CLINICAL PERSPECTIVE

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 112/12/1701    most recent CIRCULATIONAHA.104.523217v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bai, C.-X. Articles by Furukawa, T. Search for Related Content PubMed PubMed Citation Articles by Bai, C.-X. Articles by Furukawa, T. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB TESTOSTERONE Related Collections Arrythmias-basic studies Ion channels/membrane transport

(Circulation. 2005;112:1701-1710.)
© 2005 American Heart Association, Inc.

Arrhythmia/Electrophysiology

Nontranscriptional Regulation of Cardiac Repolarization Currents by Testosterone

Chang-Xi Bai, MD, PhD; Junko Kurokawa, PhD; Masaji Tamagawa, BE; Haruaki Nakaya, MD, PhD; Tetsushi Furukawa, MD, PhD

From the Department of Bio-informational Pharmacology (C.-X.B., J.K., T.F.), Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan, and the Department of Pharmacology (M.T., H.N.), Chiba University Graduate School of Medicine, Chiba, Japan.

Correspondence to Tetsushi Furukawa, MD, PhD, Department of Bio-informational Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail t_furukawa.bip{at}mri.tmd.ac.jp

Received November 22, 2004; revision received June 27, 2005; accepted July 1, 2005.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Women have longer QT_c intervals than men and are at greater risk for arrhythmias associated with long QT_c intervals, such as drug-induced torsade de pointes. Recent clinical and experimental data suggest an important role of testosterone in sex-related differences in ventricular repolarization. However, studies on effects of testosterone on ionic currents in cardiac myocytes are limited.

Methods and Results— We examined effects of testosterone on action potential duration (APD) and membrane currents in isolated guinea pig ventricular myocytes using patch-clamp techniques. Testosterone rapidly shortened APD, with an EC₅₀ of 2.1 to 8.7 nmol/L, which is within the limits of physiological testosterone levels in men. APD shortening by testosterone was mainly due to enhancement of slowly activating delayed rectifier K⁺ currents (I_Ks) and suppression of L-type Ca²⁺ currents (I_Ca,L), because testosterone failed to shorten APD in the presence of an I_Ks inhibitor, chromanol 293B, and an I_Ca,L inhibitor, nisoldipine. A nitric oxide (NO) scavenger and an inhibitor of NO synthase 3 (NOS3) reversed the effects of testosterone on APD, which suggests that NO released from NOS3 is responsible for the electrophysiological effects of testosterone. Electrophysiological effects of testosterone were reversed by a blocker of testosterone receptors, a c-Src inhibitor, a phosphatidylinositol 3-kinase inhibitor, and an Akt inhibitor. Immunoblot analysis revealed that testosterone induced phosphorylation of Akt and NOS3.

Conclusions— The nontranscriptional regulation of I_Ks and I_Ca,L by testosterone is a novel regulatory mechanism of cardiac repolarization that can potentially contribute to the control of QT_c intervals by androgen.

Key Words: nitric oxide • long-QT syndrome • testosterone • potassium current • ion channels

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Sex-related differences have been reported in the propensity for cardiac arrhythmias related to prolonged cardiac repolarization.^1–3 Female gender is an independent risk factor for syncope and sudden death in the congenital long-QT syndrome.^1–3 Women are more prone to develop torsade de pointes than men in response to QT-prolonging drugs, with 65% to 75% of drug-induced torsade de pointes occurring in women.^4–6 Higher susceptibility of females to the congenital long-QT syndrome and drug-induced torsade de pointes is associated with a sex-related difference in ventricular repolarization in the hearts.^7–9

Sex differences in cardiac repolarization are influenced by age. At birth, the QT_c interval is quite similar between men and women.^8–10 On arrival of puberty, the duration of QT_c intervals in boys shortens, which leaves adult women with longer QT_c intervals than adult men. The QT_c interval in men then gradually increases until approximately 60 years of age, when QT_c intervals approach those of women.^8–10 As sex hormone level elevates during puberty, the influence of sex hormones on differences in QT_c intervals between men and women has been documented.^8–10 Bidoggia et al,¹¹ in fact, showed that women with virilization exhibit shorter and faster repolarization times than normal women and castrated men. Pham et al¹² showed in male rabbits that a higher serum testosterone level, but not an estradiol level, is associated with shorter action potential duration (APD) and a lower incidence of proarrhythmias by a K⁺ channel blocker, dofetilide. They also showed in female rabbits that testosterone diminishes the proarrhythmic effects of dofetilide.¹³ Taken together, these data suggest that testosterone is an important regulator of sex-related differences in ventricular repolarization and the propensity to arrhythmias. However, the mechanisms that underlie the effects of testosterone on ventricular repolarization remain unknown.

Signaling of gonadal steroids such as testosterone and estradiol has traditionally been identified as a transcriptional control of target genes via the binding of complexes of nuclear receptors and ligands to the genomic consensus sequence in reproductive organs ("transcriptional mechanism").^14,15 Recently, several biological actions of gonadal steroids that are too rapid to be compatible with transcriptional mechanisms have been identified in nonreproductive organs.^16,17 In cardiac myocytes, testosterone increases expression of the ß₁-adrenergic receptor, the L-type Ca²⁺ channels, and the Na⁺-Ca²⁺ exchanger in mRNA.¹⁸ On the other hand, testosterone does not change mRNA expression of rat ERG (ether-a-go-go-related gene) but increases the current density of the rapidly activating component of the delayed rectifier K⁺ current (I_Kr) with changes in channel gating kinetics.¹⁹ Thus, testosterone modulates cardiac repolarization via both a transcriptional and a nontranscriptional mechanism. Testosterone rapidly induces dilatation of blood vessels and positive inotropism of cardiac muscle, which are abolished by pretreatment with a nitric oxide synthase (NOS) inhibitor, N^G-nitro-L-arginine methyl ester. These data implicate the interaction of the nongenomic action of testosterone and the nitric oxide (NO) system in cardiovascular regulation.¹⁶ We recently reported that NO shortens APD by enhancing the slowly activating component of delayed rectifier K⁺ currents (I_Ks) and inhibiting L-type Ca²⁺ currents (I_Ca,L).^20,21 These findings prompt us to examine whether testosterone also modulates cardiac repolarization via an NO-dependent mechanism. Our data indicate that testosterone shortens APD by modulating both I_Ks and I_Ca,L via a nontranscriptional mechanism.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
The investigation was conducted in accordance with the rules and regulations of the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University.

Patch Clamp
Single ventricular myocytes were harvested from hearts of adult guinea pigs of either sex (n=36, white Hartrey) as described previously.²² Action potentials and membrane currents were recorded with the perforated configuration of the patch-clamp technique with an Axopatch 200B amplifier (Axon Instruments). Action potentials were elicited by passing depolarizing current pulses (<2 ms in duration) of suprathreshold intensity with a frequency of 1 Hz. To record I_Ca,L, a prepulse (100 ms) was applied to –40 mV from a holding potential (V_h) of –80 mV to inactivate the Na⁺ channels and the T-type Ca²⁺ channels, followed by a 200-ms test pulse (V_t) to 0 mV at 1 Hz. I_Ks were elicited by 3.5-second V_t to 50 mV from a V_h of –40 mV at 0.1 Hz or by 0.5-second V_t at 1 Hz in some experiments. All experiments were done at 36±1°C. Compositions of pipette solutions and bath solutions used are described in the Data Supplement (available at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.104.523217/DC1). Amphotericin B (600 µg/mL; Sigma-Aldrich) was used in pipette solution to achieve patch perforation. The series resistance was 15.7±1.7 M, the capacitance time constant was 2.5±0.3 ms, and the membrane capacitance was 150±13 pF (n=119).

Recordings of Monophasic Action Potentials and Surface ECG in Isolated Langendorff-Perfused Hearts
Recordings of monophasic action potentials (MAPs) and surface ECGs in isolated guinea pig hearts were done as described previously.²³ Retrograde perfusion was maintained at a constant flow (10 to 12 mL/min) with modified Krebs-Henseleit solution containing (in mmol/L) NaCl 119, KCl 4.8, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.0, glucose 10, and NaHCO₃ 24.9 and equilibrated with 95% O₂/5% CO₂ (pH 7.4, 37°C). MAP was recorded with a suction electrode, and the ECG was obtained through a contact Ag-AgCl lead on the left ventricle.

Immunoblot Analysis
Immunoblot analyses were performed as described previously (see Data Supplement).²⁴ Briefly, cell lysates or tissue homogenates that contained 20 µg of protein were applied to SDS/7.5% acrylamide gel, transferred to PVDF membranes, and subjected to immunoblot analysis by incubation with an anti-NOS3 antibody (Zymed), an anti-Akt antibody (Cell Signaling), an anti-phospho-NOS3 antibody (Zymed), or an anti-phospho-Akt antibody (Cell Signaling) followed by incubation with a horseradish peroxidase–conjugated anti-mouse IgG (DAKO Japan Co. Ltd) or an anti-rabbit IgG (DAKO Japan). Proteins were detected with the advance enhanced chemiluminescence system (Amersham Biosciences).

Reagents
Chromanol 293B was supplied by Hoechst. E-4031 was purchased from Eisai Co. Ltd; testosterone and W7 from Wako; nisoldipine, S-methylisothiourea (SMTU), N-acetyl-L-cysteine (LNAC), 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide (carboxy-PTIO), S-methyl-L-thiocitrulline (SMTC), L-N₅-(1-lminoethy)ornithine (L-NIO), sodium nitroprusside (SNP), and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-1 hydrochloride (LY-294,002), and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-1 (ODQ) from Sigma-Aldrich; SH-6 from Merck; and nilutamide from TOCRIS. E-4031, nilutamide, W7, SMTU, LNAC, SMTC, L-NIO, and SNP were dissolved in distilled water. Testosterone was prepared as a 5-mmol/L stock solution in ethanol. Chromanol 293B, nisoldipine, carboxy-PTIO, SH-6, LY-294,002, and ODQ were stored as 100-, 10-, 200-, 20-, 60-, and 20-mmol/L stock solutions in DMSO, respectively. They were dissolved in bath solution to achieve a final concentration described in the text. The final concentration of DMSO (0.05% [vol/vol]) or ethanol (0.01% [vol/vol]) was confirmed to have no significant effects on membrane currents.

Data Analysis
All values are presented as mean±SE. Statistical significance among repeated measures in the control experiments, after testosterone, and after washout or addition of various inhibitors was evaluated by repeated-measure nonparametric Friedman test, those in electrophysiological parameters between males and females by unpaired nonparametric Mann-Whitney test, those between control and various drugs by paired nonparametric Wilcoxon test, those against time-dependent changes in current amplitude (time control) by 2-way ANOVA, and others by multiple comparison with Kruskal-Wallis test followed by Dunn’s multiple comparison test. A probability value less than 0.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Testosterone Shortened APD in a Dose-Dependent Manner
In ventricular myocytes isolated from female guinea pigs, testosterone shortened APD in a dose-dependent manner (Figure 1A). Figure 1B depicts dose-response curve for APD at 20% repolarization (APD₂₀; Figure 1B, panel a) and that for APD at 90% repolarization (APD₉₀; Figure 1B, panel b). The EC₅₀ value was 5.5±0.3 nmol/L for APD₂₀ (n=5) and 5.8±0.3 nmol/L for APD₉₀ (n=5). After the effects of testosterone (100 nmol/L) on APD had reached a quasi-steady state on 10-minute treatment with testosterone, application of a blocker of testosterone receptors, nilutamide (1 µmol/L), fully reversed APD shortening (Figure 1C), which indicates that testosterone-induced shortening of APD was via testosterone receptors.

View larger version (28K): [in this window] [in a new window]   Figure 1. Effects of testosterone on APD. A, Representative traces of action potentials in the absence or presence of testosterone at concentrations of 0.1, 1, 10, and 300 nmol/L. cont. indicates control. B, Dose-response curves for shortening of APD20 (a) and APD90 (b) by testosterone. Continuous lines are results of Hill’s fitting in the form of the following equation: Relative current=Ax[1+ (EC50xtestosterone)–n]–1, where A is the maximum response and n is the Hill coefficient. C, Effects of an inhibitor of testosterone receptors, nilutamide (1 µmol/L), on APD shortening. Left panels in this and subsequent figures for APD data represent averaged APD20 (closed circles) or APD90 (open circles) plotted against time after start of experiments. The time of drug application is shown by the lines at the top of the graph. *P<0.05 vs control; P<0.05 vs testosterone. Right panel depicts representative traces of action potentials at the times indicated by italic lowercase letters (a, b, c) in the left panel.

Effects of Testosterone on I_Ca,L, I_Ks, and I_Kr
To explore which ion currents are targets of testosterone, we examined effects of testosterone on I_Ca,L, I_Ks, and I_Kr, major ionic currents determining APD in guinea pig ventricles. Testosterone (100 nmol/L) suppressed I_Ca,L (Figure 2A) and enhanced I_Ks (Figure 2B), whereas it barely affected I_Kr (data not shown). Although we recorded I_Ks elicited by 3.5-second test pulses at 0.1 Hz to exaggerate the amplitudes unless otherwise noted, we confirmed similar enhancement of I_Ks elicited by shorter pulses (0.5 second) at 1 Hz, which is a more physiologically normal condition (Figure 2C). Suppression of I_Ca,L and enhancement of I_Ks by testosterone were completely inhibited by nilutamide (1 µmol/L; Figure 2). I_Ca,L suppression and I_Ks enhancement by testosterone were dose-dependent, with a significantly lower EC₅₀ value for enhancement of I_Ks than for suppression of I_Ca,L (Figure 3).

View larger version (29K): [in this window] [in a new window]   Figure 2. Effects of testosterone on membrane currents. A, Effects of testosterone (100 nmol/L) and subsequent application of nilutamide (1 µmol/L) on ICa,L. Left panels in this and following figures for ICa,L data represent averaged current density of inward peak ICa,L plotted against time after start of experiments. ICa,L was continuously elicited at 1 Hz as indicated in Methods. *P<0.05 vs control; P<0.05 vs testosterone. Right panels depict representative superimposed current traces recorded at the times indicated by italic lowercase letters in the left panel. B, C, Effects of application of testosterone (100 nmol/L) and subsequent application of nilutamide (1 µmol/L) on IKs. Left panels in this and following figures for IKs data represent averaged current density of IKs plotted against time after start of experiments. IKs was continuously elicited at 0.1 Hz (B) or 1 Hz (C) as indicated in Methods. *P<0.05 vs control; P<0.05 vs testosterone. Right panels depict representative current traces recorded at the times indicated by italic lowercase letters in the left panel.

View larger version (23K): [in this window] [in a new window]   Figure 3. Dose-dependence of ICa,L suppression and IKs enhancement by testosterone. A, Dose-response curve for suppression of ICa,L by testosterone in 5 experiments. Continuous line is the result of Hill’s fitting. Obtained EC50 value was 40.3±5.9 nmol/L. Inset depicts representative traces of ICa,L recording in the absence or presence of testosterone at 10 or 300 nmol/L. B, Dose-response curve for enhancement of IKs by testosterone in 5 experiments. EC50 value was 1.1±0.1 nmol/L. Inset depicts representative traces of IKs recording.

Next, we examined effects of testosterone on APD and membrane currents in cardiomyocytes isolated from male guinea pigs. In cells from males, baseline parameters and dose-dependent effects of testosterone on APD or membrane currents (I_Ca,L,I_Ks) were not significantly different from those in female cells (Data Supplement Tables I and II and Figure I). In the following experiments, therefore, only data obtained from female guinea pig cells are presented.

Relative Contribution of I_Ca,L Suppression and I_Ks Enhancement to APD Shortening by Testosterone
To examine the relative contribution of I_Ca,L suppression and I_Ks enhancement to APD shortening by testosterone, we used an I_Ca,L inhibitor, nisoldipine, and/or an I_Ks inhibitor, chromanol 293B. Nisoldipine is reported not to affect delayed rectifier K⁺ currents at 10 µmol/L.²⁵ Chromanol 293B is also reported to almost completely inhibit I_Ks, with minor effects on the transient outward current (I_to), at 10 µmol/L.²⁶ We confirmed that chromanol 293B at 10 µmol/L barely affected I_Ca,L or I_Kr in our experimental conditions (Data Supplement Figure II). Cardiac myocytes were preincubated with nisoldipine (3 µmol/L) and/or chromanol 293B (10 µmol/L), and subsequently, the effects of testosterone were examined (Figure 4; Data Supplement Figure III). For testosterone at 1 nmol/L, preincubation with nisoldipine did not affect the magnitude of APD shortening, whereas preincubation with chromanol 293B alone or chromanol 293B plus nisoldipine abolished APD shortening by testosterone (Figures 4A and 4C). For testosterone at 100 nmol/L, preincubation with nisoldipine diminished APD shortening by 37.2±1.1% for APD₂₀ and 38.6±2.2% for APD₉₀, and preincubation with chromanol 293B diminished it by 59.8±1.5% for APD₂₀ and 56.7±1.4% for APD₉₀. Both blockers together almost completely abolished APD shortening by testosterone (89.2±0.8% for APD₂₀ and 90.1±1.4% for APD₉₀; Figures 4B and 4C). These data suggest that testosterone-dependent modulation of I_Ks and I_Ca,L accounts for APD shortening induced by testosterone; at a low concentration of testosterone, APD shortening is mainly due to enhancement of I_Ks, whereas at a high concentration, both I_Ks enhancement and I_Ca,L suppression contribute to APD shortening.

View larger version (31K): [in this window] [in a new window]   Figure 4. Effects of nisoldipine and chromanol 293B on APD shortening. A, B, Representative recordings of action potentials in the absence or presence (arrows) of testosterone at 1 nmol/L (A) and 100 nmol/L (B). Panels marked "a" are in the control state, "b" panels are in the presence of nisoldipine (3 µmol/L), "c" panels are in the presence of chromanol 293B (10 µmol/L), and "d" panels are in the presence of both nisoldipine and chromanol 293B. C, Percent shortening in APD20 (panel a) and APD90 (panel b) obtained from 7 experiments. *P<0.05.

Effects of Testosterone Are Mediated by Actions of NO
We have previously reported that NO shortens APD by suppressing I_Ca,L and enhancing I_Ks,²¹ as does testosterone. We hypothesized that the electrophysiological effects of testosterone are mediated by NO actions. After APD shortening by testosterone (100 nmol/L) had reached a quasi-steady state, it was completely reversed by an NOS inhibitor, SMTU (1 µmol/L; Figure 5A), and by the NO scavengers LNAC (1 mmol/L; Figure 5B) and carboxy-PTIO (100 µmol/L; Data Supplement Figure IV). Similarly, I_Ca,L suppression and I_Ks enhancement by testosterone were reversed by SMTU (1 µmol/L; Figures 5C and 5E), LNAC (1 mmol/L; Figures 5D and 5F), and carboxy-PTIO (100 µmol/L; Data Supplement Figure VI). These data suggest that the effects of testosterone on APD, I_Ca,L, and I_Ks are mediated by an increase in cytosolic NO.

View larger version (34K): [in this window] [in a new window]   Figure 5. Role of NO in testosterone-induced APD shortening, ICa,L suppression, and IKs enhancement. A, Effects of an NOS inhibitor, SMTU (1 µmol/L), on APD shortening by testosterone (100 nmol/L). B, Effects of an NO scavenger, LNAC (1 mmol/L), on APD shortening. C, Effects of an NOS inhibitor, SMTU (1 µmol/L), on ICa,L suppression. D, Effects of an NO scavenger, LNAC (1 mmol/L), on ICa,L suppression. E, Effects of an NOS inhibitor, SMTU (1 µmol/L), on IKs enhancement. F, Effects of an NO scavenger, LNAC (1 mmol/L), on IKs enhancement. *P<0.05 vs control; P<0.05 vs testosterone.

Modulation of I_Ca,L and I_Ks Is Mediated by Akt-Dependent Activation of NOS3
Amplitudes of I_Ca,L and I_Ks were increased rapidly (within 5 minutes) after application of testosterone, which argues against activation of inducible NOS (NOS2). Therefore, we tested whether testosterone activated NOS1 or NOS3 to affect I_Ca,L and I_Ks. We used 2 different NOS inhibitors, SMTC, which is more sensitive to NOS1 (IC₅₀=0.31 µmol/L) than to NOS3 (IC₅₀=5.4 µmol/L),²⁷ and L-NIO, which is more sensitive to NOS3 (IC₅₀=0.5 µmol/L) than to NOS1 (IC₅₀=3.9 µmol/L).²⁸ Application of SMTC (3 µmol/L) did not alter suppression of I_Ca,L by testosterone, whereas L-NIO (1 µmol/L) reversed testosterone-induced suppression of I_Ca,L to its initial levels (Figure 6A). Similarly, SMTC (3 µmol/L) did not alter enhancement of I_Ks by testosterone, whereas L-NIO (1 µmol/L) reversed the testosterone-induced enhancement of I_Ks to its initial levels (Figure 6B). These findings suggest that the effects of testosterone on I_Ca,L and I_Ks are mediated by NO released from NOS3.

View larger version (18K): [in this window] [in a new window]   Figure 6. Effects of inhibitors of the signal molecules in the nongenomic pathway of testosterone. A, Effects of various inhibitors on ICa,L suppression by testosterone (100 nmol/L). The percent reversal of ICa,L suppression by various inhibitors was calculated as 100x[ICa,L(testosterone)–ICa,L(inhibitors)]/[ICa,L(testosterone)–ICa,L(control)] with 5 experiments. *P<0.05 vs control. B, Effects of various inhibitors on IKs enhancement by testosterone (100 nmol/L). The percent reversal of IKs enhancement by various inhibitors was calculated as 100x[IKs(testosterone)–IKs(inhibitors)]/[IKs(testosterone)–IKs(control)] with 5 experiments. *P<0.05 vs control.

NOS3 is activated via at least 2 distinct pathways, a phosphorylation-dependent pathway involving the Ser/Thr kinase, Akt, or a Ca²⁺-dependent pathway involving the Ca²⁺-binding protein, calmodulin (CaM).^29,30 To test which pathway is involved in testosterone-induced NOS3 activation, we used an Akt inhibitor (SH-6) and a CaM inhibitor (W7). SH-6 (10 µmol/L) reversed suppression of I_Ca,L and enhancement of I_Ks by testosterone back to the initial levels, whereas W7 (5 µmol/L) did not alter either I_Ca,L suppression or I_Ks enhancement (Figures 6A and 6B). Testosterone activates NOS3 through its nongenomic (nonnuclear) pathway, in which binding of testosterone to membrane-localized testosterone receptors activates tyrosine kinase, c-Src, followed by sequential activation of phosphatidylinositol 3-kinase (PI-3 kinase), Akt, and then NOS3.³¹ Therefore, we further tested involvement of c-Src and PI-3 kinase in testosterone-induced effects on I_Ca,L and I_Ks using a c-Src inhibitor, PP2, and a PI-3 kinase inhibitor, LY-294,002. Both PP2 (10 µmol/L) and LY-294,002 (30 µmol/L) reversed suppression of I_Ca,L and enhancement of I_Ks by testosterone back to their initial levels (Figures 6A and 6B).

Effects of Testosterone on Phosphorylation of Akt and NOS3
Akt is phosphorylated at ⁴⁷³Ser on its activation. Full activation of NOS3 is achieved by phosphorylation of ¹¹⁷⁷Ser by Akt. We therefore investigated whether testosterone induced phosphorylation of Akt at ⁴⁷³Ser and NOS3 at ¹¹⁷⁷Ser by immunoblot analysis using antibodies against phospho-Akt and phospho-NOS3. Incubation of cardiac myocytes with testosterone for 10 minutes increased phosphorylation of both Akt and NOS3 in a concentration-dependent manner (Figure 7A). Phosphorylation of Akt and NOS3 was inhibited by nilutamide, PP2, LY-294,002, and SH-6 but not by W7 (Figure 7B). These immunoblot results were consistent with our electrophysiological data.

View larger version (52K): [in this window] [in a new window]   Figure 7. Phosphorylation of Akt and NOS3 by testosterone. A, Dose-dependent phosphorylation of Akt or NOS3 by testosterone. Left panel depicts immunoblot (IB) analysis by anti-phospho-Akt (first panel), anti-Akt (second panel), anti-phospho-NOS3 (third panel), or anti-NOS3 (bottom panel) antibody in the absence or presence of testosterone at various concentrations. The right panel shows densitometric analysis averaged from 3 experiments. *P<0.05 vs control for Akt; P<0.05 vs control for NOS3. B, Effects of various blockers on phosphorylation of Akt or NOS3. Left panel depicts immunoblot analysis in the control state (lane 1), in the presence of 100 nmol/L testosterone alone (lane 2), or in the presence of 100 nmol/L testosterone and each of various inhibitors (lanes 3 to 7). Right panel shows densitometric analysis averaged from 3 experiments. *P<0.05 vs control for Akt; P<0.05 vs control for NOS3.

Effects of Testosterone on MAPs and QT Interval in Isolated Hearts
Effects of testosterone in isolated whole hearts were examined in Langendorff-perfused hearts. Perfusion of testosterone reversibly shortened duration of MAPs at 20% repolarization and at 90% repolarization and shortened the QTc interval within 15 minutes (Figures 8A and 8B).

View larger version (18K): [in this window] [in a new window]   Figure 8. Effects of testosterone on isolated Langendorff-perfused hearts. A, Effects of retrograde perfusion of coronary artery with solution containing testosterone (100 nmol/L) on APD20 and APD90 of MAP and QTc interval. *P<0.05 vs control; P<0.05 vs testosterone. B, Representative recordings of MAP (left panel) and ventricular ECG in the control state (a), in the presence of testosterone (100 nmol/L; b), and during washout (c). Vertical lines represent the beginning of MAP or QRS wave, 90% repolarization of MAP, and the end of T wave. C, Phosphorylation of Akt and NOS3 by testosterone (100 nmol/L) in isolated hearts. Right panel shows densitometric analysis averaged from 3 experiments. IB indicates immunoblot. *P<0.05 between control and testosterone.

Left ventricles were homogenated after the same amount of time (15 minutes) of perfusion with or without testosterone as functional experiments, and immunoblot analyses were performed. Immunodensity of phospho-Akt and phospho-NOS3 was higher in hearts with testosterone perfusion than in nontreated hearts (Figure 8C).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Although recent clinical and basic studies suggest important regulatory roles of testosterone in cardiac repolarization,^8–13 underlying mechanisms remain unknown. We have demonstrated the following in the present study: (1) Testosterone shortens APD at an EC₅₀ of 4.5±0.6 nmol/L for APD₂₀ and 2.7±0.6 nmol/L for APD₉₀, which is within the physiological range of serum testosterone levels in men. Serum testosterone level is reported to be 10.4 to 34.7 nmol/L in men³² and 0.6 to 2.7 nmol/L in women.³³ (2) Testosterone-induced APD shortening is mainly due to enhancement of I_Ks, in part with a contribution of I_Ca,L suppression at a high concentration of testosterone. (3) I_Ks enhancement and I_Ca,L suppression are due to NOS3 activation and NO production through a nongenomic pathway, in which c-Src, PI-3 kinase, Akt, and NOS3 are sequentially activated.

Effects of Testosterone on Cardiac Ion Currents
In the present study, APD shortening by testosterone up to 100 nmol/L was fully abolished by preincubation with chromanol 293B and nisoldipine, which suggests that testosterone-induced APD shortening was mainly caused by its effects on I_Ks and I_Ca,L in guinea pig ventricular myocytes. There are several studies investigating the effects of androgens on cardiac currents. Epiandrosterone is a metabolite of the testosterone precursor dehydroepiandrosterone. Gupte et al³⁴ demonstrated that epiandrosterone suppressed I_Ca,L in rat ventricular myocytes. Pham et al¹³ have suggested involvement of the I_Ks component in the effects of testosterone. Shuba et al³⁵ reported in the Xenopus laevis heterologous expression system that testosterone produced a 35% reduction in expressed human ERG (HERG) currents. The concentration of testosterone they used (1 µmol/L) was beyond the range of its serum concentration in normal male mammals, including humans. Chronic application of androgen in vivo exhibited enhancement of the inward rectifier K⁺ current (I_K1) and I_Kr in rabbit.¹⁹ In the present study, we found no apparent effects of testosterone on I_Kr at concentrations up to 100 nmol/L. Although we have no clear explanation for the difference, it may reflect different modes of testosterone application (chronic versus acute) or species differences.

Signaling Pathway of Testosterone-Dependent Current Modulation
In cardiac myocytes, testosterone has been shown to exhibit acute effects independently of a transcriptional mechanism, in addition to the conventional genotropic action.³¹ Androgen receptors have no intrinsic transmembrane domain, and it has not been clarified how androgen receptors are localized to the plasma membrane. Although interaction with a scaffolding protein, caveolin-1, is postulated to act for membrane localization of androgen receptors,³⁶ no direct interaction between testosterone receptors and heart-type caveolin (caveolin-3) has been determined. Nevertheless, binding of testosterone to receptors on the plasma membrane is suggested to sequentially activate c-Src, PI-3 kinase, and Akt.^31,37 In the present study, each inhibitor for testosterone receptors, c-Src, PI3-kinase, Akt, and NOS3, reversed testosterone-induced I_Ks enhancement and I_Ca,L suppression. To confirm that effects of these inhibitors were via interference with effects of testosterone, we performed additional control experiments: in the absence of testosterone, application of nilutamide, PP2, LY-294,002, SH-6, and L-NIO affected neither I_Ks nor I_Ca,L, and subsequent application of testosterone did not exhibit I_Ks enhancement or I_Ca,L suppression (Data Supplement Figures VII, VIII, and IX). Testosterone treatment induced phosphorylation of both Akt and NOS3, which were inhibited by these reagents. Thus, the present study provides evidence of testosterone regulation of cardiac ion channels through a nontranscriptional mechanism (Figure 9).

View larger version (38K): [in this window] [in a new window]   Figure 9. Proposed schematic model for regulatory mechanism of testosterone on ICa,L and IKs. A, In the ligand-free condition, NOS3 is interacted with membrane-associated caveolin-3 and is in an inactive state. The rapid effect of testosterone on ICa,L and IKs suggests an involvement of testosterone receptor on the plasma membrane, although molecular identity and mechanism of plasma membrane localization for testosterone receptor have not yet been clarified. B, When testosterone binds to its receptors, the testosterone receptor, c-Src, and p85 of PI3-kinase form a macromolecular complex,37 which renders p110 of PI3-kinase free and in an active state, and active PI3-kinase converts PIP2(4,5) to PIP3(3,4,5).31 Subsequently, PIP3(3,4,5), Akt, and NOS3 form a complex, which results in NOS3 activation and NO production.31 Produced NO then inhibits ICa,L via a cGMP-dependent manner and enhances IKs via a cGMP-independent manner.

Differential Dose Dependence of Testosterone on I_Ca,L and I_Ks
Testosterone-induced APD shortening is mainly caused by enhancement of I_Ks at a low concentration (1 nmol/L), whereas at a high concentration of testosterone, it is caused by the effects of testosterone on both I_Ks and I_Ca,L. These data are consistent with the findings that testosterone enhanced I_Ks with an EC₅₀ of 1.1±0.2 nmol/L, whereas testosterone required a relatively higher concentration to suppress I_Ca,L (IC₅₀=38.8±3.5 nmol/L).

Although suppression of I_Ca,L and enhancement of I_Ks by testosterone are mediated through the same pathway from activation of c-Src to NOS3 activation, the concentration of testosterone to modulate I_Ca,L and I_Ks differ. This could be explained by differential mechanisms of NO to regulate I_Ca,L and I_Ks. In our previous report, suppression of I_Ca,L by NO was dependent on cGMP, which suggests that phosphorylation by protein kinase G is involved.²⁰ By contrast, enhancement of I_Ks by NO is not dependent on cGMP.²⁰ In the present study, effects of testosterone on I_Ca,L, but not on I_Ks, were also inhibited by a guanylate cyclase inhibitor, ODQ (Data Supplement Figure X). Furthermore, the concentration of an NO donor, SNP, required to modulate I_Ca,L was significantly higher than that required to modulate I_Ks (Data Supplement Figure XI), which suggests that sensitivity to NO is different between I_Ca,L and I_Ks. However, direct measurement of the sensitivity of NO on guanylate cyclase and protein s-nitrosylation has not been addressed.

Clinical Implications and Study Limitations
Virilized women have shorter JT intervals than castrated men. Men have longer JT intervals after orchiectomy.¹¹ In men, QT_c intervals shorten at puberty.^9,10 Furthermore, there is a tendency toward an age-dependent reduction in the number of male patients with long-QT syndrome who manifest QT_c intervals >440 ms.³⁸ These clinical findings implicate a unique modulatory role of testosterone in ventricular repolarization. However, it is currently unproved whether the nontranscriptional regulation of cardiac repolarization currents by testosterone demonstrated in the present study underlies sex-related differences in QT interval in humans. Although we demonstrated that testosterone shortens APD and QTc interval in isolated intact hearts, the contribution of this regulatory mechanism in the physiological condition remains unknown. Because testosterone is suggested to modulate cardiac repolarization via both a transcriptional and a nontranscriptional mechanism in cardiac myocytes,^18,19,39 transcriptional regulation by testosterone should be tested in addition to nontranscriptional regulation to investigate its contribution in physiological conditions. In the present study, we used guinea pig hearts that lack I_to, which is different from human hearts. Thus, the contribution of I_to to sex differences in cardiac repolarization has not been addressed in the present study. Estradiol also acutely activates NOS3 independently of genotropic action.^40,41 In fact, in preliminary experiments, we found that estradiol modulates I_Ca,L and I_Ks via NOS3 activation (Data Supplement Figure XII). However, our data suggested the presence of crucial qualitative and quantitative differences in electrophysiological effects between testosterone and estradiol (Data Supplement Figure XII). Further studies are certainly required to clarify the role of nontranscriptional regulation of testosterone on the gender-dependent difference in the QTc interval and propensity to arrhythmias.

	Acknowledgments

 
This work was supported in part by a research grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and a research grant from Takeda Science Foundation.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Moss AJ, Schwartz PJ, Crampton RS, Tzivoni D, Locati EH, MacCluer J, Hall WJ, Weitkamp L, Vincent GM, Garson A Jr. The long QT syndrome: prospective longitudinal study of 328 families. Circulation. 1991; 84: 1136–1144.[Abstract/Free Full Text]
   
2. Hashiba K. Sex differences in phenotypic manifestation and gene transmission in the Romano-Ward syndrome. Ann N Y Acad Sci. 1992; 644: 142–156.[Medline] [Order article via Infotrieve]
   
3. Lehmann MH, Timothy KW, Frankovich D, Fromm BS, Keating M, Locati EH, Taggart RT, Towbin JA, Moss AJ, Schwartz PJ, Vincent GM. Age-gender influence on the rate-corrected QT interval and the QT-heart rate relation in families with genotypically characterized long QT syndrome. J Am Coll Cardiol. 1997; 29: 93–99.[Abstract]
   
4. Makkar RR, Fromm BS, Steinman RT, Meissner MD, Lehmann MH. Female gender as a risk factor for torsades de pointes associated with cardiovascular drugs. JAMA. 1993; 270: 2590–2597.[Abstract]
   
5. Lehmann MH, Hardy S, Archibald D, Quart B, MacNeil DJ. Sex difference in risk of torsade de pointes with d,l-sotalol. Circulation. 1996; 94: 2535–2541.[Abstract/Free Full Text]
   
6. Drici MD, Knollmann BC, Wang WX, Woosley RL. Cardiac actions of erythromycin: influence of female sex. JAMA. 1998; 280: 1774–1776.[Abstract/Free Full Text]
   
7. Bazett H. An analysis of the time-relations of electrocardiograms. Heart. 1920; 7: 353–370.
   
8. Merry M, Benzoin J, Alberta M, Locati E, Moss AJ. Electrocardiographic quantification of ventricular repolarization. Circulation. 1989; 80: 1301–1308.[Abstract/Free Full Text]
   
9. Rautaharju PM, Zou SH, Wong S, Calhoun HP, Berens on GS, Primes R, Avignon A. Sex differences in the evolution of the electrocardiographic QT interval with age. Can J Cardiol. 1992; 8: 690–695.[Medline] [Order article via Infotrieve]
   
10. Stamba-Badiale M, Spagnolo D, Bosi G, Schwartz PJ. Are gender differences in QTc present at birth? MISNES investigators: Multicenter Italian Study on Neonatal Electrocardiography and Sudden Infant Death Syndrome. Am J Cardiol. 1995; 75: 1277–1278.[CrossRef][Medline] [Order article via Infotrieve]
    
11. Bidoggia H, Maciel JP, Capalozza N, Mosca S, Blaksley EJ, Valverde E, Bertran G, Arini P, Biagetti MO, Quinteiro RA. Sex differences on the electrocardiographic pattern of cardiac repolarization: possible role of testosterone. Am Heart J. 2000; 140: 678–683.[CrossRef][Medline] [Order article via Infotrieve]
    
12. Pham TV, Sosunov EA, Gainullin RZ, Danilo P Jr, Rosen MR. Impact of sex and gonadal steroids on prolongation of ventricular repolarization and arrhythmias induced by I_K-blocking drugs. Circulation. 2001; 103: 2207–2212.[Abstract/Free Full Text]
    
13. Pham TV, Sosunov EA, Anyukhovsky EP, Danilo P Jr, Rosen MR. Testosterone diminishes the proarrhythmic effects of dofetilide in normal female rabbits. Circulation. 2002; 106: 2132–2136.[Abstract/Free Full Text]
    
14. Evans RM. The steroid and thyroid hormone receptor superfamily. Science. 1988; 240: 889–895.[Abstract/Free Full Text]
    
15. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P. The nuclear receptor superfamily: the second decade. Cell. 1995; 83: 835–839.[CrossRef][Medline] [Order article via Infotrieve]
    
16. Falkenstein E, Tillmann HC, Christ M, Feuring M, Wehling M. Multiple actions of steroid hormones: a focus on rapid, nongenomic effects. Pharmacol Rev. 2000; 52: 513–556.[Abstract/Free Full Text]
    
17. Farrar RS, Rodnick KJ. Sex-dependent effects of gonadal steroids and cortisol on cardiac contractility in rainbow trout. J Exp Biol. 2004; 207: 2083–2093.[Abstract/Free Full Text]
    
18. Golden KL, Marsh JD, Jiang Y. Testosterone regulates mRNA levels of calcium regulatory proteins in cardiac myocytes. Horm Metab Res. 2004; 36: 197–202.[CrossRef][Medline] [Order article via Infotrieve]
    
19. Liu X-K, Katchman A, Whitfield BH, Wan G, Janowski EM, Woosley RL, Ebert SN. In vivo androgen treatment shortens the QT interval and increases the densities of inward and delayed rectifier potassium currents in orchiectomized male rabbits. Cardiovasc Res. 2003; 57: 28–36.[Abstract/Free Full Text]
    
20. Bai C-X, Takahashi K, Masumiya H, Sawanobori T, Furukawa T. Nitric oxide-dependent modulation of the delayed rectifier K⁺ current and the L-type Ca²⁺ current by ginsenoside Re, an ingredient of Panax ginseng, in guinea-pig cardiomyocytes. Br J Pharmacol. 2004; 142: 567–575.[CrossRef][Medline] [Order article via Infotrieve]
    
21. Bai C-X, Namekata I, Kurokawa J, Tanaka H, Shigenobu K, Furukawa T. Role of nitric oxide in Ca²⁺-sensitivity of the slowly activating delayed rectifier K⁺ current in cardiac myocytes. Circ Res. 2005; 96: 64–72.[Abstract/Free Full Text]
    
22. Bai C-X, Sunami A, Namiki T, Sawanobori T, Furukawa T. Electrophysiological effects of ginseng and ginsenoside Re in guinea pig ventricular myocytes. Eur J Pharmacol. 2003; 476: 35–44.[CrossRef][Medline] [Order article via Infotrieve]
    
23. Suzuki M, Sasaki N, Miki T, Sakamoto N, Ohmoto-Sekine Y, Tamagawa M, Seino S, Marban E, Nakaya H. Role of sarcolemmal K_ATP channels in cardioprotection against ischemia/reperfusion injury in mice. J Clin Invest. 2002; 109: 509–516.[CrossRef][Medline] [Order article via Infotrieve]
    
24. Zheng Y-J, Furukawa T, Ogura T, Tajimi K, Inagaki N. M phase-specific expression and phosphorylation-dependent ubiquitination of the ClC-2 channel. J Biol Chem. 2002; 277: 32268–32273.[Abstract/Free Full Text]
    
25. Kass RS. Nisoldipine: a new, more selective calcium current blocker in cardiac Purkinje fibers. J Pharmacol Exp Ther. 1982; 223: 446–456.[Abstract/Free Full Text]
    
26. Bosch RF, Gaspo R, Busch AE, Lang HJ, Li G-R, Nattel S. Effects of the chromanol 293B, a selective blocker of the slow, component of the delayed rectifier K⁺ current, on repolarization in human and guinea pig ventricular myocytes. Cardiovasc Res. 1998; 38: 441–450.[Abstract/Free Full Text]
    
27. Narayanan K, Griffith OW. Synthesis of L-thiocitrulline, L-homothiocitrulline, and S-methyl-L-thiocitrulline: a new class of potent nitric oxide synthase inhibitors. J Med Chem. 1994; 37: 885–887.[CrossRef][Medline] [Order article via Infotrieve]
    
28. McCall TB, Feelisch M, Palmer RM, Moncada S. Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells. Br J Pharmacol. 1991; 102: 234–238.[Medline] [Order article via Infotrieve]
    
29. Kone BC. Protein-protein interactions controlling nitric oxide synthases. Acta Physiol Scand. 2000; 168: 27–31.[CrossRef][Medline] [Order article via Infotrieve]
    
30. Goligorsky MS, Li H, Brodsky S, Chen J. Relationships between caveolae and eNOS: everything in proximity and the proximity of everything. Am J Physiol. 2002; 283: F1–F10.
    
31. Baron S, Manin M, Beaudoin C, Leotoing L, Communal Y, Veyssiere G, Morel L. Androgen receptor mediates non-genomic activation of phosphatidylinositol 3-OH kinase in androgen-sensitive epithelial cells. J Biol Chem. 2004; 279: 14579–14586.[Abstract/Free Full Text]
    
32. Dorgan JF, Fears TR, McMahon RP, Aronson Friedman L, Patterson BH, Greenhut SF. Measurement of steroid sex hormones in serum: a comparison of radioimmunoassay and mass spectrometry. Steroids. 2002; 67: 151–158.[CrossRef][Medline] [Order article via Infotrieve]
    
33. Burger HG. Androgen production in women. Fertil Steril. 2002; 77: S3–S5.[Medline] [Order article via Infotrieve]
    
34. Gupte SA, Tateyama M, Okada T, Oka M, Ochi R. Epiandrosterone, a metabolite of testosterone precursor, blocks L-type calcium channels of ventricular myocytes and inhibits myocardial contractility. J Mol Cell Cardiol. 2002; 34: 679–688.[CrossRef][Medline] [Order article via Infotrieve]
    
35. Shuba YM, Degtiar VE, Osipenko VN, Naidenov VG, Woosley RL. Testosterone-mediated modulation of HERG blockade by proarrhythmic agents. Biochem Pharmacol. 2001; 62: 41–49.[CrossRef][Medline] [Order article via Infotrieve]
    
36. Lu ML, Schneider MC, Zheng Y, Zhang X, Richie JP. Caveolin-1 interacts with androgen receptor: a positive modulator of androgen receptor mediated transactivation. J Biol Chem. 2001; 276: 13442–13451.[Abstract/Free Full Text]
    
37. Migliaccio A, Castoria G, Di Domenico M, de Falco A, Bilancio A, Lombardi M, Barone MV, Ametrano D, Zannini MS, Abbondanza C, Auricchio F. Steroid-induced androgen receptor-oestradiol receptor ß-Src complex triggers prostate cancer cell proliferation. EMBO J. 2000; 20: 5406–5417.[CrossRef]
    
38. Locati EH, Zareba W, Moss AJ, Schwartz PJ, Vincent GM, Lehmann MH, Towbin JA, Priori SG, Napolitano C, Robinson JL, Andrews M, Timothy K, Hall WJ. Age- and sex-related differences in clinical manifestations in patients with congenital long-QT syndrome: findings from the International LQTS Registry. Circulation. 1998; 97: 2237–2244.[Abstract/Free Full Text]
    
39. Drici MD, Burklow TR, Haridasse V, Glazer RI, Woosley RL. Sex hormones prolong the QT interval and downregulate potassium channel expression in the rabbit heart. Circulation. 1996; 94: 1471–1474.[Abstract/Free Full Text]
    
40. Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem. 2001; 276: 36869–36872.[Free Full Text]
    
41. Ho KJ, Liao JK. Nonnuclear actions of estrogen. Arterioscler Thromb Vasc Biol. 2002; 22: 1952–1961.[Abstract/Free Full Text]
    

---

 

CLINICAL PERSPECTIVE

Polymorphic ventricular tachycardias associated with QT prolongation, known as torsade de pointes, occur as a manifestation of the congenital long-QT syndrome or can be acquired from any agent that induces QT prolongation. Gender influences the phenotypic manifestations of congenital long-QT syndrome and the susceptibility to acquired long-QT syndrome, with women known to be at greater risk. Torsade de pointes occurs with similar frequency in boys and girls with congenital long-QT syndrome until puberty. After puberty, it is less common in boys, whereas women have a greater risk. This observation fits with the gender difference in QT interval observed in the general population. The QT_c interval is similar between boys and girls until puberty, when it shortens only in men and then remains shorter in men than in women until around the age of 50 years. Because this period of life coincides with the highest circulating levels of testosterone in men, it seems plausible that testosterone modulates the QT_c interval. In the present study, we demonstrated that testosterone applied to isolated hearts or single cardiac myocytes rapidly shortened QT_c interval and APD. Effects of gonadal steroids occur either via transcription of genes with hormone-responsive elements or by mechanisms that do not require gene transcription ("nongenomic action"). Testosterone-induced modulation of cardiac repolarization currents found in the present study is attributable to a nongenomic action, in which nitric oxide produced by an Akt/endothelial nitric oxide synthase–dependent pathway is a main effector. In addition to the genomic regulation of ion channel expression by testosterone reported elsewhere, this novel regulatory mechanism of cardiac ion currents by testosterone may shorten the QT_c interval in men during the reproductive age, providing a potential mechanism of gender differences in QT intervals and torsade de pointes risk.

	Footnotes

 
The online-only Data Supplement can be found at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.104.523217/DC1.

This article has been cited by other articles:

				C. van Noord, W. M van der Deure, M. C J M Sturkenboom, S. M J M Straus, A. Hofman, T. J Visser, J. A Kors, J. C M Witteman, and B. H C. Stricker High free thyroxine levels are associated with QTc prolongation in males J. Endocrinol., July 1, 2008; 198(1): 253 - 260. [Abstract] [Full Text] [PDF]

				J. Kurokawa, M. Tamagawa, N. Harada, S.-i. Honda, C.-X. Bai, H. Nakaya, and T. Furukawa Acute effects of oestrogen on the guinea pig and human IKr channels and drug-induced prolongation of cardiac repolarization J. Physiol., June 15, 2008; 586(12): 2961 - 2973. [Abstract] [Full Text] [PDF]

				M. D Tobin, M. Kahonen, P. Braund, T. Nieminen, C. Hajat, M. Tomaszewski, J. Viik, R. Lehtinen, G A. Ng, P. W Macfarlane, et al. Gender and effects of a common genetic variant in the NOS1 regulator NOS1AP on cardiac repolarization in 3761 individuals from two independent populations Int. J. Epidemiol., May 29, 2008; (2008) dyn091v1. [Abstract] [Full Text] [PDF]

				H. Nakamura, J. Kurokawa, C.-X. Bai, K. Asada, J. Xu, R. V. Oren, Z. I. Zhu, C. E. Clancy, M. Isobe, and T. Furukawa Progesterone Regulates Cardiac Repolarization Through a Nongenomic Pathway: An In Vitro Patch-Clamp and Computational Modeling Study Circulation, December 18, 2007; 116(25): 2913 - 2922. [Abstract] [Full Text] [PDF]

				S. Viskin Brugada Syndrome in Children: Don't Ask, Don't Tell? Circulation, April 17, 2007; 115(15): 1970 - 1972. [Full Text] [PDF]

				T. Furukawa, C.-X. Bai, A. Kaihara, E. Ozaki, T. Kawano, Y. Nakaya, M. Awais, M. Sato, Y. Umezawa, and J. Kurokawa Ginsenoside Re, a Main Phytosterol of Panax ginseng, Activates Cardiac Potassium Channels via a Nongenomic Pathway of Sex Hormones Mol. Pharmacol., December 1, 2006; 70(6): 1916 - 1924. [Abstract] [Full Text] [PDF]

				G. D. Norata, G. Tibolla, P. M. Seccomandi, A. Poletti, and A. L. Catapano Dihydrotestosterone Decreases Tumor Necrosis Factor-{alpha} and Lipopolysaccharide-Induced Inflammatory Response in Human Endothelial Cells J. Clin. Endocrinol. Metab., February 1, 2006; 91(2): 546 - 554. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF)