Nontranscriptional Regulation of Cardiac Repolarization Currents by Testosterone
http://www.100md.com
《循环学杂志》
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.
Abstract
Background— Women have longer QTc intervals than men and are at greater risk for arrhythmias associated with long QTc 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 (IKs) and suppression of L-type Ca2+ currents (ICa,L), because testosterone failed to shorten APD in the presence of an IKs inhibitor, chromanol 293B, and an ICa,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 IKs and ICa,L by testosterone is a novel regulatory mechanism of cardiac repolarization that can potentially contribute to the control of QTc intervals by androgen.
Key Words: nitric oxide long-QT syndrome testosterone potassium current ion channels
Introduction
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 QTc interval is quite similar between men and women.8–10 On arrival of puberty, the duration of QTc intervals in boys shortens, which leaves adult women with longer QTc intervals than adult men. The QTc interval in men then gradually increases until approximately 60 years of age, when QTc intervals approach those of women.8–10 As sex hormone level elevates during puberty, the influence of sex hormones on differences in QTc 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 1-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 (IKr) 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, NG-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 (IKs) and inhibiting L-type Ca2+ currents (ICa,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 IKs and ICa,L via a nontranscriptional mechanism.
Methods
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 ICa,L, a prepulse (100 ms) was applied to –40 mV from a holding potential (Vh) of –80 mV to inactivate the Na+ channels and the T-type Ca2+ channels, followed by a 200-ms test pulse (Vt) to 0 mV at 1 Hz. IKs were elicited by 3.5-second Vt to 50 mV from a Vh of –40 mV at 0.1 Hz or by 0.5-second Vt 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
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.
Effects of Testosterone on ICa,L, IKs, and IKr
To explore which ion currents are targets of testosterone, we examined effects of testosterone on ICa,L, IKs, and IKr, major ionic currents determining APD in guinea pig ventricles. Testosterone (100 nmol/L) suppressed ICa,L (Figure 2A) and enhanced IKs (Figure 2B), whereas it barely affected IKr (data not shown). Although we recorded IKs elicited by 3.5-second test pulses at 0.1 Hz to exaggerate the amplitudes unless otherwise noted, we confirmed similar enhancement of IKs elicited by shorter pulses (0.5 second) at 1 Hz, which is a more physiologically normal condition (Figure 2C). Suppression of ICa,L and enhancement of IKs by testosterone were completely inhibited by nilutamide (1 μmol/L; Figure 2). ICa,L suppression and IKs enhancement by testosterone were dose-dependent, with a significantly lower EC50 value for enhancement of IKs than for suppression of ICa,L (Figure 3).
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 (ICa,L,IKs) 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 ICa,L Suppression and IKs Enhancement to APD Shortening by Testosterone
To examine the relative contribution of ICa,L suppression and IKs enhancement to APD shortening by testosterone, we used an ICa,L inhibitor, nisoldipine, and/or an IKs 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 IKs, with minor effects on the transient outward current (Ito), at 10 μmol/L.26 We confirmed that chromanol 293B at 10 μmol/L barely affected ICa,L or IKr 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 IKs and ICa,L accounts for APD shortening induced by testosterone; at a low concentration of testosterone, APD shortening is mainly due to enhancement of IKs, whereas at a high concentration, both IKs enhancement and ICa,L suppression contribute to APD shortening.
Effects of Testosterone Are Mediated by Actions of NO
We have previously reported that NO shortens APD by suppressing ICa,L and enhancing IKs,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, ICa,L suppression and IKs 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, ICa,L, and IKs are mediated by an increase in cytosolic NO.
Modulation of ICa,L and IKs Is Mediated by Akt-Dependent Activation of NOS3
Amplitudes of ICa,L and IKs 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 ICa,L and IKs. 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 ICa,L by testosterone, whereas L-NIO (1 μmol/L) reversed testosterone-induced suppression of ICa,L to its initial levels (Figure 6A). Similarly, SMTC (3 μmol/L) did not alter enhancement of IKs by testosterone, whereas L-NIO (1 μmol/L) reversed the testosterone-induced enhancement of IKs to its initial levels (Figure 6B). These findings suggest that the effects of testosterone on ICa,L and IKs are mediated by NO released from NOS3.
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 ICa,L and enhancement of IKs by testosterone back to the initial levels, whereas W7 (5 μmol/L) did not alter either ICa,L suppression or IKs 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 ICa,L and IKs 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 ICa,L and enhancement of IKs 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.
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).
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
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 IKs, in part with a contribution of ICa,L suppression at a high concentration of testosterone. (3) IKs enhancement and ICa,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 IKs and ICa,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 ICa,L in rat ventricular myocytes. Pham et al13 have suggested involvement of the IKs 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 (IK1) and IKr in rabbit.19 In the present study, we found no apparent effects of testosterone on IKr 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 IKs enhancement and ICa,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 IKs nor ICa,L, and subsequent application of testosterone did not exhibit IKs enhancement or ICa,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).
Differential Dose Dependence of Testosterone on ICa,L and IKs
Testosterone-induced APD shortening is mainly caused by enhancement of IKs at a low concentration (1 nmol/L), whereas at a high concentration of testosterone, it is caused by the effects of testosterone on both IKs and ICa,L. These data are consistent with the findings that testosterone enhanced IKs with an EC50 of 1.1±0.2 nmol/L, whereas testosterone required a relatively higher concentration to suppress ICa,L (IC50=38.8±3.5 nmol/L).
Although suppression of ICa,L and enhancement of IKs by testosterone are mediated through the same pathway from activation of c-Src to NOS3 activation, the concentration of testosterone to modulate ICa,L and IKs differ. This could be explained by differential mechanisms of NO to regulate ICa,L and IKs. In our previous report, suppression of ICa,L by NO was dependent on cGMP, which suggests that phosphorylation by protein kinase G is involved.20 By contrast, enhancement of IKs by NO is not dependent on cGMP.20 In the present study, effects of testosterone on ICa,L, but not on IKs, were also inhibited by a guanylate cyclase inhibitor, ODQ (Data Supplement Figure X). Furthermore, the concentration of an NO donor, SNP, required to modulate ICa,L was significantly higher than that required to modulate IKs (Data Supplement Figure XI), which suggests that sensitivity to NO is different between ICa,L and IKs. 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, QTc 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 QTc 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 Ito, which is different from human hearts. Thus, the contribution of Ito 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 ICa,L and IKs 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
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.
Hashiba K. Sex differences in phenotypic manifestation and gene transmission in the Romano-Ward syndrome. Ann N Y Acad Sci. 1992; 644: 142–156.
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.
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.
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.
Drici MD, Knollmann BC, Wang WX, Woosley RL. Cardiac actions of erythromycin: influence of female sex. JAMA. 1998; 280: 1774–1776.
Bazett H. An analysis of the time-relations of electrocardiograms. Heart. 1920; 7: 353–370.
Merry M, Benzoin J, Alberta M, Locati E, Moss AJ. Electrocardiographic quantification of ventricular repolarization. Circulation. 1989; 80: 1301–1308.
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.
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.
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.
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 IK-blocking drugs. Circulation. 2001; 103: 2207–2212.
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.
Evans RM. The steroid and thyroid hormone receptor superfamily. Science. 1988; 240: 889–895.
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.
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.
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.
Golden KL, Marsh JD, Jiang Y. Testosterone regulates mRNA levels of calcium regulatory proteins in cardiac myocytes. Horm Metab Res. 2004; 36: 197–202.
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.
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.
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.
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.
Suzuki M, Sasaki N, Miki T, Sakamoto N, Ohmoto-Sekine Y, Tamagawa M, Seino S, Marban E, Nakaya H. Role of sarcolemmal KATP channels in cardioprotection against ischemia/reperfusion injury in mice. J Clin Invest. 2002; 109: 509–516.
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.
Kass RS. Nisoldipine: a new, more selective calcium current blocker in cardiac Purkinje fibers. J Pharmacol Exp Ther. 1982; 223: 446–456.
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.
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.
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.
Kone BC. Protein-protein interactions controlling nitric oxide synthases. Acta Physiol Scand. 2000; 168: 27–31.
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.
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.
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.
Burger HG. Androgen production in women. Fertil Steril. 2002; 77: S3–S5.
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.
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.
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.
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 -Src complex triggers prostate cancer cell proliferation. EMBO J. 2000; 20: 5406–5417.
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.
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.
Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem. 2001; 276: 36869–36872.
Ho KJ, Liao JK. Nonnuclear actions of estrogen. Arterioscler Thromb Vasc Biol. 2002; 22: 1952–1961.(Chang-Xi Bai, MD, PhD; Ju)
Abstract
Background— Women have longer QTc intervals than men and are at greater risk for arrhythmias associated with long QTc 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 (IKs) and suppression of L-type Ca2+ currents (ICa,L), because testosterone failed to shorten APD in the presence of an IKs inhibitor, chromanol 293B, and an ICa,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 IKs and ICa,L by testosterone is a novel regulatory mechanism of cardiac repolarization that can potentially contribute to the control of QTc intervals by androgen.
Key Words: nitric oxide long-QT syndrome testosterone potassium current ion channels
Introduction
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 QTc interval is quite similar between men and women.8–10 On arrival of puberty, the duration of QTc intervals in boys shortens, which leaves adult women with longer QTc intervals than adult men. The QTc interval in men then gradually increases until approximately 60 years of age, when QTc intervals approach those of women.8–10 As sex hormone level elevates during puberty, the influence of sex hormones on differences in QTc 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 1-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 (IKr) 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, NG-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 (IKs) and inhibiting L-type Ca2+ currents (ICa,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 IKs and ICa,L via a nontranscriptional mechanism.
Methods
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 ICa,L, a prepulse (100 ms) was applied to –40 mV from a holding potential (Vh) of –80 mV to inactivate the Na+ channels and the T-type Ca2+ channels, followed by a 200-ms test pulse (Vt) to 0 mV at 1 Hz. IKs were elicited by 3.5-second Vt to 50 mV from a Vh of –40 mV at 0.1 Hz or by 0.5-second Vt 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
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.
Effects of Testosterone on ICa,L, IKs, and IKr
To explore which ion currents are targets of testosterone, we examined effects of testosterone on ICa,L, IKs, and IKr, major ionic currents determining APD in guinea pig ventricles. Testosterone (100 nmol/L) suppressed ICa,L (Figure 2A) and enhanced IKs (Figure 2B), whereas it barely affected IKr (data not shown). Although we recorded IKs elicited by 3.5-second test pulses at 0.1 Hz to exaggerate the amplitudes unless otherwise noted, we confirmed similar enhancement of IKs elicited by shorter pulses (0.5 second) at 1 Hz, which is a more physiologically normal condition (Figure 2C). Suppression of ICa,L and enhancement of IKs by testosterone were completely inhibited by nilutamide (1 μmol/L; Figure 2). ICa,L suppression and IKs enhancement by testosterone were dose-dependent, with a significantly lower EC50 value for enhancement of IKs than for suppression of ICa,L (Figure 3).
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 (ICa,L,IKs) 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 ICa,L Suppression and IKs Enhancement to APD Shortening by Testosterone
To examine the relative contribution of ICa,L suppression and IKs enhancement to APD shortening by testosterone, we used an ICa,L inhibitor, nisoldipine, and/or an IKs 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 IKs, with minor effects on the transient outward current (Ito), at 10 μmol/L.26 We confirmed that chromanol 293B at 10 μmol/L barely affected ICa,L or IKr 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 IKs and ICa,L accounts for APD shortening induced by testosterone; at a low concentration of testosterone, APD shortening is mainly due to enhancement of IKs, whereas at a high concentration, both IKs enhancement and ICa,L suppression contribute to APD shortening.
Effects of Testosterone Are Mediated by Actions of NO
We have previously reported that NO shortens APD by suppressing ICa,L and enhancing IKs,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, ICa,L suppression and IKs 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, ICa,L, and IKs are mediated by an increase in cytosolic NO.
Modulation of ICa,L and IKs Is Mediated by Akt-Dependent Activation of NOS3
Amplitudes of ICa,L and IKs 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 ICa,L and IKs. 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 ICa,L by testosterone, whereas L-NIO (1 μmol/L) reversed testosterone-induced suppression of ICa,L to its initial levels (Figure 6A). Similarly, SMTC (3 μmol/L) did not alter enhancement of IKs by testosterone, whereas L-NIO (1 μmol/L) reversed the testosterone-induced enhancement of IKs to its initial levels (Figure 6B). These findings suggest that the effects of testosterone on ICa,L and IKs are mediated by NO released from NOS3.
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 ICa,L and enhancement of IKs by testosterone back to the initial levels, whereas W7 (5 μmol/L) did not alter either ICa,L suppression or IKs 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 ICa,L and IKs 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 ICa,L and enhancement of IKs 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.
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).
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
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 IKs, in part with a contribution of ICa,L suppression at a high concentration of testosterone. (3) IKs enhancement and ICa,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 IKs and ICa,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 ICa,L in rat ventricular myocytes. Pham et al13 have suggested involvement of the IKs 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 (IK1) and IKr in rabbit.19 In the present study, we found no apparent effects of testosterone on IKr 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 IKs enhancement and ICa,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 IKs nor ICa,L, and subsequent application of testosterone did not exhibit IKs enhancement or ICa,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).
Differential Dose Dependence of Testosterone on ICa,L and IKs
Testosterone-induced APD shortening is mainly caused by enhancement of IKs at a low concentration (1 nmol/L), whereas at a high concentration of testosterone, it is caused by the effects of testosterone on both IKs and ICa,L. These data are consistent with the findings that testosterone enhanced IKs with an EC50 of 1.1±0.2 nmol/L, whereas testosterone required a relatively higher concentration to suppress ICa,L (IC50=38.8±3.5 nmol/L).
Although suppression of ICa,L and enhancement of IKs by testosterone are mediated through the same pathway from activation of c-Src to NOS3 activation, the concentration of testosterone to modulate ICa,L and IKs differ. This could be explained by differential mechanisms of NO to regulate ICa,L and IKs. In our previous report, suppression of ICa,L by NO was dependent on cGMP, which suggests that phosphorylation by protein kinase G is involved.20 By contrast, enhancement of IKs by NO is not dependent on cGMP.20 In the present study, effects of testosterone on ICa,L, but not on IKs, were also inhibited by a guanylate cyclase inhibitor, ODQ (Data Supplement Figure X). Furthermore, the concentration of an NO donor, SNP, required to modulate ICa,L was significantly higher than that required to modulate IKs (Data Supplement Figure XI), which suggests that sensitivity to NO is different between ICa,L and IKs. 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, QTc 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 QTc 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 Ito, which is different from human hearts. Thus, the contribution of Ito 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 ICa,L and IKs 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
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.
Hashiba K. Sex differences in phenotypic manifestation and gene transmission in the Romano-Ward syndrome. Ann N Y Acad Sci. 1992; 644: 142–156.
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.
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.
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.
Drici MD, Knollmann BC, Wang WX, Woosley RL. Cardiac actions of erythromycin: influence of female sex. JAMA. 1998; 280: 1774–1776.
Bazett H. An analysis of the time-relations of electrocardiograms. Heart. 1920; 7: 353–370.
Merry M, Benzoin J, Alberta M, Locati E, Moss AJ. Electrocardiographic quantification of ventricular repolarization. Circulation. 1989; 80: 1301–1308.
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.
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.
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.
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 IK-blocking drugs. Circulation. 2001; 103: 2207–2212.
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.
Evans RM. The steroid and thyroid hormone receptor superfamily. Science. 1988; 240: 889–895.
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.
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.
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.
Golden KL, Marsh JD, Jiang Y. Testosterone regulates mRNA levels of calcium regulatory proteins in cardiac myocytes. Horm Metab Res. 2004; 36: 197–202.
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.
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.
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.
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.
Suzuki M, Sasaki N, Miki T, Sakamoto N, Ohmoto-Sekine Y, Tamagawa M, Seino S, Marban E, Nakaya H. Role of sarcolemmal KATP channels in cardioprotection against ischemia/reperfusion injury in mice. J Clin Invest. 2002; 109: 509–516.
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.
Kass RS. Nisoldipine: a new, more selective calcium current blocker in cardiac Purkinje fibers. J Pharmacol Exp Ther. 1982; 223: 446–456.
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.
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.
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.
Kone BC. Protein-protein interactions controlling nitric oxide synthases. Acta Physiol Scand. 2000; 168: 27–31.
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.
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.
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.
Burger HG. Androgen production in women. Fertil Steril. 2002; 77: S3–S5.
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.
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.
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.
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 -Src complex triggers prostate cancer cell proliferation. EMBO J. 2000; 20: 5406–5417.
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.
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.
Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem. 2001; 276: 36869–36872.
Ho KJ, Liao JK. Nonnuclear actions of estrogen. Arterioscler Thromb Vasc Biol. 2002; 22: 1952–1961.(Chang-Xi Bai, MD, PhD; Ju)