MD Elodie Surget
email: elodie.surget@ihu-liryc.fr

MD Ghassen Cheniti

MD F Daniel Ramirez

MD Antoine Leenhardt

MD Akihiko Nogami

Estelle Gandjbakhch

PhD Fabrice Extramiana

MD Françoise Hidden-Lucet

PhD Xavier Pillois

PhD David Benoist

MD Philipp Krisai

MD Yosuke Nakatani

MD Takashi Nakashima

MD Takamitsu Takagi

MD Tsukasa Kamakura

MD Clémentine André

MD Nicolas Welte

MD Rémi Chauvel

MD Romain Tixier

MD Josselin Duchateau

MD Thomas Pambrun

MD Nicolas Derval

MD Pierre Jaïs

MD Frédéric Sacher

PhD Olivier Bernus

MD Mélèze Hocini

MD Michel Haïssaguerre

Sex differences in the origin of Purkinje ectopy initiating idiopathic ventricular fibrillation First author: Elodie Surget

Keywords: sudden death, idiopathic ventricular fibrillation, Purkinje, catheter ablation, sex differences

Background :

Purkinje ectopics (PurkE) are major triggers of idiopathic ventricular fibrillation (VF).

Identifying clinical factors associated with specific PurkE characteristics could yield insights into the mechanisms of Purkinje-mediated arrhythmogenicity.

Objective :

To examine the associations of clinical, environmental, and genetic factors with PurkE origin in patients with PurkE-initiated idiopathic VF.

Methods :

Consecutive patients from four arrhythmia referral centers with PurkE-initiated idiopathic VF were included. We evaluated demographics, medical history and clinical circumstances associated with index VF events, and electrophysiologic characteristics of PurkE. An electrophysiological study was performed in most patients to confirm the Purkinje origin.

Results :

Eighty three patients were included (age 38 ± 14 years, 44 women) among whom 32 had a previous history of syncope. Fourty four patients had VF at rest. PurkE originated from the right ventricle (RV) in 41 cases (49%), from the left ventricle (LV) in 36 (44%) and from the both ventricles in 6 (7%). Seasonal and circadian distributions of VF episodes were similar according to PurkE origin. Clinical characteristics of patients with RV vs. LV PurkE origins were similar except for sex. RV PurkE were more frequent in men than women (76% vs 24%) whereas LV and biventricular PurkE were more frequent in women (81% vs 19% and 83% vs 17% respectively, p<0.0001).

Conclusion

PurkE triggering idiopathic VF originate dominantly from RV in men and from LV or both ventricles in women adding to other sex-related arrhythmias as Brugada syndrome or long QT. Sex-based factors influencing Purkinje arrhythmogenicity warrant investigation.

Introduction

Sudden cardiac death (SCD) is an important cause of mortality in developed countries, claiming approximately 300,000 lives per year in Europe [START_REF] Waldmann | Characteristics and clinical assessment of unexplained sudden cardiac arrest in the real-world setting: focus on idiopathic ventricular fibrillation[END_REF] . The principal mechanism of SCD is ventricular fibrillation (VF), which is usually attributable to chronic and structural cardiac diseases in older individuals [START_REF] Priori | ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death: The Task Force for the Management of Patients with Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death of the European Society of Cardiology (ESC). Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC)[END_REF] . Idiopathic VF is a diagnosis of exclusion and reserved for patients who survived VF episode without evidence of underlying structural or electrical heart disease [START_REF] Waldmann | Characteristics and clinical assessment of unexplained sudden cardiac arrest in the real-world setting: focus on idiopathic ventricular fibrillation[END_REF][START_REF] Stiles | APHRS/HRS expert consensus statement on the investigation of decedents with sudden unexplained death and patients with sudden cardiac arrest, and of their families[END_REF] .

A subset of idiopathic VF is induced by premature ventricular complexes [START_REF] Anderson | Sudden Cardiac Death and Ventricular Arrhythmias: State of the Art in 2018-2019 -Catheter Ablation of Ventricular Fibrillation[END_REF] , often with a short-coupled interval (CI), which can arise from the Purkinje system [START_REF] Viskin | Mode of onset of malignant ventricular arrhythmias in idiopathic ventricular fibrillation[END_REF][START_REF] Haïssaguerre | Mapping and ablation of idiopathic ventricular fibrillation[END_REF] , the right ventricular outflow tract (RVOT) [START_REF] Ashida | Abolition of Torsade de Pointes after radiofrequency catheter ablation at right ventricular outflow tract[END_REF] , the papillary muscles [START_REF] Van Herendael | Catheter ablation of ventricular fibrillation: importance of left ventricular outflow tract and papillary muscle triggers[END_REF] , or the ventricular myocardium [START_REF] Saliba | Ventricular fibrillation: ablation of a trigger?[END_REF] . Purkinje ectopics (PurkE) arising from the right ventricle (RV) or the left ventricle (LV) are the predominant triggers, however, accounting for 87-93% [START_REF] Haïssaguerre | Mapping and ablation of idiopathic ventricular fibrillation[END_REF][START_REF] Leenhardt | Short-coupled variant of torsade de pointes. A new electrocardiographic entity in the spectrum of idiopathic ventricular tachyarrhythmias[END_REF] of cases, and their ablation has been associated with effective VF burden suppression [START_REF] Haïssaguerre | Mapping and ablation of idiopathic ventricular fibrillation[END_REF] . In addition, the Purkinje system has been implicated in different phases of VF maintenance [START_REF] Berenfeld | Purkinje-muscle reentry as a mechanism of polymorphic ventricular arrhythmias in a 3-dimensional model of the ventricles[END_REF] . Accordingly, developmental, genetic, and electrophysiologic studies seeking to better understand the mechanisms underlying its arrhythmogenic potential have been undertaken [START_REF] Van Eif | Gradual differentiation and confinement of the cardiac conduction system as indicated by marker gene expression[END_REF] . Thus far, however, data on the association between clinical factors and characteristics of Purkinje triggers are lacking.

We therefore sought to characterize the clinical phenotype of patients presenting with Purkinje-triggered idiopathic VF.

Methods

Study population

Consecutive patients from Bordeaux University Hospital, Paris University Hospital (Bichat, Pitié-Salpêtrière) and Tsukuba University Hospital who survived VF between 2001 and 2019 were reviewed. Patients were classified as having idiopathic VF based on extensive diagnostic testing as per published guidelines 2 . Structural heart disease was ruled out using echocardiography, coronary angiography with or without ergonovine infusion, and cardiac magnetic resonance imaging (MRI). Channelopathies were excluded using exercise and pharmacological testing with isoproterenol, adrenaline, and ajmaline infusions. Eighty three patients with structurally normal hearts, no identifiable channelopathy, and PurkE initiating VF were selected for analysis. Genetic testing was performed in 23 patients by sequencing a set of 98 cardiomyopathy-and arrhythmia-related genes using the Haloplex capturing system.

The five-tier terminology system of the American College of Medical Genetics and Genomics Committee and all patients gave written informed consent.

Idiopathic VF characteristics

Clinical data, including demographics, medical history, and circumstances of the VF event,

were collected. The circumstances surrounding the VF episode were classified as: occurrence during moderate to high physical exertion, low physical exertion (equivalent to usual nonstrenuous daily activities), emotional stress, at rest (no physical exertion), and during sleep.

The seasonal distribution of VF episodes was examined using the following month cut-offs:

spring (March to May), summer (June to August), fall (September to November), and winter (December to February). Additional variables, such as fever (oral temperature of ≥38°C or axillary temperature ≥37°C), hypokalemia (serum potassium ≤3.5 mmol/l), and alcohol or drug abuse were also collected. As markers of hormonal status among women at the time of VF episodes, we collected the following data on menopausal status (including age of menopause) and contraceptive or other hormonal drug use.

Localization of arrhythmogenic triggers

ECG morphology of PurkE. As 12-lead ECGs were performed frequently, we selected the first 12-lead ECGs following the VF event. Anatomical sites of PurkE origins were estimated based on their ECG morphologies (Figure 1). PurkE QRS complexes have a sharp and rapid onset due to fast conduction through the Purkinje system. When originating from the left Purkinje network, PurkE are narrow (<120 ms) with a right bundle branch block (RBBB) morphology. They exhibit right or left deviation when originating from the anterior or the posterior Purkinje fibers respectively. When originating from the right Purkinje system, PurkE QRS complexes are usually wide (>140ms) [START_REF] Haïssaguerre | Mapping and ablation of idiopathic ventricular fibrillation[END_REF] and have a left bundle branch block (LBBB) pattern in V1 with a fast initial deflexion. PurkE origins were confirmed in most cases by electrophysiological mapping. Six patients had PurkE originating from both ventricles.

Electrophysiologic study. Electrophysiological (EP) studies and ablation were performed when PurkE burden was relatively high to facilitate mapping. Two to four intracardiac catheters were used to map the Purkinje system and the electrical substrate. The RV was accessed using a venous approach and the LV by a transseptal or retrograde aortic approach. PurkE origins were mapped endocardially, usually with decapolar catheters placed along the interventricular septum. Care was taken in the RV to avoid "bumping" the right bundle branch. PurkE were identified by the presence of sharp potentials (≤10ms) preceding the QRS by >5ms in sinus rhythm and preceding the onset of clinical ectopic QRS complexes.

In the absence of spontaneous PurkE, provocative maneuvers were performed, including atrial or ventricular pacing and the administration of isoproterenol (1-2 mcg/kg/min). Pacemapping using the lowest pacing output required for local capture was also used when required.

Statistical analysis

Continuous variables are expressed as mean ± standard deviation or median and range in cases of non-normal distributions and were compared using paired t-tests, analysis of variance or Mann-Whitney U tests where appropriate. Categorical variables were compared using Fisher's exact test. P<0.05 was used to define statistical significance.

Results

Patient characteristics

Eighty-three patients met inclusion criteria (age 38 ±14 years, 44 women). Patient clinical characteristics are summarized in Table 1. Thirty-two patients had a prior history of unexplained syncope and 17 presented with electrical storm (range of 3 to 64 VF episodes).

Patient's descent was predominantly caucasian (65 Caucasian (78%), 13 Asian (16%) 5 Afro-Caribbean (6%)). There was a family history of SCD in 28% (first-degree relative in 5, second-degree relative in 6, and third-degree relative in 3). Among these patients, 6 (26%) had an identified genetic variant included SCN5A in 2, DPP6 in 2, TNNI3K in one and HCN4 in one. The prevalence of identified genetic variant was not significantly different between male and female (33% vs 13% respectively, p=0.37).

Eighteen patients had additional diagnoses or cardiovascular risk factors, including hypertension in 2, diabetes mellitus in 2, smoking in 4, dyslipidemia in 5, bronchial asthma in 2, cancer in 3 (breast, testicular and leukemia), and depression in 2. Among the 44 women with PurkE-initiated VF, 6 were post-menopausal, 5 were on an oral contraceptive, and 1 was on hormonal therapy for breast cancer.

The

Characteristics of the study population according to the origin of PurkE

PurkE originated from the RV in 41 cases (49%), from the LV in 36 (44%) and from both ventricles in 6 (7% 2 and3). PurkE originated from the LV in 61% (23/38) of cases among non-menopausal women and in 100% (6/6) of cases among menopausal women. However, due to the low number menopausal women in our study, the difference of PurkE origin between non-menopausal and menopausal women was not statistically significant (p=0.2). Male sex was associated with an odds ratio of 14 (95% CI, 4.8-37.5, p<0.0001) for right PurkE. Pathogenic variants found in patients with RV PurkE included SCN5A in 2 and DPP6 in 2, all of whom were male. Pathogenic variants found in patients with LV PurkE included TNNI3K in one female and HCN4 in one male.

Literature review on the origins of PurkE

We systematically reviewed the available literature. We entered the MeSH term 'short coupled torsade de pointes' or 'idiopathic VF' into Pubmed. Fifty seven published cases of patients with PurkE origins identified by QRS morphology on 12-lead ECG or intracardiac mapping were identified (Table 4 and Supplemental references). PurkE origin was determined on 12-lead ECG with the same criteria used in our study. The mean age of patients was 38 ± 12 years. When reported, a family history of SCD was noted in 3/22 (14%).

Twenty three (40%) were women and 13 (23%) presented with electrical storm. PurkE originated from the RV in 37 cases (65%) and from the LV in 20 (35%). The literature survey corroborates our results and demonstrates that PurkE origin differed significantly by sex. RV PurkE were more frequent in men than women (81% vs 19%) whereas LV PurkE were more frequent in women (80% vs 20%, p<0.0001)

Discussion

This study examined clinical factors associated with PurkE characteristics in patients with idiopathic VF. We demonstrate that the origin of PurkE differ considerably between men and women with RV origins predominating in men (76%) and LV origins predominating in women (81%). These findings strongly suggest that sex-related factors may influence the arrhythmogenicity of right-versus left-sided Purkinje networks.

RV and LV Purkinje

Specific embryological phenomena may underlie differences between RV and LV Purkinje cells. Recent genome-wide association studies [START_REF] Sano | Genome-Wide Association Study of Absolute QRS Voltage Identifies Common Variants of TBX3 as Genetic Determinants of Left Ventricular Mass in a Healthy Japanese Population[END_REF] have highlighted that developmental genes could be involved in arrhythmogenesis regulating conduction and repolarization properties with a sexual dimorphism in their expression [START_REF] Gershoni | The landscape of sex-differential transcriptome and its consequent selection in human adults[END_REF] or regulatory processes [START_REF] Chen | Sexual dimorphism in gene expression and regulatory networks across human tissues[END_REF] .The development of the ventricular conduction system is coordinated by cardiac transcription factors Tbx5 and Nkx2.5 which interact with key regulatory genes, such as Id2 and Irx3 [START_REF] Kim | Irx3 is required for postnatal maturation of the mouse ventricular conduction system[END_REF] .

Tbx5 is differentially expressed in the LV and RV [START_REF] Hatcher | Specification of the cardiac conduction system by transcription factors[END_REF] and between males and females [START_REF] Deegan | The developmental origins of sexbiased expression in cardiac development[END_REF] . These molecular pathways regulate SCN5A and activate calcium handling proteins (RyR2 and Atp2a2) during the embryonic and postnatal development of the Purkinje network [START_REF] Van Eif | Transcriptional regulation of the cardiac conduction system[END_REF] .

Mutations of these transcription factors can lead to conduction disorders and arrhythmias [START_REF] Jay | Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system[END_REF] .

Mutations in Irx3, SCN5A or DPP6 have been reported in cases of idiopathic VF [START_REF] Koizumi | Genetic defects in a His-Purkinje system transcription factor, IRX3, cause lethal cardiac arrhythmias[END_REF] . In our study, the two patients with DPP6 variant were male and presented right PurkE. Previous studies on this variant have reported a clear male predominance (95%) of VF episodes [START_REF] Sande | Detailed characterization of familial idiopathic ventricular fibrillation linked to the DPP6 locus[END_REF][START_REF] Postema | Founder mutations in the Netherlands: familial idiopathic ventricular fibrillation and DPP6[END_REF] .

Interestingly, all patients with DPP6 variants presented right PurkE. This predominance of right PurkE in patients with DPP6 variant can be explained by the higher expression of DDP6 in right Purkinje fibers compared to left [START_REF] Atkinson | Anatomical and molecular mapping of the left and right ventricular His-Purkinje conduction networks[END_REF] .

This asymmetry in the development and function of left and right Purkinje networks may underlie the differences observed in the origins of PurkE that trigger VF.

Sex-related arrhythmia manifestations

A recent review [START_REF] Asatryan | Sex-Related Differences in Cardiac Channelopathies: Implications for Clinical Practice[END_REF] re-emphasizes sex differences in arrhythmias -especially in Brugada syndrome and long QT -and their implications for clinical practice. Our study strongly suggest sex differences in PurkE-initiated idiopathic VF.

The different expression of male/female genes involved in cardiac depolarization and repolarization explain clinical differences in the incidence of ventricular arrhythmias between sexes. Females have reduced expression of various potassium ion channels that may account for their reduced repolarization reserve, their longer QT intervals, and their susceptibility to develop torsades de pointes [START_REF] Gaborit | Gender-related differences in ion-channel and transporter subunit expression in non-diseased human hearts[END_REF] . The higher risk of drug-induced torsades de pointes [START_REF] Gaborit | Gender-related differences in ion-channel and transporter subunit expression in non-diseased human hearts[END_REF] or LQT2related arrhythmias [START_REF] Zareba | Modulating effects of age and gender on the clinical course of long QT syndrome by genotype[END_REF] is also explained by the increase of triggered activity by oestradiol secondary to its effects on calcium cycling proteins [START_REF] Odening | How do sex hormones modify arrhythmogenesis in long-QT syndrome? -Sex hormone effects on arrhythmogenic substrate and triggered activity[END_REF] . Triggered activities -as early afterdepolarizations or delayed afterdepolarization -are the main mechanisms of PurkE which could explain the predominance of biventricular PurkE in women.

Purkinje arrythmogenicity can manifest in other forms of clinical phenotype as in multifocal ectopic Purkinje-related premature contractions syndrome (MEPPC), catecholaminergic polymorphic ventricular tachycardia (CPVT), focal or reentrant fascicular VT or bundle branch reentry [START_REF] Wilde | Role of the Purkinje system in heritable arrhythmias[END_REF] . The arrhythmogenic mechanism underlying MEPCC and CPVT is likely triggered activity. In MEPPC and CPVT patients, the highly polymorphic character of ectopies indicates a multifocal origin from both Purkinje and myocardium sources [START_REF] Haissaguerre | Ventricular arrhythmias and the His-Purkinje system[END_REF] . No sex predominance has been reported in CPVT patients even if some sex differences exist with more RYR2 found variants and earlier symptoms in male [START_REF] Offerhaus | Epidemiology of inherited arrhythmias[END_REF] . By combining results from eight studies with 65 patients affected by MEPPC, female sex slightly predominates (60%). Creta et al [START_REF] Creta | Catheter ablation for fascicular ventricular tachycardia: A systematic review[END_REF] showed in the largest review of 953 patients with fascicular VT undergoing catheter ablation, that male sex was predominant (82%) ; which may appear in discrepancy with LV PurkE predominating in female. However, fascicular VT are mainly attributable to reentry with conduction disturbance in the Purkinje fascicles and myocardium ; while ectopic triggers mainly involve triggered activity mechanisms [START_REF] Haissaguerre | Ventricular arrhythmias and the His-Purkinje system[END_REF] .

Study limitations

Only 23 patients had genetic testing in our study. A more systematic genetic analysis could have yield additional insights into potential sex-based mechanisms influencing Purkinje arrhythmogenicity. Relevant data regarding the hormonal status at the time of VF events were not available in all patients. Sex hormone assays were also not performed, therefore their direct association with PurkE characteristics could not be explored. Among the 83 patients, 15

did not undergo electrophysiological mapping and the PurkE origin was determined on 12lead ECG. However, RV PurkE demonstrated a similar morphology between patients with and without EP study.

Conclusion

Our study demonstrates that the sites of PurkE origins responsible for VF in patients without structural disease differ substantially between men and women. Studies to explore the basis for these phenotypic differences are warranted.

13

Sources of funding :

This study received financial support from the French Government as part of the

(

  ACMG) was used for variant classification including: Pathogenic, Likely Pathogenic, Variant of Unknown Significance, Likely Benign and Benign. Pathogenicity of variants was //gnomad.broadinstitute.org/), in silico prediction tools, review of the literature and of variants databases. The study was approved by the Institutional Clinical Research and Ethics

  first episode of documented VF occurred at rest in 44, during sleep in 6, during moderate to high physical exertion in 4, during low physical exertion in 7, and during emotional stress in 1. VF episodes were equally distributed between seasons (26% in spring, 16% in summer,

28% in fall, and 30% in winter). Fourteen patients were hypokaliemic at the time of the index VF episode (serum potassium 3.2 ± 0.3 mmol/l) and two patients experienced VF during febrile illnesses. All patients received an implantable-cardioverter defibrillator and 68 underwent electrophysiological study. During a follow-up of 64±70 months, 1.4 ± 1.1 antiarrhythmic drugs were trialed, including sodium channel blockers in 22, β blockers in 23, amiodarone in 7, and non-dihydropyridine calcium channel blockers in 16.

  ). PurkE QRS durations were shorter when originating from the LV (113 ± 15 vs 127 ± 19 ms, p=0.01). RV PurkE presented a similar morphology among patients with or without EP study with an initial deflexion in V1 of 40±8ms and 30±14ms respectively (p=0.35). The circumstances of VF episode were not statistically different according to the origin of PurkE (p= 0.8). Patients with biventricular PurE had more familial histories of

sudden death (p=0.02). PurkE origin differed by sex. RV PurkE were more frequent in men than women (76% vs 24%) whereas LV and biventricular PurkE were more frequent in women (81% vs 19% and 83% vs 17% respectively, p<0.0001) (Tables

Table 1 : Population study

 1

	Investments of the Future program managed by the National Research Agency (ANR; grant
	number ANR-10-IAHU04-LIRYC). Elodie Surget is supported for PhD scholarship by the
	Medical	Research	Foundation	(FRM ;	FDM201906008561).

Figure1 : Examples of PurkE morphologies in patients with IVF. PurkE origins confirmed by electrophysiological mapping are circled in green and those non confirmed by electrophysiological mapping are circled in blue.

Table 4 : Literature review on the origins of PurkE

 4

	References	Patient history	Familial	Origin of
			history	PurkE
	Rosu et al 2015	39 y.o female	No	RV
		Electrical storm
	Chan et al 2016	40 y.o female	No	LV
		VF
	Saba et al 2011	10 y.o male	No	LV
		ICD shocks for VF
	Kohsaka et al 2007	21 y.o female	NA	RV
		Electrical storm
	Kleissner et al 2014	Male	NA	RV
		Electrical storm
	Naik et al 2008	24 y.o male	NA	RV
		Syncope
	Samo Ayou et al 2014	54 y.o female	NA	RV
		Electrical storm
	Martin et al 2017	32 y.o male	QT prolongation	LV
		Cardiac arrest	in his mother
	Choo et al 2009	30 y.o male	No	RV
		Electrical storm
	Nagase et al 2014	29 y.o female	NA	LV
		Multiple ICD shocks
		for VF episodes
	Ho et al 2017	44 y.o male	NA	RV
		Electrical storm
	Sadek et al 2015	5 patients with IVF, 1	NA	RV
		female and 4 males
	Anter et al 2013	59 y.o male	NA	RV
		Electrical storm
	Wang et al 2016	3 females, mean age	NA	LV in all
		21±11 years		cases
		Frequent PVCs
	Srivathsan et al 2007	39 y.o female	No	LV
		Cardiac arrest
	Luebbert et al 2016	52 y.o male	NA	RV
		Recurrent VF
	Xiao et al 2013	5 male patients	NA	RV in all
		Cardiac arrest	All had DPP6	cases
			mutation
	Belhassen et al 1981	28 y.o male	No	RV
		Electrical storm
	Golian et al 2011	36 y.o male	NA	RV
		ICD shocks for
		polymorphic VT
	Viskin et al 1998	50 y.o woman	NA	LV