Cellular basis of sex disparities in human cardiac electrophysiology

Cellular basis of sex disparities in human cardiac

electrophysiology

A. O. Verkerk,1,2* R. Wilders,2* W. de Geringel2 and H. L. Tan1

1 Department of Clinical and Experimental Cardiology, Academic Medical Center, University of Amsterdam, Amsterdam, The

Netherlands

2 Department of Physiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Received 15 September 2005,

revision requested 14 December

2005,

revision received 28 February

2006,

accepted 21 March 2006

Correspondence: A. Verkerk,

Department of Physiology,

Academic Medical Center, Room

M01-217, Meibergdreef 15, 1105

AZ Amsterdam, The Netherlands.

E-mail: [email protected]

*Both authors contributed equally

to the study.

Abstract

Aim: Sex disparities in electrocardiogram variables and dysrhythmia sus-

ceptibility exist, notably in long QT syndrome (LQTS) and Brugada syn-

drome, but the underlying mechanisms in man are unknown. We studied the

cellular basis of sex distinctions in human cardiac electrophysiology and

dysrhythmia susceptibility using mathematical models of human ventricular

myocytes.

Methods: We implemented sex differences in the Priebe–Beuckelmann and

ten Tusscher–Noble–Noble–Panfilov human ventricular cell models by

modifying densities of the L-type Ca2+ current (ICa,L), transient outward K+

current (Ito), and rapid delayed rectifier K+ current (IKr), according to

experimental data from male and female hearts of various species. Sex dis-

parities in transmural repolarization were studied in transmural strands of

cells with ion current densities based on canine experimental data.

Results: Female cells have longer action potential duration (APD), steeper

APD-heart rate relationship, larger transmural APD heterogeneity, and a

greater susceptibility to pro-dysrhythmogenic early afterdepolarizations

(EADs) than male cells. Conversely, male cells have more prominent phase-1

repolarization and are more susceptible to all-or-none repolarization.

Conclusion: Sex differences in ICa,L, Ito and IKr densities may explain sex

disparities in human cardiac electrophysiology. Female cells exhibit a limited

‘repolarization reserve’ as demonstrated by their larger susceptibility to

EADs, which, combined with their larger transmural electrical heterogeneity,

renders them more vulnerable to tachydysrhythmias in LQTS. Conversely,

male cells have a limited ‘depolarization reserve’, as shown by their larger

susceptibility to all-or-none repolarization, which facilitates tachydysrhyth-

mias in Brugada syndrome. These general principles may also apply to

dysrhythmia susceptibility in common disease.

Keywords action potentials, dysrhythmias, Brugada syndrome, computer

simulation, electrophysiology, ionic currents, long QT syndrome.

Sex differences exist in physiology and pathophysiology

of the cardiovascular system (Mendelsohn & Karas

2005), including human electrocardiogram (ECG) vari-

ables (Pham & Rosen 2002, James et al. 2006). Women

have faster resting heart rates and longer rate-corrected

QT intervals (QTc) than men (Bazett 1920, Merri et al.

1989, Rautaharju et al. 1992) and also manifest a

greater lengthening of the QT interval at longer cycle

lengths, rendering sex-related differences in QT inter-

vals most prominent at slow heart rates (Kligfield et al.

1996, Stramba-Badiale et al. 1997). Moreover, there

are also striking sex disparities in the incidence of

Acta Physiol 2006, 187, 459–477

� 2006 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2006.01586.x 459

cardiac dysrhythmias (James et al. 2006). The most

notable examples are inherited long QT syndrome

(LQTS) and Brugada syndrome, where prominent sex

disparities in clinical expressivity exist, despite equal

genetic transmission (autosomal dominant inheritance).

Thus, women are more likely to sustain ‘Torsade de

Pointes’ (TdP) ventricular tachycardia in LQTS (Zareba

et al. 1995, Locati et al. 1998). Excessive action

potential (AP) prolongation culminating in early after-

depolarizations (EADs) is involved in TdP (Tan et al.

1995). Conversely, women are less susceptible to

dysrhythmias in Brugada syndrome (Matsuo et al.

2001). This syndrome encompasses sudden cardiac

death due to ventricular tachydysrhythmias, and a

distinctive ECG signature (ST-segment elevation in

right precordial leads). These features have been

attributed in part to severe repolarization at the end

of phase-1 of the subepicardial AP, leading to excessive

AP shortening (all-or-none repolarization) (Yan &

Antzelevitch 1999, Tan et al. 2003).

Clinical observations suggest that male sex hormones

are involved in these sex disparities. While QTc is

indistinguishable between boys and girls before the age

of 15, it shortens in men from puberty through

adulthood, but remains unchanged in women

(Rautaharju et al. 1992). Furthermore, the sex differ-

ence in QTc in adults was abolished by castration of

men, and restored by subsequent application of testos-

terone (Bidoggia et al. 2000). Similar developmental

components exist in the dysrhythmias of LQTS and

Brugada syndrome. While boys and girls before age 15

have similar incidences of TdP in LQTS, men have a

reduced incidence of TdP from puberty through adult-

hood (Zareba et al. 1995). Conversely, the incidence of

dysrhythmias in Brugada syndrome is higher in men

than in women aged 20–60 (Matsuo et al. 2001).

Interestingly, castration of men abolished the Brugada

syndrome ECG pattern (Matsuo et al. 2003).

The cellular basis of these sex-related distinctions in

human cardiac electrophysiology is unresolved. Animal

studies demonstrate the importance of sex-related

differences in membrane current densities in sex dis-

tinctions in cardiac electrophysiology (Pham & Rosen

2002). We propose that sex disparities in sarcolemmal

ion current densities may also account for the sex

differences in ECG variables and dysrhythmia suscept-

ibility in humans. However, to date, human data on sex

differences in membrane current densities of undiseased

hearts are not available (Verkerk et al. 2005), in large

part because of limited availability of human cardio-

myocytes for experimental research (ten Tusscher et al.

2006). We studied the cellular basis of sex distinctions

in human cardiac electrophysiology and dysrhythmia

susceptibility using mathematical models of human

ventricular myocytes in which we modified ion current

densities based on experimental data obtained from

male and female hearts of various species. Thus, we

studied AP characteristics, transmural electrophysiolog-

ical heterogeneity, and susceptibility to EADs (LQTS)

and all-or-none repolarization (Brugada syndrome).

Materials and methods

Human ventricular cell models

We studied the cellular basis of sex distinctions in

human cardiac electrophysiology and dysrhythmia sus-

ceptibility using mathematical models of human ven-

tricular myocytes. To date, three distinct human

ventricular cell models exist: the Priebe–Beuckelmann

(PB) model (Priebe & Beuckelmann 1998), the

ten Tusscher–Noble–Noble–Panfilov (TNNP) model

(ten Tusscher et al. 2004), and the Iyer–Mazhari–

Winslow model (Iyer et al. 2004). The large number

of variables (67) and the small time step required for

integration make the Iyer–Mazhari–Winslow model

computationally extremely demanding, with simula-

tions running about 900 times slower than either the PB

or TNNP model (ten Tusscher et al. 2006). Therefore,

we refrained from using this model in our study.

The PB model and the TNNP model provide the basis

for the simulations in this study. The PB model was the

first model for human ventricular myocytes. It is largely

based on the Luo–Rudy phase II model for guinea pig

ventricular cells (Luo & Rudy 1994) in which formula-

tions for the major ionic currents were adjusted to the

scarce data available for human ventricular cells at that

time (Bernus et al. 2002; ten Tusscher et al. 2006). The

TNNP model is a recently developed human ventricular

cell model, which uses new formulations for all major

ionic currents based on a now much wider basis of

experimental data, largely from human ventricular cell

experiments but also from ion channel expression

experiments (for overview about human ventricular cell

models, see ten Tusscher et al. 2006). Consequent to the

differences in membrane current formulations, AP

shape and duration, AP restitution, and relative import-

ance of ionic currents in the AP generation differs

between the PB and TNNP model (ten Tusscher et al.

2006). To study sex distinctions in human cardiac

electrophysiology rather than model-based effects, we

used both the PB and TNNP model.

The PB model and the TNNP model are both

importantly based on experimental data from single

ventricular myocytes isolated from explanted human

hearts. As these myocytes were generally isolated from

midmyocardial areas of the left ventricle (LV) of male

patients (Priebe & Beuckelmann 1998), the PB model

and the midmyocardial variant of the TNNP model

(ten Tusscher et al. 2004) are, in effect, models of male

460 � 2006 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2006.01586.x

Sex and human cardiac electrophysiology Æ A O Verkerk et al. Acta Physiol 2006, 187, 459–477

midmyocardial ventricular myocytes. To study sex

disparities in human cardiac electrophysiology and the

susceptibility to dysrhythmias, we incorporated experi-

mentally observed disparities in ion current densities

between sexes and myocardial layers of various species

into these models. These disparities are discussed below

and summarized in Table 1 as conductances relative to

male midmyocardial myocytes. In the midmyocardial

TNNP model, we replaced the relatively simple intra-

cellular calcium handling by the more sophisticated

calcium dynamics of the updated Luo–Rudy model

(Faber & Rudy 2000) to increase its stability, especially

in the right ventricular (RV) models.

Sex disparities in ion current densities

Sex distinctions in human cardiac electrophysiology and

dysrhythmia susceptibility were studied using the PB and

TNNP human ventricular myocyte models in which we

modified ion current densities based on experimental

data obtained from male and female hearts of various

species. Therefore, we reviewed all studies into sex

disparities in ion current densities, conducted in single

ventricular myocytes (Table 1). These data were pub-

lished for dog, rabbit, guinea pig and mouse hearts, but

not for human hearts. L-type Ca2+ current (ICa,L) in

female subepicardium and midmyocardium is 1.32 times

that of males (Pham et al. 2002). Transient outward K+

current (Ito) in females is 0.75 times that of males in all cell

layers (Di Diego et al. 2002, Wu & Anderson 2002),

although this is not a consistent finding (Leblanc et al.

1998, Trepanier-Boulay et al. 2001). The rapid compo-

nent of the delayed rectifier K+ current (IKr) in females is

0.83 times that of males (Liu et al. 1998).

Although the ultrarapid component of the delayed

rectifier K+ current (IKur) and the ATP-regulated K+

current (IK,ATP) also exhibit sex disparities (Ranki et al.

2001, Trepanier-Boulay et al. 2001), these disparities

were not incorporated, because IKur and IK,ATP are not

present or functional under normal conditions in human

ventricular myocytes (Li et al. 1996). Similarly, we did

not modify Na+ current (INa), slow component of the

delayed rectifier K+ current (IKs), inward rectifier K+

current (IK1) and Na+-Ca2+ exchange current (INaCa),

because they are equal in male and female hearts

(Leblanc et al. 1998, Liu et al. 1998, Trepanier-Boulay

et al. 2001, Li et al. 2002) [equal INaCa was inferred

from equal baseline intracellular Na+ and Ca2+ concen-

trations (Ranki et al. 2001, Sugishita et al. 2001, Chen

et al. 2003)].

Transmural heterogeneity in ion current densities

Ventricular dysrhythmias in LQTS (Akar et al. 2002)

and Brugada syndrome (Yan & Antzelevitch 1999,

Tan et al. 2003) are in large part due to electrophys-

iological heterogeneities across the ventricular wall. To

study sex disparities in the susceptibility to these

dysrhythmias, we incorporated the differences in ion

current densities between subepicardium, midmyocar-

dium and subendocardium, as summarized in Table 1,

according to data from isolated canine ventricular

myocytes. These data were used, because data from

human are limited. ICa,L in subepicardium and mid-

myocardium is 1.32 times that in subendocardium in

females, but not in males (Pham et al. 2002). In both

sexes, Ito in subepicardium and midmyocardium is

twice that in subendocardium (Liu et al. 1993, Li

et al. 2002), while IKs in subepicardium and subendo-

cardium is 1.42 times that in midmyocardium

(Li et al. 2002). INaCa in subendocardium is 0.69

times that in the other layers (Zygmunt et al. 2000).

Although a late component of INa is 27% smaller in

subepicardium and subendocardium than in midmyo-

cardium (Zygmunt et al. 2001), this transmural het-

erogeneity was not incorporated, because such a

component is not present in the PB and TNNP

models. Similarly, no transmural heterogeneities in

INa, IKr, and IK1 were reported (Liu et al. 1993,

Zygmunt et al. 2001, Li et al. 2002).

Table 1 Membrane current magnitude of male and female myocytes

Male Female

Epicardial Midmyocardial Endocardial Epicardial Midmyocardial Endocardial

INa 1 1 1 1 1 1

ICa,L 1 1 1 1.32 1.32 1

Ito 1 1 0.5 0.75 0.75 0.375

IKr 1 1 1 0.83 0.83 0.83

IKs 1.42 1 1.42 1.42 1 1.42

IK1 1 1 1 1 1 1

INaCa 1 1 0.69 1 1 0.69

Values are current density relative to male midmyocardial myocytes.


Acta Physiol 2006, 187, 459–477 A O Verkerk et al. Æ Sex and human cardiac electrophysiology

Simulation protocols

In our single cell simulations, APs were elicited with a

2-ms, 3-nA (c. 20% suprathreshold) stimulus pulse.

Transmural conduction was studied using a heteroge-

neous linear strand of 600 transversally coupled ven-

tricular cells as diagrammed in Figure 1. This strand

represents a LV wall thickness of 1.2 cm [normal range

0.9–1.4 cm (Feigenbaum 1994)], with the transverse

orientation of cells based on data from human and

canine LV (Streeter & Hanna 1973, Greenbaum et al.

1981). The strand was stimulated from the endocardial

side by injecting a c. 20% suprathreshold stimulus of

2 ms duration into the first of 300 cells of the

endocardial type. The next 240 cells of the strand were

M type cells and the final 60 cells were epicardial type

cells. This distribution of 50% endocardial type cells,

40% M type cells and 10% epicardial type cells was

based on data from human LV (Drouin et al. 1995).

The intercellular coupling conductance (Gc) was set to

5 or 10 lS, which is well within the estimated range of

3–12 lS for the gap junctional conductance between

human ventricular myocytes (Jongsma & Wilders

2000). The cytoplasmic resistivity was set to 150 Xcm.

Models were coded using Compaq Visual Fortran

6.6C (Hewlett-Packard, Palo Alto, CA, USA) and run

on a 3-GHz Intel Pentium-4 processor workstation as a

32-bit Windows application, applying a simple and

efficient Euler-type integration scheme with a 5-ls time

step. The spatial discretization step in the strand

simulations was 20 lm, i.e. the width of a single cell,

which was treated as isopotential. Stimulus and end

effects, which were restricted to <10 cells, were

minimized by discarding data obtained from cells

1–10 and 591–600 when assessing dispersion in

repolarization time (RT). All figures show steady-state

AP characteristics, obtained at 1–7 min after onset of

stimulation.

Results

We used our ‘male’ and ‘female’ models of subepicardial,

midmyocardial, and subendocardial myocytes to assess

sex disparities in the action potential duration (APD)-

heart rate relationship, transmural APD dispersion and

susceptibility to EADs and all-or-none repolarization.

Sex differences in AP duration

In both PB and TNNP based models, simulations at

different stimulation frequencies revealed significant sex

differences in APD. Figure 2 shows superimposed APs

at 1 Hz in the female and male PB (left) and TNNP

(right) cells of subepicardial, midmyocardial, and sub-

endocardial origin. In all cell types, the APs were

significantly longer in females than males. Figure 3

summarizes the frequency dependence of APD at 90%

repolarization (APD90) of the male and female PB (left)

and TNNP (right) cells. At all frequencies, APD90 of

subepicardial, midmyocardial and subendocardial cells

was longer in females than in males. Moreover, APD90

decreased with increasing stimulus frequency in all cell

types. Linear regression analysis indicated that APD-

heart rate relationship was steeper in females, except in

subendocardial cells.

Ionic basis of sex differences in response to rate

The combined effect of the differences in ionic current

densities between male and female cells (Table 1) is a

longer AP and a steeper APD-heart rate relationship in

female subepicardial and midmyocardial but not sub-

endocardial cells (Fig. 3). To investigate the contribu-

tion of the individual ion currents involved, we carried

out simulations in which we selectively increased ICa,L

density, decreased IKr density, or decreased Ito density.

As illustrated in Figure 4 for midmyocardial cells, the

APD was importantly increased by both an increased

ICa,L density and a decreased IKr density, but not by a

decrease in Ito density. Also illustrated in Figure 4, the

steepness of the APD–frequency relationship is largely

determined by ICa,L. Changes in either IKr or Ito density

had only little effect on the steepness of this relation-

ship. This readily explains the similar slopes in male and

female cells of endocardial origin, as these cells have

identical ICa,L density (Fig. 3). The larger sensitivity to

changes in ICa,L can be explained by the high membrane

Figure 1 Diagram of linear strand model. The transmural

strand is composed of 600 human left ventricular myocytes,

which are arranged transversally and coupled by an intercel-

lular coupling conductance Gc. Cytoplasmic resistivity is set to

150 Xcm. Action potential propagation is initiated by applying

an external stimulus at the endocardial side of the strand. See

Materials and methods for details.



resistance during the early plateau phase (phase-2) of

the AP, especially at low frequency. As ICa,L is an

important current during that particular phase of the

AP, as opposed to Ito and IKr which are most important

during phase-1 and phase-3 of the AP, changes in ICa,L

result in relatively large changes in APD at lower

Figure 2 Superimposed male and female action potentials (1 Hz) in the Priebe–Beuckelmann (left) and ten Tusscher–Noble–

Noble–Panfilov (right) model of subepicardial (a), midmyocardial (b) and subendocardial (c) cells.



Figure 3 Action potential duration at 90% repolarization (APD90) vs. stimulus frequency in Priebe–Beuckelmann (left) and

ten Tusscher–Noble–Noble–Panfilov (right) model of subepicardial (a), midmyocardial (b), and subendocardial (c) male and female

cells. Straight lines are linear fits to the data (r2 > 0.85) with slope factor � SE as indicated. Note differences in ordinate scales.



frequencies and thus in a relatively steep APD–fre-

quency relationship. In addition, ICa,L does not only

exhibit voltage-dependent inactivation but also Ca2+-

dependent inactivation, which is most prominent when

calcium levels are high, i.e. at high frequencies. Thus,

the Ca2+-dependent inactivation of ICa,L further steep-

ens the APD–frequency relation (data not shown).

Sex differences in transmural electrical heterogeneity

There were significant APD90 differences between sub-

epicardial, midmyocardial and subendocardial cells in

both sexes at all stimulation frequencies (0.1–3 Hz,

Fig. 3). Figure 5 shows APs at 0.5 Hz of male (Fig. 5a)

and female (Fig. 5b) subepicardial, midmyocardial and

subendocardial PB (left) and TNNP (right) cells. In both

sexes, AP repolarization was completed first in subendo-

cardial, then subepicardial, and, finally, midmyocardial

cells. The delay in final AP repolarization between

shortest and longest AP (DAPD90) was larger in female

than in male PB (left) and TNNP (right) cells (Fig. 5c).

To investigate how these cellular differences in

(intrinsic) APD90 would translate into differences in

APD90 and RT in the LV wall, we carried out

simulations with our transmural strand model (Fig. 1).

Intercellular coupling conductance was set to 5 or

10 lS. At 10 lS, transmural conduction time was 31 or

2.6 ms mm)1, which compares well with the experi-

mental values of c. 30 and 2.1 � 0.3 ms mm)1

(mean � SEM) in canine (Akar et al. 2002) and human

(Taggart et al. 2000) hearts, respectively. Figure 6

illustrates the effects of the electrotonic interactions on

AP configuration in strands of male (Fig. 6a) or female

(Fig. 6b) PB model cells. To allow comparison, APs of

the central cells of each of the three layers of the strand

have been superimposed. Compared with isolated PB

model cells (left panels of Fig. 5a,b), the AP peak is less

pronounced and AP duration is reduced. Although

considerably smaller than in isolated cells, differences in

APD90 are still present, even at high intercellular

coupling conductance (Gc ¼ 10 lS, right panels). Fig-

ure 6c shows RT of individual cells within the strand at

a stimulus frequency of 1 Hz. RT was obtained by

summing local activation time and local APD90. The

longer APD90 of the female cells results in later

repolarization, particularly in the midmyocardial and

epicardial layers of the female strand (transmural

distance 0.6–1.2 cm). Consequently, the female strand

shows a larger dispersion in RT (DRT, defined as latest

minus earliest RT within the strand), which is also

observed at other stimulus frequencies (Fig. 6d). Similar

results were obtained in simulations with strands

composed of TNNP model cells (Fig. 7).

Sex differences in susceptibility to EADs

Early afterdepolarizations typically occur at slow heart

rates (Tan et al. 1995). In isolated ventricular myocytes,

EADs may result from moderately enhanced ICa,L

(Veldkamp et al. 2001) or reduced IKr (Pham et al.

2001). Simulations with 25% increased ICa,L or 50%

reduced IKr densities revealed significant sex differences

in AP prolongation in the PB model. Figure 8 shows

superimposed APs at 0.1 Hz in the female and male

Figure 4 Action potential duration at 90% repolarization (APD90) of midmyocardial cell vs. stimulus frequency in Priebe–

Beuckelmann (a) and ten Tusscher–Noble–Noble–Panfilov model (b). Data for ‘male’ and ‘female’ cell as in Figure 3b. Compared

with the male cell, the female cell has increased ICa,L density, decreased IKr density, and decreased Ito density (Table 1). Individ-

ual effects of increasing ICa,L density, decreasing IKr density, or decreasing Ito density are shown as open squares and triangles.

Straight lines are linear fits to the data (r2 > 0.90) with slope factor � SE as indicated.



Figure 5 (a, b) Superimposed action potentials (0.5 Hz) in Priebe–Beuckelmann (PB) (left) and ten Tusscher–Noble–Noble–Pan-

filov (TNNP) (right) model of subepicardial, midmyocardial, and subendocardial male (a) and female (b) cells. (c) Difference

between longest and shortest action potential duration (DAPD90) vs. stimulus frequency in male and female PB (left) and TNNP

(right) cells.



Figure 6 Action potential propagation in transmural strand of Priebe–Beuckelmann model cells with an intercellular coupling

conductance of 5 (left) or 10 lS (right). (a, b) Superimposed action potentials (0.5 Hz) of male (a) and female (b) cells in the centre

of the subepicardial, midmyocardial or subencocardial layer of the strand. (c) Repolarization time (RT) vs. transmural distance from

endocardium in male and female strands paced at 1 Hz. (d) Dispersion in repolarization time (DRT) vs. stimulus frequency in male

and female strands.



midmyocardial PB (left) and TNNP (right) cell resulting

from increased ICa,L (Fig. 8a) or reduced IKr (Fig. 8b).

In the PB model, the excessive prolongation in females

culminated in subthreshold ‘humps’ during phase-3

repolarization (arrows), which are markers of suscep-

tibility to the formation of EADs (Cranefield 1977,

Binah & Rosen 1992). The TNNP model did not

exhibit such humps, most likely due to its particular

ICa,L inactivation kinetics that effectively prevent

reactivation of ICa,L (see Discussion). Multicellular

simulations at physiological pacing rate (1 Hz) and

intercellular coupling conductance (10 lS) demonstra-

ted an increased sex disparity in dispersion of RT with

enhanced ICa,L (Fig. 8c), both with PB (left) and TNNP

(right) model cells. In the midmyocardial and epicardial

layers of transmural strands of female, but not male, PB

model cells EADs occurred with enhanced ICa,L (Fig. 8c,

left inset, arrows).

Figure 9 illustrates the mechanism underlying the

subthreshold humps during phase-3 repolarization

observed in isolated female PB model cells (Fig. 8a) as

well as the EADs observed in strands of female PB cells

(Fig. 8c, inset). Although Ca2+-induced inactivation of

ICa,L is more prominent in the female cell because of the

larger intracellular free calcium concentration ([Ca2+ ]i)

in the female cell (Fig. 9b), the current reactivates

(Fig. 9c) and thus produces the subthreshold humps in

the single cell (Fig. 9a) and the EADs in the strand

(Fig. 9a, inset). In the male cell (Fig. 9, left), there is also

AP prolongation, but this prolongation is not large

enough to result in subthreshold humps in the single cell

(Fig. 9a) or EADs in the strand (Fig. 9a, inset). The

increased calcium release of the female cell not only

contributes to the AP shape by modulating ICa,L, but

also by increasing the sodium–calcium exchange current

(INaCa), which results in an additional inward current

during the AP plateau phase, in particular at higher

frequencies as in the strand simulations (Fig. 9d, insets).

Additional simulations in which we selectively increased

ICa,L density, decreased IKr density, or decreased Itodensity demonstrate that the smaller female IKr facili-

tates AP prolongation, but is not crucial to the prefer-

ential occurrence of subthreshold humps and EADs in

the female cell, and that Ito only plays a minor role (data

not shown).

Sex differences in susceptibility to all-or-none

repolarization

The basis of Brugada syndrome resides in the RV

(Tukkie et al. 2004). One proposed cellular mechanism

revolves around accentuation of the AP notch (end of

phase-1) and loss of the AP dome (all-or-none repolar-

ization) in RV subepicardial myocytes with concomitant

preservation of the AP dome in subendocardium. The

resulting transmural voltage gradient causes ECG ST

elevations and reentrant tachydysrhythmias (Yan &

Antzelevitch 1999). These changes may become man-

ifest when net depolarizing current is reduced [Na+

channel mutations (Tan et al. 2003)] or net repolarizing

current is enhanced during the early phases of the AP.

Repolarizing forces in RV are larger than in LV,

because IKs density is two times larger, and Ito density

is 2.5 to four times larger in RV (Volders et al. 1999,

Di Diego et al. 2002). Accordingly, we implemented

RV models of both sexes by amplifying IKs and Itodensities by factors 2 and 4, respectively. Our simula-

tions revealed significant differences in AP configura-

tions between RV and LV. Figure 10a shows

superimposed APs at 0.5 Hz in male (left) and female

(right) TNNP models of subepicardial and subendo-

cardial RV and LV myocytes. AP duration was shorter

and phase-1 repolarization was more pronounced in

RV than LV subepicardium. The AP notch clearly was

more prominent in males than females in subepicardial

RV.

An experimental study demonstrated that more

prominent phase-1 repolarization may result in all-or-

none repolarization, when an IK,ATP agonist is

administered (Di Diego et al. 2002). Accordingly, we

incorporated IK,ATP into our models to study suscepti-

bility to all-or-none repolarization. Figure 10b shows

superimposed APs from male and female TNNP cells

before and after introduction of IK,ATP. Introducing

IK,ATP (ohmic conductance: 0.8 nS) hardly affected the

AP dome in LV of both sexes (not shown). However, in

the male RV subepicardial cell (left), IK,ATP caused all-

or-none repolarization, while it evoked only a slight

depression of the AP plateau in females (right). Because

experimental studies have reported that IK,ATP density is

2.5 times larger in females than males (Ranki et al.

2001), we repeated these simulations with increased

IK,ATP (2.0 nS) in females. These simulations still

resulted in a less prominent reduction of the dome

and abbreviation of the AP than simulations with the

small IK,ATP in males. Introduction of IK,ATP into RV

subendocardial cells (Fig. 10c) hardly affected the AP

dome in males (left) and females (right). Similar results

were obtained with the PB model (not shown). To

investigate the contribution of the individual ion

currents involved in the sex disparity in susceptibility

to all-or-none repolarization, we carried out simulations

in which we selectively increased ICa,L density,

decreased IKr density, or decreased Ito density. We

found that the occurrence of all-or-none repolarization

was critically dependent on the high Ito density in the

male subepicardial cell, and facilitated by the lower

ICa,L density and higher IKr density in the male, which

both enhance the repolarizing forces of the AP (data not

shown).



Figure 7 Action potential propagation in transmural strand of ten Tusscher–Noble–Noble–Panfilov model cells with an intercel-

lular coupling conductance of 5 (left) or 10 lS (right). (a, b) Superimposed action potentials (0.5 Hz) of male (a) and female (b) cells

in the centre of the subepicardial, midmyocardial or subencocardial layer of the strand. (c) Repolarization time (RT) vs. transmural

distance from endocardium in male and female strands paced at 1 Hz. (d) Dispersion in repolarization time (DRT) vs. stimulus

frequency in male and female strands.



Figure 8 (a, b) Superimposed action potentials of midmyocardial Priebe–Beuckelmann (PB) (left) and ten Tusscher–Noble–Noble–

Panfilov (TNNP) (right) cells (0.1 Hz) with enhanced ICa,L (a) or reduced IKr (b). Arrows indicate excessive action potential

prolongation in female cells predisposing to early afterdepolarizations. (c) Dispersion in repolarization time (DRT) vs. ICa,L current

density in transmural strands of male or female PB (left) or TNNP (right) model cells paced at 1 Hz (Gc ¼ 10 lS). Insets show

action potentials of the middle cell of the subepicardial layer at 250% ICa,L current density. Arrows indicate early after

depolarizations.



To test whether preferential all-or-none repolariza-

tion of subepicardial cells in response to enhanced net

repolarizing current was still possible in case of

electrotonic interactions occurring in the well-coupled

myocardium, we also carried out simulations with

strands composed of RV male or female cells. For these

simulations, we reduced the number of cells in the

transmural strand from 600, as used for LV, to 210 (70

in each layer), based on the RV wall thickness of

4 � 1 mm observed in the human heart (Gottdiener

et al. 1985). With the IK,ATP conductance increased to

4 nS, we found preferential loss of the AP dome in the

subepicardium of the male strand (Fig. 10d).

Discussion

Our studies provide insights into the cellular basis of sex

disparities in cardiac electrophysiology and dysrhyth-

mia susceptibility. Of note, there was general consis-

tency between the PB and TNNP human ventricular cell

models.

Sex differences in AP duration

We found that female LV cells had longer intrinsic AP

duration than their male counterparts (Figs 2 and 3),

resulting in later RT (Figs 6 and 7). Although experi-

mental findings of sex disparity in AP duration of

undiseased human hearts are not available, our results

are in agreement with experimental findings in animals

and diseased human myocardium. In mouse subepicar-

dial myocytes (Trepanier-Boulay et al. 2001, Wu &

Anderson 2002), rabbit LV subendocardial myocytes

(Valverde et al. 2003), and human failing LV myocytes

(Verkerk et al. 2005), APs were significantly longer in

females than in males. Our results are also in agreement

with the clinical observations that women have longer

QTc intervals (Bazett 1920, Merri et al. 1989,

Rautaharju et al. 1992). The longer APD of the female

cell is importantly due to its larger ICa,L density and

smaller IKr density (Fig. 4). Decreased Ito density hardly

affected the APD, consistent with the findings of several

studies using mathematical modelling (Priebe & Beuck-

elmann 1998, Winslow et al. 1999; Gima & Rudy

2002; ten Tusscher et al. 2004), and a ‘dynamic clamp’

study in which a computed Ito was introduced in real

myocytes (Sun & Wang 2005).

The female LV cells had steeper APD–frequency

relationships than males (Fig. 3), largely due to their

larger ICa,L (Fig. 4). Although not studied in detail,

experimental findings in animals provide supportive

results. In rabbits, APD at 20%, 50% and 90%

repolarization is similar in males and females at a cycle

length of 300 ms, but different at longer cycle lengths

(Valverde et al. 2003). Again, these results are in

keeping with clinical observations: a steeper QT–heart

rate relationship in women than men (Kligfield et al.

1996, Stramba-Badiale et al. 1997).

Sex differences in TdP

We found that female cells exhibit greater differences in

intrinsic APD90 between subendocardium, midmyocar-

dium and subepicardium (Fig. 5), resulting in greater

transmural dispersion of repolarization (Figs 6d and 7d).

Moreover, we found that increasing ICa,L or reducing IKr

prolonged APs significantly more in females than males

(Fig. 8a,b). Increasing ICa,L resulted in EAD formation in

female, but not male, PB cells (Fig. 8c). These findings are

in agreement with experimental findings in rabbits (Pham

et al. 2001) and support the concept of limited ‘repolar-

ization reserve’ in females (Roden 2004). In our simula-

tions, the limited ‘repolarization reserve’ of female cells

was due to their larger ICa,L density and smaller IKr

density. Taken together, these observations may explain

why female sex is an independent risk factor for TdP

(Zareba et al. 1995, Locati et al. 1998). Clinical

(Schwartz et al. 1975) and experimental (Antzelevitch

& Sicouri 1994, Akar et al. 2002) studies have produced

two theories regarding the electrophysiological basis of

TdP. One theory holds that TdP arises from triggered

activity in competing ventricular foci. Evidence stems

from experimental observations (Antzelevitch & Sicouri

1994) and computer models (Zeng & Rudy 1995)

demonstrating an enhanced potential of cardiac myo-

cytes for EADs in response to factors that prolong APD.

Under circumstances of AP prolongation, the AP may

remain long enough at plateau level to permit recovery

from ICa,L inactivation during early repolarization, with

subsequent reactivation of ICa,L, resulting in interruption

of final repolarization and transient depolarization

(January & Riddle 1989, Zeng & Rudy 1995). The other

theory emphasizes dispersion of repolarization, suggest-

ing involvement of reentrant excitation (Akar et al.

2002). Our study links the observed female predomin-

ance in TdP to both theories by demonstrating both an

enhanced susceptibility to EAD formation and a larger

transmural dispersion of repolarization in female cells.

Sex influence on the clinical phenotype of Brugada

syndrome

In RV cells, we documented more negative notch

potentials in subepicardial than subendocardial APs.

Furthermore, the most negative notch potential was

found in males. Incorporation of IK,ATP caused all-or-

none repolarization in the male, but not the female,

subepicardial cell (Fig. 9). This finding is consistent

with experimental findings in dogs, where the IK,ATP

agonist pinacidil resulted in loss of the AP dome in male



Figure 9 (a, b) Superimposed action potentials of midmyocardial male (left) or female (right) Priebe–Beuckelmann (PB) model cells

(0.1 Hz) with enhanced ICa,L (125% current density). (b) Associated intracellular free calcium concentration ([Ca2+ ]i). (c) Asso-

ciated L-type Ca2+ current. Arrows indicate peak current. (d) Associated sodium–calcium exchange current (INaCa). Insets show

action potentials of the middle cell of the subepicardial layer at 250% ICa,L current density in transmural strands of male (left) or

female (right) PB model cells paced at 1 Hz (Gc ¼ 10 lS).



but not in female ventricular myocytes (Di Diego et al.

2002). IK,ATP itself is not believed to be involved in the

dysrhythmogenesis of the Brugada syndrome, but its

introduction into our simulations mimics the experi-

mental approach of blocking inward currents or

enhancing outward currents to unmask the Brugada

syndrome (Fish & Antzelevitch 2004).

Our findings demonstrate that males are exquisitely

sensitive to disruptions of the balance between depolar-

izing and repolarizing forces during the early phases of

the AP. The loss of the AP dome following small

reductions in net depolarizing force reveals that males

have a limited ‘depolarization reserve’. In our simula-

tions, the limited ‘depolarization reserve’ of male cells

was in large part because of their high Ito density in

combination with their smaller ICa,L density and larger

IKr density. This theory of limited ‘depolarization reserve’

in males is further substantiated in Brugada syndrome,

where reductions in depolarizing force result from Na+

channel mutations which reduce INa (Tan et al. 2003)

and/or the use of Na+ channel blocking drugs (class I

antidysrhythmic drugs). Thus, the concept of limited

‘depolarization reserve’ in males may explain why the

clinical phenotype of Brugada syndrome is more prom-

inent in males than females (Matsuo et al. 2001).

Order of repolarization

In our single cell simulations, AP repolarization was

completed first in subendocardial, then subepicardial,

and, finally, midmyocardial cells (Fig. 5). Such obser-

vations, with longer APD for epicardial than endocar-

dial cells, have also been made in isolated ventricular

myocytes [e.g. by Li et al. (2002)], although the

opposite (shorter APD for epicardial than endocardial

cells) has also been reported [e.g. by Liu et al. (1993)]. If

these cells are coupled together into a transmural

strand, there is only a moderate difference in APD

between the cells of the transmural layers (Figs 6a,b and

7a,b), and the repolarization wave proceeds from

endocardium to epicardium, as illustrated by the

increase in RT from endocardium to epicardium

(Figs 6c and 7c). This seems at odds with the textbook

notion of T-wave genesis, in which repolarization is

understood to begin and be completed first in the

subepicardium. However, as recently reviewed by

Conrath & Opthof (2006) with emphasis on the human

heart, there is a growing body of experimental data

suggesting that the transmural gradient in repolariza-

tion runs from endocardium to epicardium rather than

in the opposite direction. Among these data are

transmural recordings made in canine (Anyukhovsky

et al. 1996, Janse et al. 2005) and human (Conrath

et al. 2004) hearts that show little or no variation in

APD90 or activation–recovery intervals across the

ventricular wall, resulting in a repolarization wave

travelling from endocardium to epicardium. Specific-

ally, the recent experimental study by Janse et al. (2005)

demonstrates that the T wave is determined by apico-

basal gradients in RT and not by transmural gradients.

Limitations

Computer simulation studies have improved our under-

standing of the ways in which various aspects of ion

channel function (gating and expression) may alter

cardiac excitability (Kleber & Rudy 2004, Rudy 2004),

but they also have some shortcomings (Priebe &

Beuckelmann 1998, ten Tusscher et al. 2004). Our

observation that the TNNP model exhibited no EADs,

whereas the PB model did, but only with significantly

enhanced ICa,L, points to a shortcoming of all current

human ventricular cell models. As set out in detail by

Iyer et al. (2004), these models require a dominant

voltage-dependent inactivation of ICa,L to assure AP

stability. Yet, recent experimental data show that

voltage-dependent inactivation of ICa,L is slow and

incomplete, whereas Ca2+-mediated inactivation is

strong and dominates the inactivation process (Carme-

liet 2004, Iyer et al. 2004). The artificial dominant

voltage-dependent inactivation of ICa,L tends to prevent

the formation of EADs by inhibiting reactivation of

ICa,L during the plateau phase of the AP. Moreover, the

relative absence of Ca2+-mediated ICa,L inactivation

indicates that the modulatory role of Ca2+ handling

system on the AP configuration is underestimated.

In this study, we have simulated transmural conduc-

tion of the AP using linear strands with a simple and

uniform arrangement of cells. Such simulations repre-

sent the transmural conduction of broad planar activa-

tion and repolarization wavefronts during normal

ventricular excitation. However, the complex structure

of the ventricular myocardium is much simplified and

some important determinants of transmural conduc-

tion, including current-to-load mismatch and wavefront

curvature, have been excluded from our analysis.

Our recapitulation of the male predominance in the

clinical expressivity of Brugada syndrome is based on the

hypothesis that all-or-none repolarization underlies this

disorder. However, this concept is controversial

(Meregalli et al. 2005) and it has recently been demon-

strated that selective activation slowing in the RV may

also contribute to its pathophysiology (Martini 2004,

Tukkie et al. 2004). While we have not validated this

concept in this study, it is compatible with limited

‘depolarization reserve’ in males, given that conduction

velocity is primarily determined by depolarizing forces

(Tan et al. 2001). Accordingly, selective conduction

slowing in male RV may be explained by the reported

electrophysiological properties of the RV which may



Figure 10 (a) Superimposed male (left) and female (right) action potentials of left ventricular (LV) and right ventricular (RV)

subepicardial (epi) and subendocardial (endo) ten Tusscher–Noble–Noble–Panfilov (TNNP) cells (0.5 Hz). (b, c) Superimposed RV

male (left) and female (right) action potentials of subepicardial (b) and subendocardial (c) cells under control conditions, and after

implementation of 0.8 and 2.0 nS IK,ATP. (d) Superimposed action potentials of subendocardial cell (cell no. 10 from endocardium)

and subepicardial cell (cell no. 10 from epicardium) in transmural strands of male (left) or female (right) right ventricular TNNP

model cells with 4.0 nS IK,ATP. Pacing frequency and intercellular coupling conductance were 1 Hz and 10 lS, respectively.



constitute the background against which sex disparities

in net depolarizing forces produce the male predomin-

ance of disease in Brugada syndrome. Similarly, we have

recently provided evidence that RV fibrosis may be

another element in the pathophysiology of Brugada

syndrome that may be conducive to the initiation and

maintenance of reentrant tachydysrhythmias (Coronel

et al. 2005). Subsequently, in a study including 18

Brugada syndrome patients, Frustaci et al. (2005) showed

that, despite an apparently normal heart at non-invasive

evaluation, endomyocardial biopsy detected structural

alterations in all 18 patients. Thus, structural derange-

ments may also be a backdrop for the critical reduction in

depolarizing forces and the consequent increase in dys-

rhythmia susceptibility of males in Brugada syndrome.

Conclusion

The sex-related distinctions in ECG variables and

dysrhythmia susceptibility in humans may be explained

by sex disparities in ICa,L, Ito and IKr conductances,

which result in limited ‘repolarization reserve’ in

females and limited ‘depolarization reserve’ in males.

While these concepts have now been demonstrated in

model diseases (LQTS, Brugada syndrome), they may

also apply to common diseases. For instance, clinical

observations reveal that females have an increased

dysrhythmia susceptibility in common disorders where

repolarization is impaired by electrolyte disturbances or

drug use (acquired LQTS) (Lehmann et al. 1996).

Conversely, a male predominance in dysrhythmia sus-

ceptibility and sudden death may exist in heart failure

(Kannel & Schatzkin 1985), a condition which may

reduce depolarizing forces. Whether increased dysrhyth-

mia susceptibility in heart failure consistently afflicts

males, is, however, difficult to predict because of the

constellation of opposing electrophysiological changes

in heart failure. Thus, while males may be more

vulnerable to reentrant tachydysrhythmias because of

reductions in depolarizing forces, this effect may be

offset by concurrent reductions in repolarizing forces

(Armoundas et al. 2001), which may render females

more susceptible to EADs and TdP. Still, it is clear that

insights into sex disparities in dysrhythmia susceptibility

in common disease may contribute to further refine-

ments in dysrhythmia management in inherited and

common acquired diseases.

Conflict of interest

There are no conflicts of interest.

Dr Tan was supported by a fellowship of the Royal Netherlands

Academy of Arts and Sciences (KNAW), the Netherlands Heart

Foundation (NHS-2002B191), and the Bekales Foundation.

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Cellular basis of sex disparities in human cardiac electrophysiology