Camille Pichard

Amélie Nicolas

Vincent Galand

Charlène Coquard

Nathalie Behar

Céline Chérel

J.-C Daubert

Philippe Mabo

Christophe Leclercq

Dominique Pavin

Celine Chérel

Jean-Claude Daubert

Mathieu Lederlin

Raphaël P Martins
email: raphael.martins@chu-rennes.fr

Cryoballoon ablation of atrial fibrillation in patients with atypical right pulmonary vein anatomy

Keywords: Arrhythmias, Atrial fibrillation, Catheter ablation, Electrophysiology, Cryoablation 3D, three-dimensional, AF, atrial fibrillation, CT, computed tomography, LA, left atrium/atrial, PNP, phrenic nerve palsy, PV, pulmonary vein, RIPV, right inferior pulmonary vein, RMPV, right middle pulmonary vein, RSPV, right superior pulmonary vein. 4

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

Background

Cryoballoon ablation has become a widely used technique to achieve pulmonary vein (PV) isolation in patients with symptomatic atrial fibrillation (AF). Similar mid-and long-term results compared with radiofrequency ablation have been reported [START_REF] Kuck | Cryoballoon or Radiofrequency Ablation for Paroxysmal Atrial Fibrillation[END_REF][START_REF] Mugnai | Comparison of pulmonary vein isolation using cryoballoon versus conventional radiofrequency for paroxysmal atrial fibrillation[END_REF]. However, PV isolation using the cryoballoon is critically dependent on circumferential and homogeneous freezing all around the PV ostia, and anatomical factors may affect catheter stability, ablation efficacy and long-term sinus rhythm maintenance. Thus, PV anatomy is an important factor influencing cryoballoon ablation. Indeed, the efficacy of cryoballoon ablation in patients with a left common trunk has been analysed in a few studies, which reported either similar [START_REF] Heeger | Acute efficacy, safety, and long-term clinical outcomes using the second-generation cryoballoon for pulmonary vein isolation in patients with a left common pulmonary vein: A multicenter study[END_REF][START_REF] Stroker | Second-generation cryoballoon ablation in the setting of left common pulmonary veins: Procedural findings and clinical outcome[END_REF] or lower [START_REF] Beiert | Outcome in patients with left common pulmonary vein after cryoablation with second-generation cryoballoon[END_REF][START_REF] Shigeta | Clinical assessment of cryoballoon ablation in cases with atrial fibrillation and a left common pulmonary vein[END_REF] long-term efficacy compared with patients with normal left PV drainage.

A considerably more variable anatomy has been described for right PV drainage, as up to 26% of patients may present a middle lobe vein directly connected to the left atrium (LA) [START_REF] Marom | Variations in pulmonary venous drainage to the left atrium: implications for radiofrequency ablation[END_REF]. However, to the best of our knowledge, there are no studies analysing the efficacy of cryoballoon ablation in such patients. Thus, in this study, we prospectively evaluated mid-term efficacy and procedural characteristics of cryoballoon ablation in patients with typical and atypical PV anatomy, and hypothesize that ablation of the right superior PV (RSPV) and the right inferior PV (RIPV), neglecting accessory PVs, is sufficient to obtain acute right-side PV isolation and maintenance of mid-term sinus rhythm.

Methods

Patients and design

Consecutive patients referred to our tertiary centre from 01 January 2015 to 31 October 2018 for cryoballoon ablation of paroxysmal AF were enrolled prospectively. Patients were included if they presented symptomatic drug-refractory AF with at least one recurrence on antiarrhythmic drug therapy.

Patients were not included if a left atrial (LA) computed tomography (CT) scan could not be realized before the procedure. This study was approved by the institutional ethics committee, and all patients provided informed consent.

Cardiac CT acquisition and analysis of right PV anatomy

A left atrial CT scan was performed on the day before cryoballoon ablation to analyse PV anatomy.

Image acquisition was performed on a 64-row multidetector CT scanner (Discovery™ CT750 HD; GE Healthcare, Milwaukee, WI, USA) during a single breath-hold, without electrocardiogram gating. Typical acquisition parameters were: field of view 20-25 cm; tube voltage 100-120 kV; tube current 350 mAs with a dose modulation protocol; slice thickness 0.625 mm; and pitch 0.984. Z-axis coverage was limited to the cardiac volume, from the carina to the caudal part of the LA. A bolus of 60-100 mL of iobiditridol (Xenetix® 350; Guerbet, Roissy, France) was injected at 4 mL/s, followed by a 40 mL saline chaser bolus. An automated bolus tracking system was used to synchronize the arrival of the contrast material with the initiation of the scan, with a threshold set at 150 Hounsfield units in the LA.

A blind analysis of right PV anatomy was performed by two independent observers (one thoracic radiologist and one cardiologist) using Telemis PACS software, version 4.7 (Telemis SA, Louvain-la-Neuve, Belgium) and through a postprocessing three-dimensional (3D) reconstruction using CARTO-Merge software (Biosense Webster, Diamond Bard, CA, USA). Discordance between readers was adjudicated by a third reader to determine the exact type of anatomy. Ostial architecture (using both external and endoluminal projections) was analysed, identifying PV drainage pattern. For each patient, PV drainage pattern, number of PV ostia and maximal and minimal right PV ostial diameters were assessed. The classification proposed by Marom et al. [START_REF] Marom | Variations in pulmonary venous drainage to the left atrium: implications for radiofrequency ablation[END_REF] was used to classify right PV anatomy (Fig. 1); thus, R1, R2, R3, R4 and R5 patterns of right PV anatomy were used to describe patients with one, two, three, four or five right PV ostia draining to the LA, respectively. Then, each pattern was differentiated depending on the draining pattern of the middle pulmonary lobe (e.g. R2a, R2b or R2c, as shown in Fig. 1). In our study, "normal" PV anatomy was defined as patients having two PV ostia (i.e. R2a, R2b and R2c patterns), while "atypical" PV anatomy was defined as those having one, three, four or five PV ostia (i.e. R1, R3a, R3b, R3c, R4a, R4b and R5 patterns).

Cryoballoon ablation procedural characteristics

Before the procedure, transoesophageal echocardiography was performed to exclude the presence of LA thrombus. Vitamin K antagonists were continued (international normalized ratio of 2-3) if prescribed, while non-vitamin K antagonist oral anticoagulation was discontinued and replaced with intravenous heparin before ablation. Procedures were performed under conscious sedation using midazolam and fentanyl as necessary.

Venous access was obtained via the femoral vein. A 6F Xtrem® quadripolar catheter (Sorin SPA, was performed under fluoroscopic and pressure guidance, without the use of periprocedural transoesophageal or intracardiac echocardiography. A "single big cryoballoon" approach, using a 28 mm balloon (Medtronic, Minneapolis, MN, USA) was performed, as described previously [START_REF] Chun | The 'single big cryoballoon' technique for acute pulmonary vein isolation in patients with paroxysmal atrial fibrillation: a prospective observational single centre study[END_REF].

The cryoballoon catheter was introduced into the LA through a steerable, 12F inner diameter FlexCath® sheath (Medtronic, Minneapolis, MN, USA) constantly flushed with heparinized saline. An Achieve™ mapping catheter (Medtronic, Minneapolis, MN, USA) was advanced over the cryoballoon to the PV orifice and positioned inside the PV. The cryoballoon was inflated and advanced to the ostium of each PV. The quality of vein occlusion was assessed by the injection of diluted iodinated contrast agent into the PV using a semiquantitative grading, as previously described, from grade 4 (excellent with full retention of contrast medium) to grade 1 (very poor occlusion leading to rapid leakage from the PV) [START_REF] Chierchia | The Use of Cryoballoon Ablation in Atrial Fibrillation: Simplifying Pulmonary Vein Isolation?[END_REF]. Ablation duration was decided upon depending on the time to PV isolation: 180 or 240 seconds if PV isolation was documented before or after 30 seconds of freezing, respectively; if the Achieve™ catheter was pushed into the PV to obtain a better stability of the cryoballoon, leading to the inability to observe real-time PV isolation, a 240-second freezing was performed.

Ablation was first performed for the left superior PV and then for the left inferior PV. Before ablation of the right-sided PVs, the quadripolar catheter was relocated to the superior vena cava to constantly pace the right phrenic nerve at a 1500-2000 ms cycle length and a 10 mA/2 ms output during freezing. The pacing was started 30 seconds after the initiation of freezing to avoid cryoballoon dislodgment as a result of the intense right hemidiaphragmatic contractions. In case of cessation or weakening of the contraction, freezing was immediately discontinued and the cryoballoon was deflated.

For patients with atypical right PV anatomies, cryoballoon ablation was only performed for the RSPV and the RIPV. Indeed, because a 28 mm cryoballoon was used, and to avoid increasing the risk of phrenic nerve palsy (PNP), we assumed that the RSPV and RIPV applications would isolate these supplementary PVs.

Finally, the accessory right PVs were eventually catheterized with the Achieve™ catheter to assess PV isolation before cryoballoon removal.

Postablation management and follow-up

After the procedure, all antiarrhythmic drugs were stopped, and patients underwent continuous inhospital electrocardiogram monitoring for 48 hours. The first out-patient clinic visit was 4-6 weeks after the procedure. Subsequent follow-up visits consisted of a clinical interview, surface electrocardiogram and 24-hour Holter monitoring, at 3, 6 and 12 months. Recurrence of atrial arrhythmia was defined as any AF, atrial tachycardia or flutter lasting longer than 30 seconds, recorded, after a 3-month blanking period, according to the latest guidelines [START_REF] Calkins | HRS/EHRA/ECAS/APHRS/SOLAECE expert consensus statement on catheter and surgical ablation of atrial fibrillation: Executive summary[END_REF]. Procedural success was defined as freedom from any documented arrhythmia recurrence without administration of any antiarrhythmic drugs.

Endpoints

The primary endpoint was survival free from any documented atrial arrhythmia. The secondary endpoints were procedural characteristics during the first freezing application (minimal temperature, time to PV isolation, number of pulmonary vein potentials recorded during freezing) in both groups (normal and atypical right PV anatomy) and the occurrence of PNP.

Statistical analysis

Normally distributed variables are expressed as means ± standard deviations, and were compared using Student's t test. Non-normally distributed variables are expressed as medians (interquartile ranges), and were compared using the Mann-Whitney U test. Categorical variables are expressed as counts and percentages, and were compared using the χ 2 test or Fisher's exact test when needed. A P value < 0.05 was considered statistically significant. The analyses were performed with the SPSS statistical package, version 11.0 (SPSS Inc., Chicago, IL, USA).

Results

Population characteristics

From 01 January 2015 to 31 October 2018, 303 consecutive patients had cryoballoon ablation of symptomatic paroxysmal AF, and were included prospectively. Clinical characteristics are described in Table 1. Patients were mainly men, the median age was 61.0 (53.8-67.9) years and co-morbidities were rare, reflected by the low CHA2DS2-VASc score of the overall population (66.0% of all patients had a score of 0 or 1). The median left ventricular ejection fraction was 62.0% (60.0-65.0%). A median of 2.0 (1.0-2.0) antiarrhythmic drugs were tested before the ablation was scheduled.

Anatomical considerations

Right PV drainage pattern were classified as stated above (Fig. 1). As shown in Table 2, most patients (n = 254, 83.8%) had the expected normal right PV anatomy, consisting of two right atrial ostia for upper and lower lobe veins, respectively, with the middle lobe vein joining the superior lobe vein. The remaining 49 (16.2%) patients had an atypical right PV anatomy. The most common atypical drainage was an R3a pattern (n = 40, 81.6%) i.e. three atrial ostia for the superior, middle and lower lobe veins, respectively. R3b and R4 anatomies were each found in three patients, R3c anatomy was found in two patients and one patient had an R5 anatomy. None of the patients presented an R1 anatomy.

Examples of atypical right PV anatomies are depicted in Fig. 2.

RSPV short-and long-axis diameters of patients with "atypical" right PV anatomy were significantly smaller than those with the R2 pattern (P < 0.001). For patients with an R3 anatomy, the right middle PV (RMPV) long and short axes were 10.3 ± 2.3 mm and 7.9 ± 2.2 mm respectively, and its distances to the RSPV and the RIPV were 3.7 ± 2.1 and 5.3 ± 2.7 mm, respectively.

Procedural characteristics

After ablating left-side PVs, cryoballoon applications were performed in the RSPV and RIPV only, neglecting accessory right PVs. First-freeze isolation for RSPV occurred in 44 (89.8%) and 218 (85.8%) patients with atypical and typical right PV anatomy, respectively (P = 0.61; Table 3). Similar results were observed for the RIPV, with first-freeze isolation occurring in 37 (75.5%) and 217 (85.4%) patients with atypical and typical right PV anatomy, respectively (P = 0.13). There was no difference in terms of occlusion degree during freezing. Minimal temperature was lower in both the RSPV and RIPV in the group with normal anatomy (-47.6 ± 5.7 vs -49.5 ± 9.5 [P = 0.006] and -46.2 ± 7.2 vs -47.7 ± 8.1 [P = 0.046]), and more real-time recordings of PV potentials were observed in the RSPV for patients with normal anatomy. Lastly, there was no difference in procedural duration between the two groups.

Of note, once RSPV and RIPV isolation were performed, accessory right PV isolation was assessed, and was found to be isolated in all patients with an R3 anatomy pattern. Catheterization of accessory PVs in patients with R4 and R5 anatomies could not be obtained because of their small diameter.

Complications

Twenty-nine transient PNPs occurred in 26 (8.6%) patients, including 20 PNPs during RSPV isolation, three PNPs during RIPV isolation and three PNPs during both superior and inferior cryoballoon isolation. Interestingly, all PNPs occurred in patients with normal right PV anatomy, whereas none of the patients with atypical anatomy had this complication (P = 0.039). Of note, all the PNPs resolved before the end of the procedure. No other major complication occurred.

Follow-up

After a follow-up of 12.6 ± 0.2 months, 42 (13.9%) patients had symptomatic documented recurrences of atrial arrhythmias, including 37 (14.6%) and five (10.2%) patients with normal and atypical right PV anatomy, respectively (P = 0.560). Kaplan-Meier curves of survival free from atrial arrhythmias are shown in Fig. 3. No difference was observed between groups (log-rank P = 0.43). Among these patients, 22 and four patients were referred for a redo ablation, respectively. The localization of gaps encountered during redo cases are detailed in Table 4. Among the four patients with atypical anatomy, only one had a gap in the RSPV, in its posterosuperior region (i.e. not close to the accessory PV).

Discussion

Main results

The main findings of our study are as follows: (1) a significant proportion of patients have atypical right PV anatomy (16.2% in our study); (2) procedural characteristics and acute PV isolation success are similar in all patients, regardless of their right PV anatomy, although a less negative temperature was observed in patients with atypical anatomy; (3) the isolation of the RSPV and RIPV, neglecting the accessory PVs, is sufficient to obtain acute right-side PV isolation and maintenance of mid-term sinus rhythm in patients with atypical anatomy; and (4) PNPs were only observed in patients with normal anatomy.

Variations in PV anatomy

usually drains the right upper and middle pulmonary lobes, whereas the left superior PV drains the left upper lobe, including the lingula. Inferior PVs drain their respective lower lobes. This configuration is observed in 57-82% of the population [START_REF] Marom | Variations in pulmonary venous drainage to the left atrium: implications for radiofrequency ablation[END_REF][START_REF] Cronin | Normative analysis of pulmonary vein drainage patterns on multidetector CT with measurements of pulmonary vein ostial diameter and distance to first bifurcation[END_REF][START_REF] Hof | Does left atrial volume and pulmonary venous anatomy predict the outcome of catheter ablation of atrial fibrillation?[END_REF][START_REF] Kato | Pulmonary vein anatomy in patients undergoing catheter ablation of atrial fibrillation: lessons learned by use of magnetic resonance imaging[END_REF][START_REF] Mansour | Assessment of pulmonary vein anatomic variability by magnetic resonance imaging: implications for catheter ablation techniques for atrial fibrillation[END_REF][START_REF] Scharf | Anatomy of the pulmonary veins in patients with atrial fibrillation and effects of segmental ostial ablation analyzed by computed tomography[END_REF][START_REF] Sohns | Pulmonary vein anatomy predicts freedom from atrial fibrillation using remote magnetic navigation for circumferential pulmonary vein ablation[END_REF]. Anatomical variants of this typical drainage have been described in 18-53% of patients [START_REF] Cronin | Normative analysis of pulmonary vein drainage patterns on multidetector CT with measurements of pulmonary vein ostial diameter and distance to first bifurcation[END_REF][START_REF] Kato | Pulmonary vein anatomy in patients undergoing catheter ablation of atrial fibrillation: lessons learned by use of magnetic resonance imaging[END_REF][START_REF] Sohns | Pulmonary vein anatomy predicts freedom from atrial fibrillation using remote magnetic navigation for circumferential pulmonary vein ablation[END_REF][START_REF] Mclellan | Pulmonary vein isolation: the impact of pulmonary venous anatomy on long-term outcome of catheter ablation for paroxysmal atrial fibrillation[END_REF][START_REF] Wannasopha | Anatomical variations of pulmonary venous drainage in Thai people: multidetector CT study[END_REF], the most common being the presence of a left common trunk (8-59%) [START_REF] Heeger | Acute efficacy, safety, and long-term clinical outcomes using the second-generation cryoballoon for pulmonary vein isolation in patients with a left common pulmonary vein: A multicenter study[END_REF][START_REF] Stroker | Second-generation cryoballoon ablation in the setting of left common pulmonary veins: Procedural findings and clinical outcome[END_REF][START_REF] Shigeta | Clinical assessment of cryoballoon ablation in cases with atrial fibrillation and a left common pulmonary vein[END_REF][START_REF] Marom | Variations in pulmonary venous drainage to the left atrium: implications for radiofrequency ablation[END_REF][START_REF] Sohns | Pulmonary vein anatomy predicts freedom from atrial fibrillation using remote magnetic navigation for circumferential pulmonary vein ablation[END_REF][START_REF] Mclellan | Pulmonary vein isolation: the impact of pulmonary venous anatomy on long-term outcome of catheter ablation for paroxysmal atrial fibrillation[END_REF][START_REF] Wannasopha | Anatomical variations of pulmonary venous drainage in Thai people: multidetector CT study[END_REF][START_REF] Kubala | Normal pulmonary veins anatomy is associated with better AF-free survival after cryoablation as compared to atypical anatomy with common left pulmonary vein[END_REF]. Anatomical variants of the right PVs are less common (6-32%) [START_REF] Marom | Variations in pulmonary venous drainage to the left atrium: implications for radiofrequency ablation[END_REF][START_REF] Cronin | Normative analysis of pulmonary vein drainage patterns on multidetector CT with measurements of pulmonary vein ostial diameter and distance to first bifurcation[END_REF][START_REF] Hof | Does left atrial volume and pulmonary venous anatomy predict the outcome of catheter ablation of atrial fibrillation?[END_REF][START_REF] Mansour | Assessment of pulmonary vein anatomic variability by magnetic resonance imaging: implications for catheter ablation techniques for atrial fibrillation[END_REF][START_REF] Sohns | Pulmonary vein anatomy predicts freedom from atrial fibrillation using remote magnetic navigation for circumferential pulmonary vein ablation[END_REF][START_REF] Mclellan | Pulmonary vein isolation: the impact of pulmonary venous anatomy on long-term outcome of catheter ablation for paroxysmal atrial fibrillation[END_REF][START_REF] Wannasopha | Anatomical variations of pulmonary venous drainage in Thai people: multidetector CT study[END_REF], but tend to be more complex because of the presence of a third PV coming from the middle pulmonary lobe, draining either into the superior or inferior PV, or directly into the LA through a separated ostium. The presence of an RMPV has been described in 11-26% of individuals [START_REF] Marom | Variations in pulmonary venous drainage to the left atrium: implications for radiofrequency ablation[END_REF][START_REF] Cronin | Normative analysis of pulmonary vein drainage patterns on multidetector CT with measurements of pulmonary vein ostial diameter and distance to first bifurcation[END_REF][START_REF] Hof | Does left atrial volume and pulmonary venous anatomy predict the outcome of catheter ablation of atrial fibrillation?[END_REF][START_REF] Mansour | Assessment of pulmonary vein anatomic variability by magnetic resonance imaging: implications for catheter ablation techniques for atrial fibrillation[END_REF][START_REF] Mclellan | Pulmonary vein isolation: the impact of pulmonary venous anatomy on long-term outcome of catheter ablation for paroxysmal atrial fibrillation[END_REF][START_REF] Mulder | Pulmonary vein anatomy and long-term outcome after multi-electrode pulmonary vein isolation with phased radiofrequency energy for paroxysmal atrial fibrillation[END_REF]. Tsao et al. [START_REF] Tsao | Role of right middle pulmonary vein in patients with paroxysmal atrial fibrillation[END_REF] reported that the RMPV might drain directly into the LA in 23% of patients. In our study, 16.2% of the patients had an atypical right PV drainage, mainly an R3a pattern, consisting of a separate PV coming from the middle lobe and draining directly into the LA. Other types of atypical right PV anatomy were less common, and only four patients had four or five PV ostia.

Cryoballoon ablation in patients with atypical PV anatomy

Data regarding the efficacy of cryoballoon ablation in patients with a left common trunk are controversial. Some studies have demonstrated a similar AF-free survival rate compared with patients with normal left PV anatomy [START_REF] Heeger | Acute efficacy, safety, and long-term clinical outcomes using the second-generation cryoballoon for pulmonary vein isolation in patients with a left common pulmonary vein: A multicenter study[END_REF][START_REF] Stroker | Second-generation cryoballoon ablation in the setting of left common pulmonary veins: Procedural findings and clinical outcome[END_REF], whereas others have reported a worse arrhythmia-free survival rate [START_REF] Beiert | Outcome in patients with left common pulmonary vein after cryoablation with second-generation cryoballoon[END_REF][START_REF] Shigeta | Clinical assessment of cryoballoon ablation in cases with atrial fibrillation and a left common pulmonary vein[END_REF]. Shigeta et al. [START_REF] Shigeta | Clinical assessment of cryoballoon ablation in cases with atrial fibrillation and a left common pulmonary vein[END_REF] demonstrated that the presence of a left common trunk was an independent predictor of atrial tachyarrhythmia recurrence after a follow up of 454 ± 195 days (hazard ratio 3.0, 95% confidence interval 1.32-6.83; P < 0.01). Further studies will be needed to investigate whether a critical size of common trunk impairs cryoballoon ablation efficacy.

However, to the best of our knowledge, there are currently no reports specifically on the efficacy of cryoballoon ablation in patients with atypical right PV anatomy. In our study, we did not find any difference in terms of mid-term arrhythmia-free survival, although some procedural variables differed between groups. Indeed, there was no difference in terms of degree of PV occlusion, successful first freeze and total number of applications per PV. However, the nadir temperature was significantly more negative in those patients with normal PV anatomy, probably explained by their larger short-and longaxis diameters compared with patients with atypical drainage. Indeed, the middle lobe vein is usually connected to the RSPV, which drains the blood coming from the upper and the middle pulmonary lobes. In patients with a middle lobe vein directly connected to the LA, the RSPV only drains the upper pulmonary lobe, explaining its smaller size and, subsequently, the less negative nadir temperatures. Surprisingly, more applications with PV potentials were recorded in patients with normal right PV drainage during RSPV isolation compared with in those with atypical PV anatomy (P = 0.005). This observation can possibly be explained by a difference in terms of myocardial PV sleeve anatomy or a more distal positioning of the Achieve™ catheter during ablation.

Interestingly, as previously stated, because a 28 mm cryoballoon was used, and to avoid increasing the risk of PNP, we decided to not perform a freezing application specifically for the accessory PVs, assuming that the proximity to RSPV and RIPV ostia (3.7 ± 2.1 and 5.3 ± 2.7 mm, respectively) would lead to isolation of these supplementary PVs. Despite their smaller size (10.3 ± 2.3 mm x 7.9 ± 2.2 mm for the RMPV in patients with an R3 pattern), accessory PVs could be catheterized in patients with an R3 pattern to prove PV isolation, demonstrating that a specific freezing application is unnecessary for these PVs, and that they can be neglected without compromising ablation outcome. Indeed, the similar mid-term efficacy observed in our study, regardless of the right PV pattern, is a plea to avoid performing supplemental freezing applications in patients with atypical PV drainage. As described, none of the two patients requiring a redo ablation had conduction gaps in the region close to the supplemental PV.

Clinical impact of preprocedural imaging

The aim of this study was to show whether a difference in clinical outcomes after cryoballoon ablation of paroxysmal AF was observed between patients with normal and atypical right PV anatomy, ablating the RSPV and the RIPV only, and neglecting ablation of accessory PVs. Indeed, some studies [START_REF] Sorgente | Pulmonary vein ostium shape and orientation as possible predictors of occlusion in patients with drug-refractory paroxysmal atrial fibrillation undergoing cryoballoon ablation[END_REF] have shown cryoballoon ablation efficacy to be critically dependent on the degree of venous occlusion, suggesting that it would be more difficult to obtain perfect occlusion in patients with atypical PV drainage. Conversely, in this study, we did not show any difference in terms of clinical outcomes depending on PV anatomy, and patients with atypical right PV drainage should not be denied a cryoballoon ablation based only on this anatomical pattern. However, we believe that this finding should not weaken the value of preprocedural 3D imaging of the PV anatomy. Indeed, many studies have demonstrated the benefit of cardiac CT scans or magnetic resonance imaging before AF ablation [START_REF] Kato | Pulmonary vein anatomy in patients undergoing catheter ablation of atrial fibrillation: lessons learned by use of magnetic resonance imaging[END_REF][START_REF] Mansour | Assessment of pulmonary vein anatomic variability by magnetic resonance imaging: implications for catheter ablation techniques for atrial fibrillation[END_REF][START_REF] Mlcochova | Magnetic resonance angiography of pulmonary veins: implications for catheter ablation of atrial fibrillation[END_REF], allowing recognition of anatomical variants, and providing an indication about PV orientation, shape and size. Furthermore, as described above, the efficacy of cryoballoon ablation in patients with a left common trunk is still a matter of debate [START_REF] Heeger | Acute efficacy, safety, and long-term clinical outcomes using the second-generation cryoballoon for pulmonary vein isolation in patients with a left common pulmonary vein: A multicenter study[END_REF][START_REF] Stroker | Second-generation cryoballoon ablation in the setting of left common pulmonary veins: Procedural findings and clinical outcome[END_REF][START_REF] Beiert | Outcome in patients with left common pulmonary vein after cryoablation with second-generation cryoballoon[END_REF][START_REF] Shigeta | Clinical assessment of cryoballoon ablation in cases with atrial fibrillation and a left common pulmonary vein[END_REF]. Thus, we believe that 3D reconstruction is essential for procedural planning, and may help to decide whether cryoballoon-or radiofrequency-based ablation should be performed in a given patient.

Lastly, although treatment efficacy was similar, regardless of right PV anatomy, the safety profile was different. Indeed, patients with normal anatomy seemed to be at higher risk of PNP. Knowing a patient's anatomy before the procedure may lead to more careful evaluation of phrenic nerve contraction during cryoballoon ablation of right PVs in patients with a typical right PV drainage.

Impact of PV anatomy on PNP

PNP is one of the most common complications during cryoballoon ablation, occurring in 1.5-13.6% of patients [START_REF] Abugattas | Phrenic nerve injury during right inferior pulmonary vein ablation with the second-generation cryoballoon: clinical, procedural, and anatomical characteristics[END_REF][START_REF] Furnkranz | Incidence and characteristics of phrenic nerve palsy following pulmonary vein isolation with the second-generation as compared with the first-generation cryoballoon in 360 consecutive patients[END_REF][START_REF] Martins | Safety and efficacy of a second-generation cryoballoon in the ablation of paroxysmal atrial fibrillation[END_REF][START_REF] Mugnai | Incidence and characteristics of complications in the setting of second-generation cryoballoon ablation: A large single-center study of 500 consecutive patients[END_REF][START_REF] Stroker | Anatomic predictors of phrenic nerve injury in the setting of pulmonary vein isolation using the 28-mm second-generation cryoballoon[END_REF][START_REF] Su | Retrospective review of Arctic Front Advance Cryoballoon Ablation: a multicenter examination of second-generation cryoballoon (RADICOOL trial)[END_REF]. A similar rate of PNP occurrence (8.6%) was observed in our study. Several predictors of PNP occurrence have been described, including the presence of a right common ostium, large PV dimensions, early branching pattern originating from the same ostium or shorter distance from the PV to the superior vena cava [START_REF] Abugattas | Phrenic nerve injury during right inferior pulmonary vein ablation with the second-generation cryoballoon: clinical, procedural, and anatomical characteristics[END_REF][START_REF] Stroker | Anatomic predictors of phrenic nerve injury in the setting of pulmonary vein isolation using the 28-mm second-generation cryoballoon[END_REF]. More precisely, these studies described predictors of PNP occurrence for RSPV freeze (RSPV area, RSPV-LA angle) and RIPV freeze (temperature drop velocity from basal to -20 °C). Saitoh et al. [START_REF] Saitoh | Fluoroscopic position of the second-generation cryoballoon during ablation in the right superior pulmonary vein as a predictor of phrenic nerve injury[END_REF] found that the strongest predictor of phrenic nerve injury at the RSPV was the position of the balloon at least one third outside the cardiac shadow (odds ratio 119.9, 95% CI 11.6-1234.7; P = 0.001). Interestingly, in our study, all PNPs occurred in patients with normal PV anatomy, and none of the patients with atypical right PV drainage had this complication. Different hypotheses may explain such a finding. First, one may postulate that an atypical right PV anatomy may be associated with an atypical phrenic nerve position, located further from the PV ostia, making it less prone to freezing. This hypothesis was not specifically analysed in this study, and would require further investigation. Second, as demonstrated in our study, patients with normal PV anatomy have larger RSPVs, and cryoenergy may be delivered further inside the PV ostium, closer to the phrenic nerve position. Indeed, Casado-Arroyo et al. [START_REF] Casado-Arroyo | Phrenic nerve paralysis during cryoballoon ablation for atrial fibrillation: a comparison between the first-and second-generation balloon[END_REF] showed that a less vigorous wedging manoeuvre in the RSPV ostia might reduce the occurrence of PNP. The authors recommended slightly retrieving the inflated cryoballoon until a small leak is observed, before repositioning it at the beginning of the cryoenergy application to create a more proximal lesion, without compromising the quality of the occlusion. Patients with atypical anatomy, having smaller RSPVs, are probably at less risk because the lesion created by a big 28 mm cryoballoon is possibly more proximal.

Lastly, the nadir temperature was less negative in patients with atypical anatomy, a variable that may explain why no PNPs were observed in this group. Further studies will be needed to analyse the effect of PV shape, orientation and anatomy on PNP occurrence.

Study limitations

This study had several limitations that must be considered. First, it was a single-centre study. The clinical endpoint of sinus rhythm maintenance evaluated the global efficacy of cryoballoon ablation for both right and left PVs, and AF recurrence in a given patient with typical or atypical right PV anatomy may be the result of a reconnection of left PVs while right PVs remain disconnected. Most of the patients with atypical anatomy had an R3 pattern, only a limited number of patients had an R4/R5 pattern and none had an R1 pattern, somewhat limiting the results observed to patients with this type of anatomy. Lastly, PV reconnection was only assessed in those patients referred for a redo ablation, and one may argue that the reconnection rate could differ between asymptomatic and symptomatic patients not referred for a second procedure. However, Miyazaki et al. proved that, regardless of clinical recurrences, the incidence and characteristics of PV reconnections were similar in both groups [START_REF] Miyazaki | Clinical recurrence and electrical pulmonary vein[END_REF].

We focused our study on CT scans. However, PV angiography or rotational angiography may also be helpful in delineating PV anatomy. The impact of such imaging modalities in patients with atypical PV anatomies would require further studies.

Conclusions

To the best of our knowledge, this is the first study specifically reporting the safety and efficacy of cryoballoon ablation in patients with atypical right PV drainage. In addition to the isolation of left-side PVs, the isolation of the RSPV and the RIPV, neglecting accessory PVs, is sufficient to obtain acute right-side PV isolation and maintenance of mid-term sinus rhythm in patients with atypical anatomy. To note, the risk of PNP seems to be negligible in these patients, and further studies will be needed to determine whether anatomical or procedural characteristics can explain such a finding.

Figure 1 .

 1 Figure 1. Right pulmonary drainage pattern; adapted from Marom et al. [7]. RLL: right lower lobe; RML: right middle lobe; RUL: right upper lobe; SSRLL: superior segment right lower lobe; BSRLL: basilar segment right lower lobe.

Figure 2 .

 2 Figure 2. Cardiac computed tomography scan reconstructions of left atrium (posteroanterior view and endoluminal view). A. R3a pattern. B. R3b pattern. C. R3c pattern. D. R4 pattern. E. R5 pattern.

Figure 3 .

 3 Figure 3. Kaplan-Meier curves for cumulative survival free of atrial arrhythmias in patients with normal and atypical right pulmonary vein (PV) anatomy.

  are expressed as median (interquartile range) or number (%), unless otherwise indicated. AF: atrial fibrillation; CHA2DS2-VASc: Congestive heart failure, Hypertension, Age ≥ 75 years (Doubled), Diabetes, Stroke/transient ischaemic attack/thromboembolism (Doubled) -Vascular disease, Age 65-74 years and Sex category [Female]); LVEF: left ventricular ejection fraction; PV: pulmonary vein.

Table 1

 1 Patient characteristics.

		All patients	Atypical right PV anatomy	Normal right PV anatomy	P
		(n = 303)	(n = 49)	(n = 254)
	Age (years)	61.0 (53.8-67.9) 56.9 (50.7-66.8)	61.5 (54.5-68.3)	0.09
	Male/female sex (n/n)	212/91	36/13	176/78	0.68
	Hypertension	94 (31.0)	11 (22.4)	83 (32.7)	0.21
	Diabetes mellitus	9 (3.0)	2 (4.1)	7 (2.7)	0.97
	Current smoker	33 (10.9)	10 (20.4)	23 (9.05)	0.037
	Dyslipidaemia	49 (16.2)	7 (14.3)	42 (16.5)	0.87
	History of stroke	20 (6.6)	5 (10.2)	15 (5.9)	0.43
	Body mass index (kg/m 2 )	25.9 (23.5-28.7) 25.8 (22.8-27.8)	26.0 (23.5-28.7)	0.24
	CHA2DS2-VASc score
	0	128 (42.2)	20 (40.8)	108 (42.5)
	1	72 (23.8)	13 (26.5)	59 (23.2)
	2	67 (22.1)	11 (22.5)	56 (22.0)
	3	23 (7.6)	4 (8.2)	19 (7.5)	0.97
	4	7 (2.3)	1 (2)	6 (2.4)
	5	5 (1.7)	0 (0)	5 (2.0)
	6-9	1 (0.3)	0 (0)	1 (0.4)
	AF episode duration
	< 12 hours	196 (64.7)	27 (55.1)	169 (66.5)
	12-24 hours	51 (16.8)	10 (20.5)	41 (16.1)
					0.47
	24-48 hours	26 (8.6)	6 (12.2)	20 (7.9)
	> 48 hours	30 (9.9)	6 (12.2)	24 (9.5)
	Number of antiarrhythmic
	drugs tested before ablation

Table 2

 2 Anatomical considerations.

		Atypical right PV anatomy Normal right PV anatomy	P
		(n = 49)	(n = 254)
	Right PV size, mm
	RSPV long axis	17.6 ± 3.3	20.3 ± 3.0	< 0.001
	RSPV short axis	14.2 ± 3.2	17.2 ± 3.2	< 0.001
	RIPV long axis	17.5 ± 3.3	18.1 ± 3.1	0.23
	RIPV short axis	15.0 ± 3.1	15.8 ± 2.9	0.09
	RMPV anatomy
		0 (0)	NA
	R2a	NA	182 (71.6)
	R2b	NA	53 (20.9)
	R2c	NA	19 (7.5)
	R3a	40 (81.6)	NA
				NA
	R3b	3 (6.1)	NA
	R3c	2 (4.1)	NA
	R4a	3 (6.1)	NA
	R4b	0 (0)	NA
	R5	1 (2.1)	NA
	For R3 (n = 28)
	RMPV long axis (mm)	10.3 ± 2.3	NA
	RMPV short axis (mm)	7.9 ± 2.2	NA
				NA
	Distance to RSPV (mm)	3.7 ± 2.1	NA
	Distance to RIPV (mm)	5.3 ± 2.7	NA
	Data are expressed as mean ± standard deviation or number (%). NA: not applicable; PV:
	pulmonary vein; RIPV: right inferior pulmonary vein; RMPV: right middle pulmonary vein; RSPV:
	right superior pulmonary vein.

Table 3

 3 Procedural characteristics.

	Atypical right PV anatomy Normal right PV anatomy	P
	(n = 49)	(n = 254)

Data are expressed as number (%), mean ± standard deviation or median (interquartile range). PNP: phrenic nerve palsy; PV: pulmonary vein; PVP: pulmonary vein potential; RIPV: right inferior pulmonary vein; RSPV: right superior pulmonary vein.

Table 4

 4 Localization of conduction gaps during redo procedures.

Sources of funding

None.

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.


Béatrice Rocher
email: beatrice.rocher@univ-lehavre.fr.

Elise David

Arnaud Tanguy

Aimie Le Saux

Lauris Evariste

Fanny Louis

Laurence Delahaut

Damien Rioult

Sandrine Pain-Devin

Romain Péden

Damien Rioult B-E

Odile Dedourge-Geffard

Simon Devin

Florence Bultelle

Characterization of Cu/Zn-SODs in sympatric species: A comparison of zebra and quagga mussels

Keywords: Biomonitoring, Oxidative stress, Sentinel, Invasive species, Dreissena

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

Bivalves such as Dreissena polymorpha are widely used as sentinel organisms in freshwater biomonitoring programs [START_REF] Hanana | Comparative study of the effects of gadolinium chloride and gadolinium -based magnetic resonance imaging contrast agent on freshwater mussel, Dreissena polymorpha[END_REF][START_REF] Binelli | Does zebra mussel (Dreissena polymorpha) represent the freshwater counterpart of Mytilus in ecotoxicological studies? A critical review[END_REF][START_REF] Faria | Separating natural from anthropogenic causes of impairment in Zebra mussel (Dreissena polymorpha) populations living across a pollution gradient[END_REF][START_REF] Pain-Devin | Towards a better understanding of biomarker response in field survey: A case study in eight populations of zebra mussels[END_REF][START_REF] Navarro | Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults[END_REF]. They have a wide geographical distribution, a filtering and fixed adult lifestyle, significant bioaccumulation capacity, high abundance and are resistant to complex stresses. Originating from the Ponto-Caspian basin, Dreissena polymorpha is a well-known invasive species of freshwater rivers in Western Europe and North America [START_REF] Ginn | Quantifying a shift in benthic dominance from zebra (Dreissena polymorpha) to quagga (Dreissena rostriformis bugensis) mussels in a large, inland lake[END_REF][START_REF] Marescaux | Sympatric Dreissena species in the Meuse River: towards a dominance shift from zebra to quagga mussels[END_REF]. A rapid expansion of a related species, Dreissena rostriformis bugensis, quagga mussel, has been observed in Western Europe where D. rostriformis bugensis may displace D. polymorpha, a shift previously observed in North America's Great Lakes [START_REF] Sturtevant | Recent history of nonindigenous species in the Laurentian Great Lakes; An update to Mills et al., 1993 (25 years later)[END_REF][START_REF] Kerambrun | Differentiation of sympatric zebra and quagga mussels in ecotoxicological studies: A comparison of morphometric data, gene expression, and body metal concentrations[END_REF][START_REF] Potet | Differential tolerance to nickel between Dreissena polymorpha and Dreissena rostriformis bugensis populations[END_REF]Matthew et al., 2015). In Western Europe, first observations of quagga mussels were made in the Hollands Diep in 2006 [START_REF] Matthews | A dominance shift from the zebra mussel to the invasive quagga mussel may alter the trophic transfer of metals[END_REF][START_REF] Molloy | Discovery of Dreissena rostriformis bugensis (Andrusov 1897) in Western Europe[END_REF] and in the Meuse River in 2008 [START_REF] Marescaux | First recors of the quagga mussels, Dreissena rostriformis bugensis (Andrusov, 1897)[END_REF][START_REF] Vaate | Range expansion of the quagga mussel Dreissena rostriformis bugensis (Andrusov, 1897) in Western Europe: first observation from France[END_REF]. In France, D. rostriformis bugensis expansion has since been reported in the Rhône drainage and the Oise River [START_REF] Kerambrun | Differentiation of sympatric zebra and quagga mussels in ecotoxicological studies: A comparison of morphometric data, gene expression, and body metal concentrations[END_REF].

In this context, efficient discrimination of the two species became crucial. Zebra and quagga mussels indeed present morphological differences, but shell morphotypes can vary and lead to misidentification. Indeed, morphological indices based on length, width and height, have been proposed [START_REF] Teubner | Is a visuo-haptic differentiation of zebra mussel and quagga mussel based on a single external morphometric shell character possible?[END_REF][START_REF] Beggel | Shell morphological versus genetic identification of quagga mussel (Dreissena bugensis) and zebra mussel (Dreissena polymorpha)[END_REF], but the risk of erroneous classification has already been underlined [START_REF] Kerambrun | Differentiation of sympatric zebra and quagga mussels in ecotoxicological studies: A comparison of morphometric data, gene expression, and body metal concentrations[END_REF][START_REF] Beggel | Shell morphological versus genetic identification of quagga mussel (Dreissena bugensis) and zebra mussel (Dreissena polymorpha)[END_REF]. For instance, in relation with their habitats, quagga mussel exhibit different shell morphotypes through a developmental plasticity [START_REF] Peyer | Developmental plasticity of shell morphology of quagga mussels from shallow anddeep-water habitats of the Great Lakes[END_REF]. In a biomonitoring context, it appears essential to clearly and correctly identify the species in order to properly analyze ecotoxicological responses [START_REF] Madeira | Molecular assessment of wild populations in the marine realm: Importance of taxonomic, seasonal and habitat patterns in environmental monitoring[END_REF][START_REF] Vernon | Assessing relative sensitivity of marine and freshwater bivalves following exposure to copper: Application of classical and novel genotoxicological biomarkers[END_REF][START_REF] Farkas | Interspecies comparison of selected pollution biomarkers in dreissenid spp. inhabiting pristine and moderately polluted sites[END_REF].

Genetic methods based on DNA markers, therefore requiring nucleic acid extractions, have already been developed to differentiate Dreissena species [START_REF] Marescaux | Using DNA barcoding to differentiate invasive Dreissena species (Mollusca, Bivalvia)[END_REF][START_REF] Voroshilova | Natural hybridization of two mussel species Dreissena polymorpha (Pallas, 1771) and Dreissena bugensis (Andrusov, 1897)[END_REF]. Hence in general, RFLP (Restriction Fragment Length Polymorphism) analysis of the fragment of the mitochondrial gene COI allows the discrimination of the two species [START_REF] Voroshilova | Natural hybridization of two mussel species Dreissena polymorpha (Pallas, 1771) and Dreissena bugensis (Andrusov, 1897)[END_REF]. Superoxide dismutases (SODs) are classic biomarkers in ecotoxicological studies [START_REF] Geret | Molecular cloning of superoxide dismutase (Cu/Zn-SOD) from aquatic molluscs[END_REF]. These enzymes exhibit an oxido-reductase activity that catalyzes dismutation of superoxide anion into molecular oxygen and hydrogen peroxide which is in turn used by the catalase enzyme. Superoxide dismutases therefore represent first line antioxidant defenses.

Depending on their metal catalytic cofactors, SODs are classified into distinct groups, (for details see revue by [START_REF] Abreu | Superoxide dismutases -a review of the metalassociated mechanistic variations[END_REF] and only copper/zinc SODs and manganese SOD are expressed in animals, the last form being exclusively mitochondrial. Cu/Zn-SODs include extra-and intracellular SODs, each one encoded by a specific gene. The extracellular form contains a signal peptide with a cleavage site for its secretion. The intracellular form, a major SOD, is often referred to as cytosolic Cu/Zn-SOD, although it has been localized not only in the cytosol but also in all cellular compartments, including the nucleus and the internal mitochondrial membrane, excluding the mitochondrial matrix (Zelco et al., 2002). SOD activity is often measured on tissue homogenates, but it can also be measured by gel zymography, as developed by [START_REF] Beauchamp | Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels[END_REF], a method that highlights isoforms. Indeed, SODs present distinct properties including electrophoretic mobility and differential susceptibility to inhibitors such as cyanide, making possible a separate study of the different allozymes. Thus, after isoelectrofocusing in native conditions, intracellular Cu/Zn-SOD (SOD1) activity is revealed at pH between 4 and 5 while the other SODs are observed at more basic pH [START_REF] Manduzio | Characterization of an inducible isoform of the Cu/Zn superoxide dismutase in the blue mussel Mytilus edulis[END_REF].

In the present work, we chose to focus on the intracellular Cu/Zn-SOD and investigate its characteristics in the two Dreissena mussels in order to assess whether the sister species could be used interchangeably in environmental surveys. Geographically distant sites were selected for sampling and an in-depth study of intracellular Cu / Zn-SOD was carried out. Enzymatic profiles were analyzed by zymography, and we demonstrated that Cu/Zn-SOD exhibits distinct patterns in the two Dreissena species. In order to understand such differences, data mining of published sequences was performed, allowing us to build a phylogenetic tree that places the sister species side by side with clams as their closest neighbors. Finally, structure prediction for both Cu/Zn SODs was conducted and revealed very similar shapes.

Methods

All chemicals were of analytical grade and were used as received. Water for preparation of the solutions was purified using a Milli-Q purification system. Nitrobluetetrazolium (NBT), riboflavin, aprotinin and purified Cu/Zn-SOD from bovine erythrocytes and isoelectric focusing markers were purchased from Sigma.

Mussels were collected in one location in the St. Lawrence River (Quebec, Canada) and in several locations in the French territory. Sampling maps were previously published in [START_REF] Evariste | Field biomonitoring using the zebra mussel Dreissena polymorpha and the quagga mussel Dreissena bugensis following immunotoxic reponses. Is there a need to separate the two species?[END_REF] for the Canadian part and in [START_REF] Pain-Devin | Towards a better understanding of biomarker response in field survey: A case study in eight populations of zebra mussels[END_REF] for the French side. The Canadian site is located upstream of Montreal city at Pointe-des-Cascades (45°19'49.9''N; 73°58'11.8''W). This site is receiving water masses from the Laurentian Great Lakes and was previously shown to be a low-polluted area [START_REF] Evariste | Field biomonitoring using the zebra mussel Dreissena polymorpha and the quagga mussel Dreissena bugensis following immunotoxic reponses. Is there a need to separate the two species?[END_REF][START_REF] Kwan | Metal contamination in zebra mussels (Dreissena Polymorpha) along the St. Lawrence River[END_REF]. In Europe, the sampling sites are located in the rivers of north France, namely the Meuse (E5°32'04; N48°53'14), Moselle (E6°04'43; N49°03'58), Vilaine (W2°18'30; N47°31'22) and Oise (E2°30'36; N49°16'50). These sites have already been the subject of environmental monitoring under the research program IPOC (Interactions between pollution and climate changes: development of an improved monitoring strategy, ANR-12-ISV7-0004-01, 2013-2015), and the presence of Dreissena is confirmed, either as monospecific assemblages or as sympatric populations of both species [START_REF] Kerambrun | Differentiation of sympatric zebra and quagga mussels in ecotoxicological studies: A comparison of morphometric data, gene expression, and body metal concentrations[END_REF][START_REF] Kerambrun | Variations in gene expression levels in four European zebra mussel, Dreissena polymorpha, populations in relation to metal bioaccumulation: A field study[END_REF].

Hemolymph collections were withdrawn from the posterior adductor muscle sinus using a 0.5 mL syringe with a 0.33 mm x 12 mm needle and stored at -80°C. Mantles were preserved in 70% ethanol. After dissection, gills and digestive glands were immediately frozen in liquid nitrogen and stored at -80°C until further analysis.

Genetic species identification was carried by Restriction Fragment Length Polymorphism

(RFLP) analysis performed on the mitochondrial COI gene using RSA I enzyme according to a method adapted from [START_REF] Voroshilova | Natural hybridization of two mussel species Dreissena polymorpha (Pallas, 1771) and Dreissena bugensis (Andrusov, 1897)[END_REF]. For details, total genomic DNA was extracted from mussel mantle using phenol/chloroform/isoamyl alcohol (25:24:1). The fragment of COI mitochondrial DNA containing the RSA I restriction site was amplified using COI universal primers (5′-GGTCAACAAATCATAAAGATATTGG-3′ and 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) from [START_REF] Black | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF]. PCR amplifications were performed in a volume of 25 μL containing 1×Taq polymerase buffer, 2mM MgCl2, 80 μM deoxynucleotides (dNTPs), 10 pmol of each primer, 0.5 units of TAQ Pol DNA polymerase (Euromedex, France) and about 10 ng of total genomic DNA. The following steps were applied for the PCR: denaturation at 94°C for 4 min, then 35 cycles at 94°C for 50 s, 56°C for 50 s, 72°C for 1 min, and a final step at 72°C for 10 min. Resulting fragments were hydrolyzed using RSA I restriction endonuclease (Euromedex, France), following the manufacturer's instructions. Restriction fragments were visualized under UV light after electrophoresis on a 2% agarose gel in Tris Borate EDTA 0.5% buffer, and staining with ethidium bromide.

Gills and digestive glands were homogenized by Precellys grinding in an ice-cold solution of 100 mM Tris, 60 mM saccharose, 60 mM mannitol, 3 mM EDTA, 1.5 µM aprotinin, pH 7.5.

The homogenates were centrifuged at 10,000 g, at 4 °C for 15 min. After centrifugation, supernatants containing the cytosolic fraction were collected. If analyses were done the same day, samples were kept at 4°C otherwise they were stored at -80°C. Protein concentration of each extract was measured with bovine serum albumin as a standard according to the Bradford method [START_REF] Bradford | A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding[END_REF].

Isoelectric focusing (IEF) was carried out in a Multiphor II equipment (Amersham Pharmacia Biotech, France) on gels with pH range 4.0-5.0 for a better resolution of Cu/Zn-SOD isoforms. The isoelectric point values (pI) were determined in regard with an isoelectric focusing calibration kit. Electrophoreses were performed at 60 mA at 4°C for 2 hours. Current was applied on electrode strips soaked either into an anode solution (1 M H3PO4) or into a cathode solution (1 M glycin). Polyacrylamide gel electrophoresis (PAGE) was performed with a 10% polyacrylamide running gel at 150 mV at 4°C for 2 hours. In both electrophoreses, purified bovine Cu/Zn-SOD was loaded as control.

Revelation of SOD activity was performed on gel immediately after the achievement of electrophoresis using the nitrobluetetrazolium (NBT) photochemical method of Beauchamp and Fridovitch (1971). The gels were soaked for 30 min in 2.5 mM NBT, in darkness, under gentle agitation and then incubated in a solution of 1 M phosphate buffer (pH 7.8) containing 0.05 mM riboflavin and 0.4% TEMED (v/v) for 30 min, once again in the dark and under constant agitation. Finally, the gels were exposed to light for 15 min in a 10 mM phosphate buffer (pH 7.8). Achromatic bands in the gels indicated SOD activity. The gels were scanned using GS-800 densitometer, BIO-RAD and subsequently analyzed by Image Lab (version 4.1, BIO-RAD) software.

Transcripts encoding Cu/Zn-SOD sequences were searched in annotated transcriptome databases. For details, databases were constructed using either gills or digestive gland of quagga and zebra mussels found in the French rivers. Following translation using the SIB Bioinformatics Resource Portal (tool, Translate, https://web.expasy.org/translate/), the protein's local structural features were predicted from the primary sequence using NetSurfP-2.0 (http://www.cbs.dtu.dk/cgi-bin/webface2.fcgi, [START_REF] Klausen | NetSurfP-2.0: Improved prediction of protein structural features by integrated deep learning[END_REF]. For both species Cu/Zn SOD sequences, variant annotations such as Single Nuclear Polymorphisms (SNPs) and INsertion/DELetions events (INDELs) were issued from previously published transcriptomes [START_REF] Péden | Environmental transcriptomes of invasive Dreissena, a model species in ecotoxicology and invasion biology[END_REF]. As mentioned in the study, variant callings were realized with GATK following the best practices found on the GATK website [START_REF] Mckenna | The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data[END_REF].

Phylogenetic trees were generated with the maximum composite likelihood (ML) algorithm in Mega v6 software [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. The Akaike Information Criterion was used to determine the best evolution model and JTT was retained for SOD. Statistical support for the nodes was estimated used by a bootstrap with 1,000 pseudoreplicates [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF].

Genetic sequences of SOD proteins of other species used in these phylogenetic analyses were obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/).

Results and Discussion

Characterization of Cu/Zn SODs

Zymograms of Cu/Zn-SOD activity show specific contrasted patterns that clearly discriminate quagga and zebra individuals (Figure 1A). Indeed, the quagga mussel is characterized by a major band at pI 4.95. The purified bovine erythrocyte Cu/Zn-SOD loaded as a control exhibited the same pI. By contrast, the zebra mussel shows multiband profiles at more acidic isoelectric points. In detail, two characteristic patterns were obtained, one with a major band at pI 4.6, the other with 3 major bands at pI 5.0, 4.8 and 4.6, suggesting higher genetic variability in the zebra than in the quagga mussel population (Figure1B). Species identity of individuals sampled in France was confirmed morphologically and genetically (Figure 1C).

Comparison of Cu/Zn SOD profiles of geographically distant mussels

Zymogram profiles of animals collected in France were compared to those of mussels from Canada (Figure 2). All quagga mussels, at any site, exhibit the same characteristic profile with a main Cu/Zn-SOD band at pI 4.95. For the zebra mussels, the two main profiles described above were found. Thus, it appears that differences in electrophoretic mobility of Cu/Zn-SOD isoforms are clearly species-related since they are conserved in geographically remote areas.

Cu/Zn-SOD profiles as a tool for species discrimination

Because zymographic profiles are different between the two species, the question of using this characteristic to recognize the different species has arisen. Hemolymph constitutes a good candidate for this application because this tissue is minimally invasive and relatively easy to collect. The tests showed that 20 µL of hemolymph was sufficient to visualize SOD and discriminate between species after IEF (data not shown). We also checked that samples prepared for other enzymatic tests with 10mM Tris buffer pH7 or 100 mM phosphate buffer pH7.5 allow the construction of zymographic profiles. Hence samples stored at minus 20°C could be used later for species identification by SOD zymogram analyses. Such a result is consistent with the very high stability of this enzyme. Finally an element that can limit the use of such a test is the availability of hardware allowing the IEF. Therefore, SOD profiles were tested on classic non denaturating gel electrophoresis PAGE. As shown in Figure 3, zebra and quagga mussels are clearly discriminated with a separate main band for each species.

Phylogenetic analysis of Cu/Zn-SODs

A phylogenetic tree was constructed based on the intracellular Cu/Zn-SOD amino acid sequence from the two dressenid species and other invertebrates and vertebrates (Figure 4).

The tree shaped two main clades (even branch nodes show a low bootstrap value).

Interestingly, the dressenid SODs are not clustered in the marine mussels and oyster group but are closely related to sequences of SOD from clam species (Mactra quadrangularis and Meretrix meretrix). Moreover, the percentages of identities between all those SODs sequences are close to 70% showing a high level of conservation as it has already been described in other studies relating to the sequencing of SOD in Dreissena sp. or in other bivalves [START_REF] Wu | The expression of superoxide dismutase in Mytilus coruscus under various stressors[END_REF][START_REF] Anju | Molecular cloning, characterization and expression analysis of cytoplasmic Cu/Zn-superoxide dismutase (SOD) from pearl oyster Pinctada fucata[END_REF][START_REF] Gao | Intracellular Cu/Zn superoxide dismutase (Cu/Zn-SOD) from hard clam Meretrix meretrix: its cDNA cloning, mRNA expression and enzyme activity[END_REF][START_REF] Ni | The cDNA cloning and mRNA expression of cytoplasmic Cu, Zn superoxide dismutase (SOD) gene in scallop Chlamys farreri[END_REF][START_REF] Geret | Molecular cloning of superoxide dismutase (Cu/Zn-SOD) from aquatic molluscs[END_REF].

Comparison of secondary structures

Alignment of amino acid sequences were performed with the two cytosolic Cu/Zn-SOD sequences from D. polymorpha and D. bugensis. The sequences exhibited two conserved cysteine residues (Cys-58 and 146) needed for disulfide bond formation and highly conserved residues involved in copper and zinc fixation (Figure 5). Amino acid comparison shows only 11 variations among the total of 153 amino acids signifying 93% of identity between the 2 sequences. Of the 11 variations, 5 amino acids share similar properties. For instance, the switch from glutamine to glutamic acid and that of cysteine to serine don't lead to major property changes. In such a way, it raised to 96% the similarity between the cytosolic Cu/Zn-SODs of the two species. At this point, both proteins were identical at a very high level, with only single amino acid modification points. However, these differences lead to minor changes in secondary sequence with the appearance of an α-helix (position 75), a longer β-sheet (position 101) and longer α-helix between position 130 and 135 in Dreissena polymorpha comparing Dreissena rostriformis bugensis (Figure 6).

Additionally, 40 SNPs and INDELs events were found on both Cu/Zn-SOD transcript sequences (data not shown) with respectively 10 and 14 events within Dreissena polymorpha and Dreissena rostriformis bugensis coding sequences (Figure 7). Among those events, 6 were silent for Dreissena polymorpha and 10 for Dreissena rostriformis bugensis. Remarkably, only one SNP impacts the secondary protein's sequence, and it is present in the Dreissena polymorpha sequence. Moreover, these slight differences could be accompanied by changes in the quaternary structure and may explain the differences in isoelectric point observed between the two species.

Conclusions

The characterization of Cu/Zn-SODs has allowed us to identify significant differences between the isoforms of zebra and quagga mussels and propose their use as species enzymatic markers. At the nucleotide sequence level, the transcripts are also different, which allows the use of specific primer sets to measure Cu/Zn-SOD gene expression in these two species [START_REF] Kerambrun | Differentiation of sympatric zebra and quagga mussels in ecotoxicological studies: A comparison of morphometric data, gene expression, and body metal concentrations[END_REF]. Hence cytosolic Cu/Zn-SOD can be used as a discrimination marker for quagga and zebra mussels at the functional, structural and transcriptomic levels. Moreover, SOD is a stable enough enzyme to permit Dreissena species verification based on a posteriori analyses of homogenates prepared for other enzymatic assays. The study of Cu/Zn-SOD in Dreissena sp. revealed the existence of isoforms specific to zebra and quagga mussels and, incidentally, provided a tool for species discrimination using a simple enzymatic test. Finally, as superoxide dismutases are among the first defenses against ROS damages, differences in Cu/Zn-SOD isoforms in the two dreissenid species could contribute to a possible contrasting response in sensitivity to oxidative stress and metal pollutants as already observed [START_REF] Louis | Energy allocation in two dreissenid species under metal stress[END_REF]. Further studies should shed light on this issue. Cu/Zn-SOD isoforms were separated according to their pI using non-denaturing IEF. Three patterns were obtained after NBT staining. For details, in the Moselle River a unique band corresponding to quagga mussels is observed. In the Vilaine and Meuse, two distinct patterns of Cu/Zn-SOD activity were observed corresponding only to zebra mussels. In the sites where the two species of Dreissena coexist (Oise and Saint-Laurent Rivers) the three patterns of SOD activity are found, similar to those obtained for the two species in the other sampled sites. [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF].

Legends

Statistical support for the nodes was estimated used by a bootstrap with 1,000 pseudoreplicates [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF].

Figure 5: Amino acid sequence aligment of Cu/Zn-SODs. In line 1, Drpol, the primary sequence of Dreissena polymorpha; in line 2, Drbug, the primary sequence of Dreissena rostriformis bugensis. Amino acid differences were shaded; the residues required for copper fixation are in bold. The two cysteines predicted to form a disulfide bond were underlined.

Figure 1 :

 1 Figure 1: Characterization of Cu/Zn-SOD profiles by zymography.

Figure 2 :

 2 Figure 2: Distribution of Cu/Zn-SOD profiles in Dreissena samples collected from different areas. Animals were sampled in France (4 rivers) and in Canada (Saint-Laurent River).

Figure 3 :

 3 Figure 3: Zymogram of Cu/Zn-SOD allozymes after PAGE and NBT revelation from Dreissena rostriformis bugensis and from Dreissena polymorpha. Homogenates are separated in a 10% PAGE. Images of NBT staining are presented in inversed grey colors for a better visualization of the bands. Arrows indicated Cu/Zn-Sod activity.

Figure 4 :

 4 Figure4: Phylogenetic analysis of Cu/Zn-SODs. Phylogenetic trees were generated with the maximum composite likelihood algorithm in Mega v6 software[START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF].

Figure 6 :

 6 Figure 6: Secondary structure representation of Cu/Zn-SODs. Models of protein's local structural features were predicted from the primary sequences of Dreissena polymorpha (up)and Dreissena rostriformis bugensis (down) using NetSurfP-2.0.Three secondary sequence differences (black rectangles) were observed between both species, with Q -> E in position 75 creating a small α-helix in polymorpha, C -> S in position 101 leading to a smaller β-sheet in quagga, and a smaller α-helix in quagga between positions 130 and 135 with no sequence difference.

Figure 7 :

 7 Figure 7: SNP positions in Cu/Zn-SOD nucleotide sequence for Dreissena polymorpha (Dp SODC) and Dreissena rostriformis bugensis (Db SODC). SNPs are highlighted by shaded residues. In the squares, SNPs that changes the amino acid sequence without changes at the secondary structure level. The double square corresponds to the only SNP that impacts the secondary sequence of the protein.

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Acknowledgements

Aimie Le Saux was a recipient of a PhD grant from the French Ministry for Research. The study was partly financially supported by both ANR and NSERC Agencies (joined France-Canada project) as a part of the research program IPOC (Interactions between POllution and Climate changes: Development of improved monitoring strategy, ANR-12-ISV7-0004-01, CRSNG-STPGP-430539-2012, 2013-2016, https://www-iuem.univ-brest.fr/ipoc/fr) as well as the FR CNRS 3730 SCALE. We thank Mélissa Palos-Ladeiro, Elodie Kerambrun and Estelle Petiau for their help.

Data availability

Data are available on request to the corresponding author (beatrice.rocher@univ-lehavre.fr).

Declaration of interest

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.