follow-up, without demonstrating a meaningful association either with cardiac or total mortality [START_REF] Chamandi | Long-Term Outcomes in Patients With New Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement[END_REF] . In their single-center series, Jørgensen et al. reported a higher risk of HHF and all-cause mortality among TAVR recipients with 30-days PPI at a median follow-up of 2.5 years. In the present study, P-PPI and IH-PPI had no J o u r n a l P r e -p r o o f detrimental association with survival or HHF, while only FU-PPI was significantly associated with HHF and the composite of all-cause death and HHF. Thereby, there are overall significant discrepancies in the literature regarding the true impact of pacing in TAVR recipients. A number of reasons may explain these differences. First, in studies by Chamandi et al. and Jørgensen et al. the PPI group included IH-PPI and FU-PPI patients implanted within 30 days of TAVR [START_REF] Chamandi | Long-Term Outcomes in Patients With New Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement[END_REF][START_REF] Jørgensen | Mortality and Heart Failure Hospitalization in Patients With Conduction Abnormalities After Transcatheter Aortic Valve Replacement[END_REF] . Nonetheless, it can be assumed that the majority of pacemakers were implanted during the index hospitalization given the median time from TAVR to PPI of 2 (1-4) days and the median length of hospital stay of 7 (5-9) days reported by Chamandi et al [START_REF] Chamandi | Long-Term Outcomes in Patients With New Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement[END_REF] . Therefore, it is unlikely that this difference in the definition of the PPI group played a significant role in the differing association of PPI with clinical outcomes. Second, the detrimental influence of a high burden (>40%) of right ventricular pacing has been previously highlighted [START_REF] Jørgensen | Mortality and Heart Failure Hospitalization in Patients With Conduction Abnormalities After Transcatheter Aortic Valve Replacement[END_REF][START_REF] Nadeem | Impact of Right Ventricular Pacing in Patients Who Underwent Implantation of Permanent Pacemaker After Transcatheter Aortic Valve Implantation[END_REF] . These previous studies reported rates of approximately 50% of high pacing burden at follow-up among TAVR recipients who underwent early post-procedural PPI [START_REF] Chamandi | Long-Term Outcomes in Patients With New Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement[END_REF][START_REF] Jørgensen | Mortality and Heart Failure Hospitalization in Patients With Conduction Abnormalities After Transcatheter Aortic Valve Replacement[END_REF][START_REF] Nadeem | Impact of Right Ventricular Pacing in Patients Who Underwent Implantation of Permanent Pacemaker After Transcatheter Aortic Valve Implantation[END_REF] . Unfortunately, the ventricular pacing percentage at follow-up was not routinely reported in our database. However, we demonstrated in a recent study including 78 TAVR recipients who benefited from our local electrophysiological study-guided PPI strategy, that 85% of patients had >1% of pacing, which is comparable to the data of Chamandi et al., nonetheless with only 25% of patients demonstrating a high pacing burden at follow-up [START_REF] Chamandi | Long-Term Outcomes in Patients With New Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement[END_REF][START_REF] Bourenane | Electrophysiological Study-Guided Permanent Pacemaker Implantation in Patients With Conduction Disturbances Following Transcatheter Aortic Valve Implantation[END_REF] . Although a higher pacing burden may be expected among patients with unequivocal pacing indications such as HAVB, these data may also reflect a careful programming of devices to preserve the spontaneous conduction of patients. Of note, it has previously been demonstrated that up to 45% of TAVR patients who underwent PPI have sufficient atrioventricular conduction to avoid pacing at follow-up suggesting the importance of regular pacemaker interrogations and reprogramming in this population [START_REF] Marzahn | Conduction recovery and avoidance of permanent pacing after transcatheter aortic valve implantation[END_REF] . In keeping with this point, 68% of the IH-PPI patients received dual-chamber devices, which may offer the opportunity for a more physiologic pacing, compared with 57% in the study by Chamandi et al [START_REF] Chamandi | Long-Term Outcomes in Patients With New Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement[END_REF] . Third, it may also be hypothesize that significant residual confounding played a crucial role in previous studies, PPI being solely an innocent bystander of the worse outcomes observed. The fact that the association between PPI and increased 1year mortality was independent of cardiac causes of death in the recent meta-analysis by Faroux et al supports this hypothesis [START_REF] Faroux | Clinical impact of conduction disturbances in transcatheter aortic valve replacement recipients: a systematic review and meta-analysis[END_REF] . The adjustment strategy performed in the present study, especially with the J o u r n a l P r e -p r o o f inclusion of echocardiographic parameters at discharge such as pulmonary hypertension, which we previously identified as a strong predictor of HHF post-TAVR [START_REF] Auffret | Determinants and Impact of Heart Failure Readmission Following Transcatheter Aortic Valve Replacement[END_REF] , may have contributed to mitigate the association between IH-PPI and HHF. Overall, it should be stressed that our data do not support a lower threshold for IH-PPI. Rather, we believe that physicians should apply a careful selection strategy of PPI recipients post-TAVR based on class I indications in recent guidelines and rely on electrophysiological studies or ambulatory ECG monitoring, which are being further evaluated, to guide their management in equivocal cases [START_REF] Glikson | ESC Guidelines on cardiac pacing and cardiac resynchronization therapy[END_REF] .

From a pathophysiological standpoint, IH-PPI may affect patients with a direct injury to the conduction system with minimal or without significant structural heart damage whereas PPI remote from TAVR (either P-PPI or FU-PPI) may involve patients with a damage to their conduction pathways reflecting a more global degenerative impairment of their cardiac structure and function in the setting of more evolved valvular heart diseases [START_REF] Dweck | Calcific aortic stenosis: a disease of the valve and the myocardium[END_REF] . Aside from the overall cardiac involvement of severe aortic stenosis, the former group is also more susceptible to experience a recovery of the atrioventricular conduction leading to low ventricular pacing rates unlikely to promote an asynchronyinduced left ventricular dysfunction and its subsequent complications [START_REF] Elder | Pacing-induced heart disease: understanding the pathophysiology and improving outcomes[END_REF][START_REF] Zhang | Mortality risk associated with bundle branch blocks and related repolarization abnormalities (from the Women's Health Initiative [WHI])[END_REF][START_REF] Zannad | Left bundle branch block as a risk factor for progression to heart failure[END_REF][START_REF] Zhou | Different effects of abnormal activation and myocardial disease on left ventricular ejection and filling times[END_REF][START_REF] Curtis | Biventricular pacing for atrioventricular block and systolic dysfunction[END_REF] . Regarding FU-PPI, its exact role in the occurrence of HHF remains elusive despite the significant association evidenced in the present report (sub HR 3.21, 95% CI:2.02-5.11; p<0.001). Indeed the occurrence of HHF before PPI in a significant proportion of these patients and the high implantation rate of CRT (16.9%) among them, point towards an unfavorable evolution following TAVR independent of permanent pacing. Also, undiagnosed bradyarrhythmia may have precipitated these unfavorable events in some cases.

Nevertheless, almost two-third of these patients had their first HHF after PPI, which may also suggest that they represent a cohort particularly sensitive to chronic right ventricular pacing. Overall, these data suggest we should raise our awareness regarding the myocardial involvement contributing to symptoms in severe aortic stenosis, provide patients with optimal guidelines-directed medical therapies, and ensure a close follow-up of TAVR recipients, especially those requiring pacing at follow-up.

This study represents a retrospective analysis of prospectively collected data and is subject to the inherent limitations of this study design. This was a single center report, which may limit the J o u r n a l P r e -p r o o f generalizability of our results. The lack of data regarding pacing percentage at follow-up precluded the analysis of its role in the long-term outcomes post-TAVR. We also acknowledge the lack of information regarding pacing indications prior to TAVR and at follow-up. Finally, although the follow-up of the present study is among the longest in studies focusing on the effect of pacing post-TAVR, it remains somewhat limited and we cannot exclude that it had some influence upon our results.

CONCLUSION

IH-PPI has no long-term detrimental influence on the composite of all-cause mortality and HHF, whereas FU-PPI increases its risk, mainly because of a higher risk of HHF. Provided an optimal patients selection for IH-PPI, these results are of major importance to inform TAVR candidates regarding the long-term risk of the procedure. Moreover, whether FU-PPI patients represent a cohort particularly sensitive to chronic right ventricular pacing should be evaluated in future studies. In the interim, physicians should pay the utmost attention to ensure a careful follow-up and optimize heart failure therapies (drug and/or device, i.e. CRT) among these patients.

SOURCE OF FUNDING:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

J o u r n a l P r e -p r o o f

  Statistical analyses were conducted using Stata Statistical Software Release 13 (StataCorp, College Station, TX) and the Statistical Package for Social Sciences version 25 (SPSS Inc., IBM, Armonk, New York).

Figure 1 -Figure 2 -

 12 FIGURES LEGENDS

Figure 3 -

 3 Figure 3 -Cumulative incidence function of hospitalization for heart failure according to PPI status

Figure 4 -

 4 Figure 4 -Changes in left ventricular ejection fraction over time according to PPI status

  J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

in table 1. Procedural characteristics and in-hospital outcomes

  years). Baseline characteristics of the study population are shown

	Procedural and in-hospital data are available in supplementary table 1. Briefly, balloon-expandable
	devices were implanted in 66.9% of patients whereas 32.1% received self-expandable valves. Rates of
	new-onset persistent left bundle branch block were 17.3%, 6.6%, 15.2%, and 25.0% in the no PPI, P-
	PPI, IH-PPI, and FU-PPI groups, respectively. Treatments at discharge are summarized in
	supplementary table 2.
	J o u r n a l P r e -p r o o f

Table 1 -Characteristics of the study population according to the presence and timing of implantation of a permanent pacemaker

 1

	Patients without a	Patients with a	Patients with in-	Patients with	p-value
	permanent	previous permanent	hospital permanent	permanent
	pacemaker (n=894)	pacemaker (n=130)	pacemaker	pacemaker
			implantation (n=116)	implantation
				during follow-up
				(n=59)

Table 2 -Univariate and multivariable predictors of the composite of all-cause death and heart failure

 2 CI: confidence interval; HR: hazard-ratio; PPI: permanent pacemaker implantation; RAS: renin-angiotensin system. Other abbreviations as in Table1

	hospitalization
	Variable	Univariate HR	p-value	Multivariable	p-value
		(95% CI)		HR (95% CI)
	PPI
	Previous PPI	1.31 (1.00-1.72)	0.05	0.96 (0.71-1.29)	0.77
	In-hospital PPI	1.45 (1.03-2.03)	0.03	1.26 (0.88-1.81)	0.21
	PPI during follow-up	2.32 (1.55-3.49)	<0.001	2.11 (1.39-3.20)	<0.001
	Age	1.02 (1.01-1.03)	0.004	1.02 (1.00-1.03)	0.009
	Female sex	0.82 (0.70-0.97)	0.02	0.81 (0.66-1.00)	0.054
	Body surface area	1.49 (1.01-2.18)	0.04	1.25 (0.72-2.19)	0.43
	Previous aortic balloon valvuloplasty	1.29 (0.99-1.67)	0.056	0.97 (0.73-1.29)	0.86
	Previous cerebrovascular disease	1.24 (1.01-1.53)	0.04	1.23 (0.98-1.54)	0.075
	Chest radiotherapy	0.77 (0.58-1.04)	0.09	0.95 (0.69-1.30)	0.74
	Atrial fibrillation	1.82 (1.54-2.15)	<0.001 1.34 (1.11-1.62)	0.002
	NYHA III or IV NTproBNP Previous acute heart failure Chronic lung disease Previous diabetes mellitus Oral antidiabetic drugs Insulin-treated Chronic kidney disease Moderate Severe Dialysis Logistic EuroScore II Mean aortic gradient prior to TAVR Aortic regurgitation grade III or IV prior to TAVR Moderate or severe mitral stenosis prior to TAVR Approach Sub-clavian Transaortic J o u r n a l P r e -p r o o f 1.20 (1.01-1.41) 0.03 1.08 (0.90-1.29) 1.00 (1.00-1.00) <0.001 1.00 (1.00-1.00) 1.69 (1.43-2.00) <0.001 1.30 (1.08-1.58) 1.39 (1.18-1.65) <0.001 1.30 (1.08-1.56) 1.22 (0.98-1.54) 1.74 (1.24-2.43) 0.08 0.001 1.10 (0.86-1.40) 1.58 (1.11-2.25) 1.52 (1.28-1.80) 1.32 (0.86-2.03) 1.93 (0.72-5.20) <0.001 0.21 0.19 1.39 (1.14-1.69) 1.09 (0.69-1.73) 0.79 (0.25-2.51) 1.03 (1.01-1.06) 0.006 0.99 (0.96-1.02) 0.991 (0.986-0.996) 0.002 0.99 (0.99-1.00) 0.48 (0.25-0.93) 0.03 0.49 (0.27-0.92) 1.38 (1.02-1.86) 0.04 1.38 (1.00-1.90) 1.02 (0.73-1.43) 1.11 (0.75-1.65) 0.91 0.61 0.95 (0.66-1.36) 0.89 (0.58-1.38)	0.42 0.075 0.007 0.005 0.44 0.012 0.001 0.71 0.69 0.45 0.035 0.026 0.047 0.77 0.62
	Transapical	0.82 (0.57-1.17)	0.27	1.03 (0.69-1.53)	0.89
	Transcarotid	1.58 (0.99-2.51)	0.05	1.17 (0.72-1.92)	0.52
	Major vascular complications	1.50 (0.94-2.40)	0.09	1.83 (1.13-2.99)	0.015
	Acute kidney injury stage 2 or 3	2.00 (1.03-3.91)	0.04	1.26 (0.62-2.57)	0.65
	Percentage of optimal daily
	recommended dose of RAS inhibitor at
	discharge
	1% -< 25%	1.19 (0.80-1.76)	0.39	0.91 (0.60-1.39)	0.23
	25% -< 50%	0.95 (0.75-1.21)	0.68	0.91 (0.71-1.18)	0.49
	50% -< 75%	0.84 (0.66-1.06)	0.14	0.86 (0.67-1.10)	0.24
	75% -100%	0.77 (0.58-1.03)	0.08	0.92 (0.68-1.43)	0.57
	Loop diuretics at discharge
	Dose < 40 mg/d	1.25 (1.04-1.51)	0.02	1.01 (0.82-1.25)	0.90
	Dose > 40 mg/d	2.03 (1.62-2.56)	<0.001	1.27 (0.97-1.67)	0.08
	LVEF at discharge	0.988 (0.982-0.994) <0.001 0.99 (0.99-1.00)	0.23
	LVEDD at discharge	1.01 (1.00-1.02)	0.02	1.00 (0.99-1.02)	0.91
	Indexed aortic valve area at discharge	0.73 (0.55-0.98)	0.03	0.82 (0.58-1.15)	0.24
	Aortic regurgitation grade III or IV at	1.80 (0.94-3.29)	0.08	1.57 (0.78-3.18)	0.21

Dr Le Breton received lecture fees from Edwards Lifescience. Dr Donal received research grants from Abbott and General Electric Healthcare. Dr Mabo received consulting fees from Microport and research grants from Medtronic. Dr Leclercq received lecture fees from Abbott, Biotronik, Medtronic, and Boston Scientific.