How do sex hormones modify arrhythmogenesis in long-QT syndrome? – Sex hormone effects on arrhythmogenic substrate and triggered activity

Abstract

Gender differences in cardiac repolarization and the arrhythmogenic risk of patients with inherited and acquired long-QT syndromes are well appreciated clinically. Enhancing our knowledge of the mechanisms underlying these differences is critical to improve our therapeutic strategies for preventing sudden cardiac death in such patients.

This review summarizes the effects of sex hormones on the expression and function of ion channels that control cardiac cell excitation and repolarization as well as key proteins that regulate Ca²⁺ dynamics at the cellular level. Moreover, it examines the role of sex hormones in modifying the dynamic spatiotemporal (regional and transmural) heterogeneities in action potential duration (e.g., the arrhythmogenic substrate) and the susceptibility to (sympathetic) triggered activity at the tissue, organ, and whole-animal levels. Finally, it explores the implications of these effects on the management of LQTS patients.

Introduction

Women with inherited or acquired long-QT syndrome (LQTS) have longer QT intervals and are more prone to develop additional drug-induced QT prolongations, polymorphic ventricular tachycardia (pVT), and sudden cardiac death (SCD) than men^{1, 2}. Several observations suggest an important role for sex hormones in conferring these gender differences; different phases of the menstrual cycle, pregnancy, and the postpartum period are all associated with changes in QT duration and the incidence of pVTs in LQTS patients^{3, 4}.

This review explores the mechanisms underlying these gender differences and sex hormone effects at the cellular, tissue, organ, and whole-animal levels in order to identify potential novel therapeutic approaches to prevent arrhythmias and SCD in LQTS patients. Specifically, it recapitulates cellular sex hormone effects on the transcription, protein expression, post-translational modification, and function of cardiac ion channels and Ca²⁺ cycling proteins. Moreover, this review explores sex hormone effects on the arrhythmogenic substrate and triggered activity and their modulation by sympathetic stimulation.

Cellular level: Mechanisms underlying gender differences and sex hormone effects on ion channels, calcium cycling proteins, and triggered activity

Major determinants of the duration of cardiac repolarization include repolarizing voltage-gated rapid delayed rectifier current I_Kr (alpha-subunit HERG), slow delayed rectifier current I_Ks (alpha- and beta-subunits KvLQT1 and KCNE1), and inward rectifier current I_K1 (Kir2.1), as well as depolarizing L-type Ca²⁺ current I_Ca,L (alpha-subunit Cav1.2) and the activity of Na⁺/K⁺ ATPase. I_Kr and I_K1 current densities are lower in ventricular cardiomyocytes of female rabbits than in males⁵. The lower I_Kr current density in females that reduces the “repolarization reserve” is thought to be of major importance for females' higher sensitivity to I_Kr-blocking drugs and their prolonged action potential duration (APD)⁵. Increased activity of cardiac Na⁺/K⁺ ATPase in female rats⁶ that shortens the duration of cardiac repolarization, particularly at fast heart rates, may contribute to the steepening of the QT/RR slope in females. Recently, similar findings of gender differences in cardiac repolarizing ion currents have also been detected in human cardiac tissue derived from non-diseased transplant donors. Using a high-throughput quantitative approach, Gaborit et al.⁷ demonstrated lower transcript and protein expression of several repolarizing K⁺-channel subunits – such as HERG, KCNE1, and Kir2.3 – in female cardiac tissue derived from RV and LV base regions, compared with analogous male tissue.

Sex hormone effects on the (regionally heterogeneous) expression and function of these ion channels may contribute to gender differences in the arrhythmogenic substrate. Sex hormones may influence the expression and function of cardiac ion channels via sex hormone receptor-mediated genomic modulation of their expression or of their posttranslational phosphorylation state or by an acute, non-genomic regulation of ion current densities involving nitric oxide⁸ (Table 1).

Table 1.

Gender and sex hormone effects on cardiac repolarization and LQT-related arrhythmogenesis on cellular and whole-heart levels

Sex	Intervention	Ion currents	Magnitude of changes	Cardiac region	Level of changes	Species	Ca2−cycling proteins	Magnitude of changes	Cardiac region	Level of changes	Species	APD in rabbits	EADs in LQT rabbits
Male	--------	↑ IKr ↑ IK1 compared to females [5]	+ 30% + 15%	LV LV	current current	rabbit rabbit	↓ NCX compared to females [32]	−2.5-fold −55%	LV base LV base	activity protein	rabbit rabbit	↓ APD compared to females [5]
	ORCH	↓ IKr ↓ IK1 [15]	−40% −50%	LV LV	current current	rabbit rabbit	↓ RyR2 ↓ SERCA ↓ PLN-phos ↓ NCX [28]	−25% −30% −25% −15%	LV LV LV LV	activity, ISO activity, ISO Ser16-phos activity, PE	rat rat rat rat	↑ APD compared to males [15, 44]
	ORCH+DHT	↑ IKr ↑ IKs ↑ IK1 ↓ ICa,L ↑ ICa,L [8, 10, 15, 44]	+ 40% + 45% + 50–60% −40% −50% + 2.5-fold + 2-fold	LV LV LV LV LV LV LV	current current, eNOS current current, acute current, acute current, chron protein, chron	rabbit guinea pig rabbit guinea pig rat rat rat	↑ RyR2 ↑ SERCA ↑ NCX [28]	+ 25% + 30% + 15%	LV LV LV	activity, ISO activity, ISO activity, PE	rat rat rat	↓ APD compared to ORCH [15, 44]
Female	--------	↓ IKr ↓ IK1 ↑ ICa.L (grad) compared to males/OVX [5, 44, 45]	−30% −15% + 30% + 70% + 3-fold	LV LV LV base LV base LV base	current current current protein mRNA	rabbit rabbit rabbit rabbit rabbit	↑ NCX compared to males [32]	+ 2.5-fold + 55%	LV base LV base	activity protein	rabbit rabbit	↑ APD compared to males [5]	↑ EADs compared to DHT and PROG [25]
	OVX						↑ RyR2 ↓ SERCA ↓ PLN-phos ↓ NCX [27, 30, 33]	+ 2-fold −15% −25%/−3-fold −30% −40%	LV LV LV LV LV apex	activity activity protein/mRNA Thr17-phos protein	rat rat rat rat rat	↓ APD compared to females [44]	↑ EADs compared to DHT and PROG [25]
	OVX+EST	↓ IKr ↑ KCNE2 ↑ IKs ↓ KCNE1 ↑ ICa,L [8, 9, 13, 14, 16, 25, 44]	−25–35% + 4.5-fold + 35% no expr + 30% + 40–100% + 20% + 30 %	LV LV LV LV LV apex LV base LV base LV base	current mRNA current mRNA current, chron current protein mRNA	guinea pig rat guinea pig rabbit rabbit rabbit rabbit rabbit	↑ RyR2 leak ↑ PLN ↓ PLN-phos ↑ PLN-phos ↑ NCX [27, 29, 30, 31, 32, 34]	+ 50% + 30% −10-fold + 30% + 40% + 20–25% + 30%/+30% + 60%/+20%	LV LV apex LV LV LV apex - - LV base	leakiness mRNA Ser16-phos Thr17-phos protein activity, chron mRNA/protein activity/protein	rat rat rat rat rat rat H9C2 rat H9C2 rabbit	↑ APD compared to OVX [16, 44]	↑ EADs compared to DHT and PROG [25]
	OVX+DHT	↑ IKs [25]	+ 50%	LV apex	current, chron	rabbit							↑ EADs compared to females and EST [25]
	OVX+PROG	↑ IKs ↓ ICa,L [8, 25]	+ 20–35% −30%, cAMP −25%	LV LV LV apex	current, eNOS current, eNOS current, chron	guinea pig guinea pig rabbit	↑ SERCA ↑ PLN-phos [25, 27]	+ 50% + 15% + 30%	LV apex LV LV	protein activity Thr17-phos	rabbit rat rat		↓ EADs compared to females and EST [25]

APD, action potential duration; EADs, early afterdepolarizations; ORCH, orchiectomy; OVX, ovariectomy; DHT, dihydrotestosterone; EST, estradiol; PROG, progesterone; I_Kr, rapidly delayed rectifier K⁺ current; I_Ks, slow delayed rectifier K⁺ current; I_K1, inward rectifier K⁺ current; I_Ca,L, L-type Ca²⁺ current; KCNE1, beta-subunit to KvLQT1 to form I_Ks; KCNE2, beta-subunit to HERG to form I_Ks,; NCX, sodium calcium exchanger; SERCA, sarcoplasmatic reticulum ATPase; RyR, ryanodine receptor; LV, left ventricle (region not further specified); grad, base-to-apex gradient. ↑ indicates an increase; ↓ indicates a reduction; ↑ APD indicates an APD prolongation; ↓ APD indicates an APD shortening. Chron, chronic hormone effects; Phos, phosphorylation; eNOS, acute eNOS-mediated mechanism; cAMP, under cAMP stimulated condition; PE, under phenylephrine stimulation; ISO, under isoproterenol stimulation; rat H9C2, rat H9C2 cell line

However, since the concentrations of sex hormones used and durations of hormone treatment vary, it is challenging to integrate data from all available studies into one concise picture. Ideally, sex hormone effects should be studied using physiological hormone dosages; e.g., 1) Estradiol concentrations could range from 15 to 350 pg/ml corresponding to the physiological levels in post-pubertal premenopausal women during the different phases of normal menstrual cycle and could reach up to 7,000 pg/ml as during pregnancy, 2) Progesterone concentrations could range from 0.3 to 1.2 ng/ml corresponding to the physiological levels during follicular phase, from 1.7 to 27 ng/ml as during luteal phase and could reach up to 340 ng/ml as during pregnancy, 3) Dihydrotestosterone concentrations could range from 120 to 1000 pg/ml and testosterone concentrations could range from 3000 to 12.000 pg/ml corresponding to the physiological levels in healthy post-pubertal adult men (ACP reference ranges, Mayo Clinic reference ranges). However, in the different studies available, estradiol concentrations ranging from 30 pg/ml up to 2,000 ng/ml, progesterone concentrations from 0.3 up to 3,000 ng/ml, dihydrotestosterone concentrations from 200 up to 900 pg/ml, and testosterone concentrations from 300 pg/ml up to 300 ng/ml were utilized. This is particularly important, since it is known 1) that sex hormone concentrations above naturally occurring physiological levels may exert differential, partially opposing effects on ion currents and channel expression (as demonstrated for estradiol’s effects on I_Kr⁹) and 2) that acute and chronic hormone effects may counteract each other (as demonstrated for testosterone’s effects on I_Ca,L¹⁰). Since rabbits have repolarizing ion current characteristics similar to humans and mimic human gender differences in LQT-related arrhythmias and cardiac repolarization^{5, 11, 12}, this review focuses mainly on rabbit studies investigating sex hormone effects on cardiac repolarizing currents on both the whole-heart and animal levels.

I_Kr can be directly inhibited by estradiol^{9, 13} and can also be reduced indirectly by estradiol-induced increased transcription of the beta-subunit KCNE2¹⁴. Moreover, estradiol and I_Kr-blocking drugs exert a synergistic effect on I_Kr¹³. In contrast, testosterone acutely increases I_Kr and I_K1¹⁵, while other sex hormones have no direct effect on the inward rectifier current. Similarly, I_Ks can be acutely increased by testosterone or progesterone via a non-genomic pathway involving NO⁸, while estradiol indirectly reduces I_Ks by downregulating mRNA levels of the beta-subunit KCNE1¹⁶. Thus far, sex hormone effects on the expression and activity of Na⁺/K⁺ ATPase have only been investigated in arteries (not in cardiomyocytes), demonstrating an estradiol-induced increased function and mRNA expression of its isoform 2¹⁷. Whether similar sex hormone effects may be found in cardiac muscle–specific isoforms remains to be investigated. All these hormone-induced alterations of ion current densities or the expression of its subunits result in a net estradiol-induced prolongation of APD and a net testosterone- and progesterone-induced shortening of APD (Table 1).

Reactivation of I_Ca,L currents plays an important role in the formation and propagation of early afterdepolarizations (EADs)¹⁸. Additionally, Ca²⁺ cycling proteins such as the ryanodine receptor (RyR2), the sodium calcium exchanger (NCX), the sarcoplasmic reticulum ATPase 2a pump (SERCA), phospholamban (PLN), and the Ca²⁺-calmodulin-dependent protein kinase-II (CaMKII) are known to contribute to or prevent the formation of EADs by altering cytoplasmic and sarcoplasmic reticulum (SR) Ca²⁺ concentrations, spontaneous Ca²⁺ release, and Ca²⁺ transient characteristics. An increased intracellular Ca²⁺ concentration or spontaneous SR Ca²⁺ release may thereby activate NCX in its forward mode, thus prolonging action potential duration to allow the activation of the window I_Ca,L current or the re-activation of I_Na in late phase 3 repolarization thereby eliciting an EAD.

Sex hormone effects on the expression and function of Ca²⁺ cycling proteins may thus contribute to gender differences in the susceptibility to triggered activity (Table 1). Since PKA- or CamKII-mediated phosphorylation of these Ca²⁺ cycling proteins alters their function – e.g., phosphorylation of L-type Ca²⁺ channel increases I_Ca,L current, phosphorylation of PLN diminishes its inhibitory effects on SERCA, and phosphorylation of RyR2 increases its open probability – sex hormones may exert their effects on the activity of Ca²⁺ cycling proteins partly by altering their phosphorylation state. Moreover, since previous studies have demonstrated gender differences in intracellular Ca²⁺ transient characteristics with longer Ca²⁺ transient durations, as well as a slower rate of rise and a greater spatial Ca²⁺ transient variability in female rats’ cardiomyocytes^{19, 20}, sex hormone effects on Ca²⁺ cycling proteins may also account for differences in Ca²⁺ transients. Indeed, estradiol increases Ca²⁺ transient amplitudes in rat cardiomyocytes²¹, while testosterone shortens Ca²⁺ transient duration²².

Since most studies investigating sex hormone effects on Ca²⁺ cycling properties and Ca²⁺ transients were performed on rodents, we have integrated this information into the review, despite the fact that rodents have different repolarization characteristics than human subjects and lack some of the gender differences in cardiac repolarization and arrhythmia vulnerability observed in human subjects^{11, 23}. Moreover, I_Ca,L recovery from inactivation, which is important for EAD formation, requires an action potential long enough to enable the reactivation of the L-type Ca²⁺ “window current” within its voltage range²⁴. The AP in rodents, however, is too short for the reactivation of the I_Ca,L window current. Thus, data on sex hormone effects on Ca²⁺ cycling properties from rodents may not necessarily reflect (all) sex hormone effects on Ca²⁺ dynamics and triggered activity in human subjects. Nevertheless, we added this information since these data may give some idea about the effects of sex hormones on Ca²⁺ dynamics that may contribute to their pro- or anti-arrhythmic effects.

Estradiol increases I_Ca,L current density in rabbits, likely enhancing the propensity to develop EADs²⁵. Acute and chronic progesterone and acute testosterone exposure, in contrast, decrease I_Ca,L current densities^{8, 10, 25}. The acute direct I_Ca,L-blocking effects of testosterone over the physiological range of concentrations will be dominant in the short-term time scale. Chronic exposure to testosterone, however, may eventually result in a contrasting increase in channel expression and I_Ca,L current densities (at higher concentrations)^{10, 26}, though repeated acute exposure to testosterone could mask these chronic effects.

SERCA expression and activity in rats and rabbits are reduced by ovariectomy²⁷ and can be restored by progesterone and testosterone^{25, 27, 28}. This progesterone-induced increased SERCA expression and activity²⁵ may contribute to progesterone’s anti-arrhythmic effect by increasing the Ca²⁺ reuptake into the SR, thus shortening Ca²⁺ transient duration. Thr¹⁷-phosphorylation of PLN, which inhibits phospholamban’s SERCA-inhibitory effect, is reduced by ovariectomy in rats, resulting in a net reduction of SERCA activity²⁷. This effect can be partly reversed by estradiol or progesterone replacement²⁷. Ser¹⁶-phosphorylation of PLN, in contrast, is reduced by estradiol via an ERβ-mediated mechanism involving PIK3, thus partly counteracting the above-described effects²⁹. NCX expression can also be reduced by ovariectomy³⁰, while estradiol increases its transcription, protein expression, and activity in rabbits, rats, and the embryonic rat cardiomyoblast cell line H9C2^{30, 31, 32}. This estradiol-effect may contribute to the increased EAD formation observed in female rabbits. Testosterone also increases NCX transcription and activity in rats³³. However, increased function of NCX is accompanied and counteracted by testosterone-induced increased function of SERCA²⁸, thus leading to a faster removal of Ca²⁺ from the cytosol and shortened Ca²⁺ transient duration (Table 1). In ovariectomized rats, PKA-mediated increased RyR2 activity leads to increased Ca²⁺ release from the SR into the cytosol³³. Similarly, testosterone increases RyR2 activity²⁸. Estradiol, however, increases the leakiness of RyR2 via an ERβ-mediated mechanism, thereby increasing the risk of pro-arrhythmic afterdepolarizations³⁴. The net overall effects of sex hormones on Ca²⁺ transients and triggered activity, however, are hard to predict or model due to the lack of comprehensive studies that thoroughly investigated these different and partially opposing effects. There is ample evidence, however, that female gender and estradiol may at least increase I_Ca,L currents^{25, 32}, NCX activity³² and RyR2 leakiness³⁴ thus increasing the propensity for triggered activity, while testosterone and progesterone may increase SERCA activity either directly or by altering phospholamban phosphorylation^{25, 27, 28} and acutely decrease I_Ca,L^{8, 10, 25} thus reducing the likelihood for triggered activity.

There are known feedback mechanisms between Ca²⁺ transients and action potential duration, including an inactivation of I_Ca,L currents induced by SR Ca²⁺ release that results in an APD shortening as well as an increase in NCX activity induced by SR Ca²⁺ release with the opposite (prolonging) effect on APD³⁵. Consequently, any sex hormone effects on the activity of Ca²⁺ cycling proteins and Ca²⁺ transients may also indirectly affect APD and may have either synergistic or opposing effects on APD relative to the hormone’s direct effects on ion currents. The testosterone-induced increased Ca²⁺ release via RyR2²⁸ may lead to a faster inactivation of I_Ca,L, creating a synergistic effect on APD with the testosterone-induced reduction of I_Ca,L¹⁰. Similarly, testosterone-induced increased Ca²⁺ release from the SR may enhance NCX activity, creating a synergistic effect with testosterone’s direct effects on the transcription and activity of NCX²⁸, thereby partly counteracting the aforementioned APD-shortening effects. However, the effects of estradiol on the leakiness of RyR2³⁴ and the increased Ca²⁺ transient amplitudes in rat cardiomyocytes²³ may partly counteract the direct effects of estradiol on I_Ca,L and thus on APD. Future studies of the effect of sex hormones on ion currents and Ca²⁺ dynamics should include modelling of these feedback mechanisms to reconcile and integrate sex hormone effects on AP duration and triggered activity.

Sympathetic α₁-adrenergic receptor-activation facilitates EAD formation and prolongs APD by reducing I_Kr and I_K1. Any sex hormone effects on the response to sympathetic α₁-adrenergic stimulation, such as an estradiol-induced increase in α₁-adrenoceptor responsiveness³⁶, may therefore affect APD and the susceptibility to EAD formation. β-adrenergic stimulation enhances I_Ks via G-protein-mediated phosphorylation of KvLQT1 and thereby shortens APD. Additionally, β-adrenergic stimulation enhances I_Ca,L – which, because it occurs faster than that the increase of I_Ks, leads to an initial APD prolongation and increased EAD formation in a situation of short sudden sympathetic surge³⁷. In the presence of the β-sympathomimetic drug isoproterenol, APD shortens upon faster pacing in male dogs, while the opposite is seen in females³⁸, strongly suggesting gender differences in the autonomic modulation of I_Ks and I_Ca,L. In rats, ovariectomy induces an upregulation³⁹ of β₁-adrenoceptor expression, while estrogen induces its downregulation^{30, 40}. β₂-adrenoceptor expression, in contrast, is upregulated by estradiol⁴¹ and is higher in female than in male rat cardiomyocytes⁴¹, indicating that sex hormones may indeed modulate cardiac responses to sympathetic stimulation by altering cardiac expression of β₁ and β₂-adrenergic receptors.

Tissue and organ level: Mechanisms underlying gender differences and sex hormone effects on the arrhythmogenic substrate and triggered activity

Regional and transmural spatial heterogeneities in APD as well as dynamic temporal APD changes, forming the so-called “arrhythmogenic substrate,” are of major importance for re-entry formation in arrhythmias related to long QT⁴². Spatial and transmural heterogeneities in I_Kr, I_Ks, and I_Ca,L current densities underlie these pronounced APD heterogeneities in LQTS^{43, 44}. However, few studies have investigated gender differences and sex hormone effects on spatial APD heterogeneities and the underlying spatially heterogeneously expressed ion channels at the tissue and organ levels. In female rabbits but not in males, there is a transmural and base-to-apex gradient of I_Ca,L with a higher conductance of I_Ca,L in epi- than endocardial and basal than apical cells^{44, 45}. Ovariectomy eliminates the transmural gradient, which reappears upon treatment with estradiol⁴⁴. In contrast, there is no transmural gradient in orchiectomized rabbits even after exposure to estradiol⁴⁴, suggesting a potential gender difference in the mechanism of action of sex hormones. Moreover, estradiol increases the base-to-apex gradient of I_Ca,L in rabbits⁴⁶. However, to date, there are no experimental data available analyzing gender differences or sex hormone effects on transmural or base-to-apex distribution of I_Ks or I_Kr that might contribute to the observed gender differences in dispersion of repolarization.

Moreover, since sympathetic stimulation decreases the transmural dispersion of refractoriness⁴⁷, gender differences and sex hormone effects on (spatial inhomogeneities of) β₁ and β₂-adrenoceptor expression might produce marked discrepancies in APD and thereby facilitate ventricular arrhythmias. However, to date, no studies have investigated sex hormone effects on spatial differences in cardiac adrenoceptor expression and activity.

Animal models: Gender differences and sex hormone effects on the arrhythmogenic substrate in animal models of drug-induced and inherited LQTS

Similar to human subjects, rabbits exhibit pronounced gender differences in duration and dispersion of repolarization and are thus often used to explore such differences and their underlying mechanisms. Compared with males, female rabbits have longer QT intervals⁵, a steeper QT/RR slope⁴⁸, and a more pronounced I_Kr-blocker induced QT prolongation and transmural APD dispersion⁴⁴.

Experiments involving castration and subsequent exposure to sex hormones can reveal the impact of different hormones on cardiac repolarization: Ovariectomized female and castrated male rabbits exhibit similar ventricular APD₉₀⁴⁴. Moreover, orchiectomized rabbits have longer APD than intact males, while ovariectomized females have decreased drug-induced prolongation of APD compared to intact females⁴⁴, thus reversing the normal gender differences in drug-induced APD or QT prolongation. The normal female phenotype can be mimicked by treating ovariectomized or orchiectomized rabbits with estradiol^{16, 44}. Exposure to testosterone, in contrast, shortens QT and reduces the extent of drug-induced QT prolongation⁴⁴ (Tables 1 and 2).

Table 2.

Gender and sex hormone effects on cardiac repolarization and LQT-arrhythmogenesis in rabbit models in vivo

Sex	Intervention	Drug-induced QT-prolongation	Drug-induced EADs / pVT	QT/RR slope steepness in LQT rabbits	pVT / SCD in LQT rabbits
Male	--------	↓ QT-prolongation less pronounced than in females [5, 44]	↓ EADs fewer than in females [5]	↓ QT/RR less steep than in females [48]
	ORCH	↑ QT-prolongation more pronounced than in males [15, 44]	↑ EADs more than in males [15, 44]
	ORCH+DHT	↓ QT-prolongation less pronounced than in ORCH [15, 44]	↓ EADs reduced compared to ORCH [15, 44]
Female	--------	↑ QT-prolongation more pronounced than in males [5, 44]	↑ EADs more than in males [5]	↑ QT/RR steeper than in males [48]
	OVX	↓ QT-prolongation less pronounced than in females [16, 44]	↓ EADs reduced compared to females [16, 44]	↓ QT/RR less steep than in females [25]
	OVX+EST	↑ QT-prolongation more pronounced than in OVX [16, 44]	↑ EADs more than in OVX [16, 44]	↑ QT/RR steeper than in OVX [25]	↑ pVT, ↑ SCD increased incidence of arrhythmias [25]
	OVX+DHT			↓ QT/RR less steep than in females and EST [25]	no pVT, no SCD decreased incidence of arrhythmias [25]
	OVX+PROG				no pVT, no SCD decreased incidence of arrhythmias [25]

EAD, early afterdepolarization; pVT, polymorphic ventricular tachycardia; SCD, sudden cardiac death; ORCH, orchiectomy; OVX, ovariectomy; DHT, dihydrotestosterone; EST, estradiol; PROG, progesterone; QT/RR, QT-interval to RR-interval ratio; ↑ indicates an increase; ↓ indicates a reduction.

In a transgenic LQT2 rabbit model over-expressing a loss-of-function pore mutation of the human HERG channel (HERG-G628S)⁴⁹, estradiol steepens the QT/RR slope by prolonging QT particularly at slow heart rates, while testosterone decreases QT/RR steepness²⁵, thereby mimicking the gender differences observed in human LQT patients (Table 2). Moreover, sex hormones differentially affect APD in different regions of the heart in transgenic LQT2 rabbits, and thus modify spatial APD dispersion. Estradiol induces a particularly pronounced APD prolongation in the LV and RV apex, increasing VF inducibility in estradiol-treated hearts²⁵. In contrast, in prepubertal rabbits treated with the I_Kr-blocker E4031, male rabbits show a greater base-to-apex dispersion of repolarization and thus a higher incidence of EAD formation than do female rabbits⁵⁰, suggesting that other sex-related factors besides sex hormones might influence the extent of dispersion of repolarization during (prepubertal) development.

Animal models: Gender differences and sex hormone effects on triggered activity in animal models of drug-induced and inherited LQTS

Like women, female rabbits have a higher incidence of drug-induced pVTs than males¹². Castration reverses these gender differences: Orchiectomy increases the incidence of I_Kr-blocker-induced EADs, while ovariectomy has the opposite effect⁴⁴. The normal female phenotype can be mimicked by estradiol treatment¹⁶, while combined treatment with estradiol and progesterone reduces the risk of drug-induced arrhythmias⁵¹. Similarly, testosterone reduces the risk of drug-induced EADs⁴⁴ (Table 2).

In transgenic LQT2 rabbits, which develop spontaneous pVT and SCD⁴⁹, estradiol exerts a pro-arrhythmic effect in vivo, while progesterone exerts an anti-arrhythmic, protective effect and completely abolishes pVTs and SCD²⁵ (Table 2). On the organ level, a reduced rate of EAD formation in response to sympathetic stimulation has been identified as the mechanism underlying the protective effect of progesterone²⁵.

Clinical observations: Gender differences in cardiac repolarization and arrhythmogenesis in patients with inherited or acquired LQTS

Patients with inherited LQTS demonstrate pronounced gender differences in cardiac repolarization and in arrhythmogenic risk: Adult women with LQT types 1 and 2 have longer QT intervals, a more pronounced transmural QT-dispersion, and a higher risk of pVTs and SCD than men^{2, 52}. Moreover, female gender is a main independent risk factor for increased risk of life-threatening cardiac events during adulthood independent of the genotype or of the location of the mutation within the gene^{2, 53}. Interestingly, in female patients with LQT2, the arrhythmogenic risk remains elevated after the onset of menopause⁵⁴, suggesting that other gender-related factors besides sex hormones may contribute to gender differences in arrhythmogenesis. Before puberty, however, the incidence of arrhythmia (particularly in LQT1) is higher in boys than in girls, and the age at first cardiac event is younger in male than female LQT patients⁵⁵.

Similarly, drug-induced, acquired LQTS is also strongly influenced by gender. A variety of different drugs that block I_Kr currents (including antiarrhythmics, gastrokinetics, antipsychotics, antidepressants, antihistamines, and antibiotics) can cause this serious side effect (see complete list on www.QTDrugs.org). This risk and the extent of drug-induced QT prolongation and pVTs are significantly higher in women than in men, with two-thirds of reported drug-induced pVTs occurring in women^{1, 56}.

Clinical observations: Impact of sex hormones on the arrhythmogenic substrate and triggered activity in human subjects with inherited or acquired long-QT syndrome

Several observations suggest an important role for sex hormones in conferring these gender differences. The different phases of menstrual cycle, pregnancy, and the postpartum period are all associated with changes in QT duration and the incidence of pVT in patients with inherited and acquired LQTS. In acquired LQTS, drug-induced QT prolongation is more pronounced and the risk for pVTs is higher during menses and the follicular phase (with high estradiol serum levels) than during the luteal phase (with relatively higher progesterone levels)³. During pregnancy (with high progesterone levels), LQT2 patients have a reduced arrhythmogenic risk, while the risk markedly increases during the nine months postpartum⁴. These observations strongly suggest a pro-arrhythmic role for estradiol and an anti-arrhythmic effect of progesterone in humans. Additionally, other postpartum-related hormones such as oxytocin and prolactin may contribute to the heightened postpartal arrhythmogenic risk, as indicated in a recent study of transgenic LQT2 rabbits⁵⁷.

Conclusions

Observations in human subjects and animal models indicate that the sex hormones estradiol, progesterone, and testosterone are responsible for most of the gender differences observed in the arrhythmogenic substrate (cardiac repolarization), the susceptibility to (sympathetic) triggered activity, and the resulting gender differences in arrhythmia incidence in LQTS. Estradiol prolongs QT duration, increases the risk for additional drug-induced QT prolongation independently of gender, and exerts a pro-arrhythmic effect in vivo and ex vivo, whereas testosterone and progesterone shorten QT duration and exert an anti-arrhythmic effect with a reduced susceptibility to sympathetic stimuli. Some of the underlying mechanisms involved are: 1) an estradiol-induced decrease in I_Kr current densities as well as increases in I_Ca,L, NCX expression and activity, RyR2 leakiness, Ca²⁺ transient amplitude, and α1- and β2-adrenoceptor responsiveness; 2) a testosterone-induced increase in I_Kr, I_Ks, and I_K1 current densities, increased SERCA activity, and shortened Ca²⁺ transient duration; and 3) a progesterone-induced increase in I_Ks, increased SERCA expression and activity, and decreased I_Ca,L current densities with reduced Ca²⁺ oscillations upon sympathetic stimulation.

However, it is difficult to fully appreciate the complexity of sex hormone effects, not only on different single components of cardiac repolarization and excitability but also on the feedback interaction between Ca²⁺ transients and APD. This is because most studies have investigated hormone effects on only a very limited number of ion channels or Ca²⁺ cycling proteins. Moreover, comprehensive interpretation of data from studies of sex hormone effects on ion channels or Ca²⁺ cycling proteins is limited by the use of different sex hormone concentrations as well as different subject species. For example, mice and rats have different repolarization characteristics than human subjects and lack some of the gender differences in cardiac repolarization and arrhythmia vulnerability observed in human subjects^{11, 23}. Rabbits have repolarizing ion currents similar to those of humans and mimic their gender differences in cardiac repolarization and arrhythmogenesis^{5, 11, 12}. Ideally the effects of chronic sex hormone treatment should be studied using physiological hormone dosages, simultaneously targeting the activity of multiple ion currents, pumps, and Ca²⁺ dynamics in species with greater similarity to the human heart. Importantly, these studies should address such effects at the cellular, tissue, and organ levels.

Moreover, not all gender differences in cardiac repolarization and arrhythmogenesis can be completely recapitulated and explained by hormonal influences, as gender differences are also found in castrated and in prepubertal rabbits. Here, further investigations are warranted, focusing on possible genetic factors – or epigenetic modifications – responsible for these gender differences.

Clinical implications

As women with inherited LQTS are at higher risk for pVT, syncopes, and SCD than men and are particularly prone to develop lethal VTs during the postpartum phase^{2, 4}, particularly close clinical monitoring of female LQTS patients is mandatory – especially in phases associated with changing hormone levels. Similarly, as women generally have a higher risk for drug-induced pVTs than men¹, physicians should be particularly cautious when treating women with drugs with potential QT-prolonging properties.

Understanding the exact mechanisms that underlie sex hormone effects on cardiac repolarization and arrhythmogenesis in inherited and drug-induced LQTS will help to better discern the individual risk of LQTS patients and of healthy subjects treated with QT-prolonging drugs. Moreover, this understanding could help to develop future specific hormone-based anti-arrhythmic therapies. A recent community-based study determined that higher estradiol levels were associated with a higher risk for SCD in both sexes also in patients with ischemic heart disease. Those findings suggest an even broader potential impact of a deeper understanding of the exact mechanisms underlying sex hormone effects on arrhythmogenesis for risk stratification and treatment for a greater variety of arrhythmogenic diseases⁵⁸.

List of Abbreviations

APD

action potential duration

EAD

early afterdepolarization

EST

estradiol

DHT

dihydrotestosterone

I_Ca,L

L-type Ca²⁺ current

I_K1

inward rectifier K⁺ current

I_Kr

rapidly delayed rectifier K⁺ current

I_Ks

slow delayed rectifier K⁺ current

KCNE1

beta-subunit to KvLQT1 to form I_Ks

KCNE2

beta-subunit to HERG to form I_Ks

LQTS

long-QT syndrome

LV

left ventricle

NCX

sodium calcium exchanger

ORCH

orchiectomy

OVX

ovariectomy

PLN

phospholamban

PROG

progesterone

pVT

polymorphic ventricular tachycardia

RV

right ventricle

RyR

ryanodine receptor

SCD

sudden cardiac death

SERCA

sarcoplasmatic reticulum ATPase

QT/RR

QT-interval to RR-interval ratio steepness