A Combined Approach Using Patch-Clamp Study and Computer Simulation Study for Understanding Long QT Syndrome and TdP in Women.

Female sex is an independent risk factor for development of torsade de pointes (TdP)-type arrhythmias in both congenital and acquired long QT syndrome (LQTS). In females, QT(c) interval and TdP risk vary during the menstrual cycle and around delivery. Biological experiments including single-cell current recordings with the patch-clamp technique and biochemical experiments show that progesterone modulates cardiac K(+) current and Ca(2+) current via the non-genomic pathway of the progesterone receptor, and thus the cardiac repolarization duration, in a concentration-dependent manner. Incorporation of these biological findings into a computer model of single-cell and coupled-cell cardiomyocytes simulates fluctuations in QT(c) interval during the menstrual cycle with reasonable accuracy. Based on this model, progesterone is predicted to have protective effects against sympathetic nervous system-induced arrhythmias in congenital LQTS and drug-induced TdP in acquired LQTS. A combined biological and computational approach may provide a powerful means to risk stratify TdP risk in women.

INTRODUCTION

A growing body of evidence suggests that clinical arrhythmia syndromes emerge as a result of complicated interactions of multiple endogenous and environmental factors. A combined approach using patch-clamp study and computer simulation study is a powerful means for investigating the influence of multiple interacting factors on the development of clinical symptoms. In this mini-review, we will discuss our recent work using a combined biological and computational approach to predict arrhythmic risks in women.

ARRHYTHMIAS IN LONG QT SYNDROME (LQTS) IN WOMEN

LQTS is a cardiac arrhythmia syndrome characterized by prolonged QT intervals on the 12-lead surface electrocardiogram, polymorphic ventricular tachyarrhythmias with unique morphology, called torsade de pointes (TdP), and syncope and sudden death. Experiments using multicellular wedge prepatnion indicate that TdP is triggered by early afterdepolarization (EAD) followed by intramural phase 2 reentry, which is based on hetelogeneous prolongation myocardial action potential duration (APD) [3]. APD prolongation is caused by either suppression of outward currents including transient outward current (Ito), and rapidly-activating and slowly-activating delayed rectifier K+ current (IKs and IKr), or/and enhancement of inward currents including L-type Ca2+ current (ICa,L) and persistent Na+ current (INa).

LQTS occurs as a congenital form or an acquired form. In both congenital and acquired LQTS, female sex is an independent risk factor for the development of TdP [1, 2]. In females, there are dynamic fluctuations in QTc interval and the risk of TdP during the menstrual cycle [4]. Although several previous studies did not find QTc interval differences among the different menstrual phases [5, 6], a recent study analyzing various parameters of cardiac repolarization finds that repolarization duration is shorter in the luteal phase than in the follicular phase by about 10 msec [6]. Ibutilide is a class III antiarrhythmic agent that prolongs QTc interval in a dose-dependent manner, and is used for termination of atrial fibrillation and atrial flutter. QTc prolongation induced by ibutilide is the greatest during menses (63 msec), intermediate in ovulation (59 msec), and the least in the luteal phase (53 msec) [5]. In these studies [5, 7], serum sex hormone level was determined: serum progesterone level was higher in the luteal phase than in the follicular phase, during menses, and in ovulation, while serum 17β-estradiol level was not significantly different between the luteal phase and the follicular phase. Thus, progesterone is suggested to be responsible for differences in cardiac repolarization duration and in ibutilide-induced QTc prolongation during the menstrual cycle.

In post-menopausal women, although earlier studies report conflicting data for effects of hormone replacement therapy on QTc interval [8-10]. a recent study consisting of a large study population indicates that hormone replacement therapy with estrogen alone causes slight but significant prolongation of QTc interval by about 2 msec, while combinational hormone replacement therapy with estrogen and progestin consistently shortens QTc interval by about 1 msec [11]. Effects of pregnancy in LQTS patients were also examined [12, 13]. In careful survey of arrhythmia events in congenital LQTS patients around delivery, new-onset of arrhythmia events increased postpartum where progesterone level falls dramatically compared to before or during pregnancy [12]. Taken together, the luteal hormone, progesterone, is strongly suggested to have protective effects against long QT-associated arrhythmias.

GENOMIC EFFECTS OF PROGESTERONE ON CARDIAC ION CHANNELS

Progesterone belongs to lipophilic gonadal steroid hormone family, whose canonical pathway is to permeate into cell across surface membrane, binds to intracellular receptor, translocates into the nucleus as a ligand/receptor complex form, and binds to a gene containing a hormone responsive element (Fig. ) [14-16]. In addition to this “genomic action”, for the last decade sex hormones have been shown to exhibit rapid actions which cannot be explained by genomic action and are referred to as “non-genomic action” (Fig. ) [17-20]. Non-genomic action takes place in a membrane-delimited manner: PI3-kinase/Akt-dependent activation of endothelial nitric oxide synthase (eNOS) [21, 22] and activation of MAP-kinase [23, 24] are the two most well characterized signaling pathways.

Previous studies of effects of progesterone on cardiac ion channels have mostly dealt with its genomic actions. Song et al. [25] examined effects of gonadal steroids on expression of transient outward current channels, KV4.3, using a myometrium heterologous expression as a model system. They found that 4 days-injection of 17β-estradiol (50 μg/ml) decreased expression of KV4.3, whereas injection of progesterone (3 mg/ml) did not affect KV4.3 expression. The α1C subunit of the L-type Ca2+ current (ICa,L) channel can be detected as a 240 kDa long form (α1C long) and a 190 kDa short form (α1C short). In myometrium, 17β-estradiol decreased the long α1C form/short α1C form (L/S ratio), while progesterone increased the L/S ratio; in brain or heart, either 17β-estradiol or progesterone did not change the L/S ratio [26]. Thus, the genomic effects of progesterone on cardiac repolarization are currently undefined and cannot explain a protective effect of progesterone against TdP risk.

NON-GENOMIC EFFECTS OF PROGESTERONE ON CARDIAC ION CURRENTS

Major currents determining cardiac repolarizaion are IKs, IKr, and ICa,L in human and guinea-pig. Ito and ICa,L are critical in mouse and rat [27, 28]. Thus, we used cardiac myocytes isolated from guinea pig left ventricle to investigate acute effects of progesterone. Sympathetic nervous system (SNS) stimulation is a critical triggering factor for TdP in LQTS patients [29], and thus we examined both the basal condition and the SNS stimulation-mimicked condition with isoproterenol application or with intracellular dialysis of cAMP and okadaic acid (OA). Progesterone at a concentration of 100 nM shortened APD both in the basal condition and the SNS-stimulated condition.29 Progesterone-induced APD shortening is via the non-genomic pathway, since progesterone-induced APD shortening was observed within a few minutes, reached steady-state within 10 min, and was inhibited by a specific progesterone receptor inhibitor, mifepristone (1 μM).

The ionic mechanism underlying APD shortening by progesterone is to modulate IKs and ICa,L, but not IKr. In the basal condition, progesterone enhanced IKs in a concentration-dependent manner with an EC50 value of 2.7 nM, while progesterone did not significantly affect ICa,L (Fig. ) [30]. SNS-stimulation caused enhancement of both ICa,L and IKs. Further application of progesterone reduced ICa,L to the level before cAMP and OA application, while it did not significantly change IKs [30]. The IC50 value for ICa,L suppression was 29.9 nM (Fig. ).

The biophysical mechanism for regulation of IKs and ICa,L is different. The effects of progesterone on IKs were frequency- and voltage-independent [30]. In contrast, progesterone caused a positive shift in the ICa,L activation curve and a negative shift in the inactivation curve [30]. Computer simulation analysis showed that changes in current conductance without changes in current kinetics reproduced the effects of progesterone on IKs observed in biological experiments. Changes in voltage dependency alone with no change in current conductance reproduced the effects of progesterone on ICa,L with a high accuracy. Thus, effects of progesterone on IKs are mainly to alter current conductance and modulate ICa,L by affecting current kinetics.

Despite distinct biophysical mechanism for IKs and ICa,L regulation, the principal mediator for both IKs enhancement in the basal condition and ICa,L suppression in the SNS-stimulated condition appears to be nitric oxide (NO), since both were abolished by nitric oxide (NO) trappers and eNOS inhibitors [31, 32]. However, the mechanism by which NO modulates IKs and ICa,L appears to be different. ICa,L suppression by progesterone was abolished by an inhibitor of soluble guanylyl cyclase (sGC), indicating that ICa,L is regulated by progesterone via a NO/sGC/cGMP axis (Fig. ) [33]. Antagonistic action of cAMP and cGMP for ICa,L has been demonstrated, which appears to vary among species [34]. In rabbit and frog ventricular myocytes, cGMP antagonizes cAMP effects by promoting cAMP breakdown by activating cGMP-dependent phosphodiesterase (PDE2) [34]. In guinea-pig and rat ventricular myocytes, cAMP-dependent protein kinase (PKA) phosphorylates the α-subunit of ICa,L and enhances ICa,L only in the presence of A-kinase anchoring protein (AKAP) [34]. cGMP-dependent protein kinase (PKG) phosphorylates both the α-subunit and the β-subunit of ICa,L [35]. Phosphorylation of the α-subunit by PKG does not affect ICa,L, likely due to the absence of AKAP, while phosphorylation of the β-subunit antagonizes the effect of the α-subunit phosphorylation by PKA [35]. In addition, the inhibition of PDE3 by cGMP to enhance the cAMP-induced activation and facilitation of ICa,L, and activation of protein phosphatase via cGMP-PKG signaling pathway to suppress the cAMP-mediated facilitation may contribute to the complicated interaction of cAMP and cGMP in the heart.

On the other hand, IKs enhancement was not inhibited by a sGC inhibitor, but was inhibited by a thiol-alkylating reagent, N-ethylmaleimide, and a reducing reagent, di-thiothreitol [33]. These data suggest that cGMP-independent mechanisms, possibly protein s-nitrosylation, play a role for IKs enhancement (Fig. ) [33]. Protein s-nitrosylation is the direct NO transfer to the thiol residue of Cys, is highlighted as a novel mechanism of protein post-translational modification [36, 37], and occurs independent of cAMP. Thus, it is possible that progesterone regulates IKs in the basal condition and ICa,L only in the SNS-stimulated condition. However, it remains to be proven if the IKs channel is indeed s-nitrosylated. If that is the case, it is also undetermined whether the α-subunit, KCNQ1, or the β-subunit, KCNE1, is the target of s-nitrosylation, what is the underlying mechanism for specific s-nitrosylation of KCNQ1 or KCNE1, and how s-nitrosylation induces IKs channel activation.

COMPUTATIONAL SIMULATION OF THE EFFECTS OF PROGESTERONE

QTc interval and TdP risk are regulated by various factors, including SNS status, heart rate, medications, serum electrolyte level, and others. Our biological experiments suggest progesterone as an additional major factor that modulates QTc interval and TdP risk. Since progesterone level varies during the menstrual cycle and around delivery, progesterone effects may contribute to the fluctuation of QTc interval and TdP risk during the menstrual cycle and pregnancy. Since a computational approach is especially powerful to simulate these changes, our first challenge was to investigate if incorporating effects of progesterone in the cardiac APD computer model reproduces fluctuation of APD during the menstrual cycle.

We incorporated effects of progesterone obtained in our biological experiments in the Faber-Rudy model of the guinea pig myocyte [38]. Since reported progesterone level in women is ~2.5 nM in the follicular phase and ~40.6 nM in the luteal phase [39], we incorporated effects of progesterone at 2.5 nM and at 40.6 nM. The model predicts that progesterone at 40.6 nM shortens APD by 3.7 % under basal conditions and 4.6 % under SNS-stimulated conditions compared to APD at 2.5 nM progesterone (Fig. ) [30]. Clinically observed QT intervals are shorter by about 2.4%-2.8 % in the luteal phase than in follicular phase [5], and so the APD shortening predicted in the model (3.7-4.6 %) fits well with the observed fluctuation in QT interval during the menstrual cycle in women.

Effects of progesterone in a single cell do not necessarily predict the effect at the multi-cell level, organ level, or in vivo level. As a first step to simulate effects of progesterone in higher dimensions, we constructed a coupled-cells model, in which 100 cardiomyocytes are electrotonically connected with simulated resistances between them to represent gap-junctions. We then investigated the effects of progesterone and SNS in simulated coupled tissue and computed virtual electrograms from simulated gradients of depolarization and repolarization. Simulations suggest that during the luteal phase when progesterone = 40.6 nM, maximal SNS may additionally shorten QT interval by 12.2 % (Fig. ) [30]. These simulations support the notion that progesterone may exert protective QT shortening effects under conditions on SNS.

PREDICTED EFFECTS OF PROGESTERONE AGAINST ARRHYTHMIA

Since the model reproduces the effects of progesterone on APD in patch-clamp experiments and QTc variation during the menstrual cycle in women with a good accuracy, our next step was to utilize this model to predict the effects of progesterone on LQTS-associated arrhythmia susceptibility. To examine the effects on SNS-induced arrhythmias, we used the D76N KCNE1 mutation linked to congenital LQTS5. IKs exhibits accumulation in the pre-open state during the rapid heart rates, resulting in action potential adaptation [40]. SNS stimulation enhances ICa,L to increase Ca2+ influx [41]. SNS stimulation also enhances IKs [42, 43] that counter-balances ICa,L enhancement and maintains APD within a certain range [44]. In LQTS1 and LQTS5, IKs channel disturbance results in dysfunction of action potential adaptation to rapid heart rates and response to SNS stimulation. The D76N KCNE1 mutation reduces the current and renders the IKs channel insensitive to β-adrenergic stimulation [45], thus probands carrying D76N KCNE1 mutation readily develop TdP with SNS stimulation at rapid heart rates [46]. In the absence of progesterone, the mutant model cells are unable to adapt to the fast pacing frequency because IKs fails to increase in response to the SNS stimulation (Fig. ) [30]. Interestingly, both in the single-cell and coupled-cell model in the presence of progesterone at 2.5 nM, some improvement is observed; in the presence of 40.6 nM, a failed SNS stimulation response is compensated for by the action of progesterone alone to increase IKs (Fig. ) [30]. Thus, enhancement of IKs in the absence of SNS stimulation, and inhibition of cAMP-induced ICa,L by progesterone improve action potential adaptation, which is dependent on progesterone level. These simulations suggest a mechanism for SNS-related arrhythmic risk varies during the menstrual cycle in women.

Drug-induced TdP is believed to occur by blockade of the human ether-a-go-go related gene (hERG) channel by drugs with various structures [47], and at slow heart rates. In a simulation, severe EADs were induced by 50% block of IKr at a slow heart rate (30 bpm) (Fig. ). At 2.5 nM of progesterone, some improvement is observed (middle panel); at 40.6 nM of progesterone, the EADs are abolished and the action potential morphology is normalized (Fig. ). Thus, progesterone is predicted to have protective effects against drug-induced arrhythmias, which also fluctuate during the menstrual cycle. Progesterone does not have apparent effects on IKr (data not shown), and thus predicted protection against drug-induced EAD may be attributed to an increase in repolarization reserve by IKs enhancement [48].

CONCLUSION

Our patch-clamp experiment demonstrates that the non-genomic effect of the sex hormone progesterone constitutes a novel regulatory mechanism of cardiac repolarization. Serum progesterone level fluctuates during the menstrual cycle: within this level, progesterone modulates IKs and ICa,L and, therefore, is partly responsible for the cyclic changes in QTc interval and TdP risk during the menstrual cycle. A computational approach allows for simulation of multi-factorial and periodical phenomenon. Incorporation of progesterone effects observed in our biological study into the computational model reproduces cyclic changes in QTc interval, and predicts dose-dependent protective effects of progesterone against SNS-stimulation-induced and drug-induced arrhythmias. This approach provides a first step to risk stratify TdP arrhythmias in women. To improve this approach, further efforts are certainly needed, which include the elucidation of; (1) the effects of sex hormones other than progesterone, including various estrogen metabolites; (2) genomic effects of progesterone and estrogens; and (3) simulation at the organ and in vivo level.