Chang-Xi Bai

Nontranscriptional regulation of cardiac repolarization currents by testosterone.

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.

Acute effects of oestrogen on the guinea pig and human IKr channels and drug-induced prolongation of cardiac repolarization

Female gender is a risk factor for drug‐induced arrhythmias associated with QT prolongation, which results mostly from blockade of the human ether‐a‐go‐go‐related gene (hERG) channel. Some clinical evidence suggests that oestrogen is a determinant of the gender‐differences in drug‐induced QT prolongation and baseline QTC intervals. Although the chronic effects of oestrogen have been studied, it remains unclear whether the gender differences are due entirely to transcriptional regulations through oestrogen receptors. We therefore investigated acute effects of the most bioactive oestrogen, 17β‐oestradiol (E2) at its physiological concentrations on cardiac repolarization and drug‐sensitivity of the hERG (IKr) channel in Langendorff‐perfused guinea pig hearts, patch‐clamped guinea pig cardiomyocytes and culture cells over‐expressing hERG. We found that physiological concentrations of E2 partially suppressed IKr in a receptor‐independent manner. E2‐induced modification of voltage‐dependence causes partial suppression of hERG currents. Mutagenesis studies showed that a common drug‐binding residue at the inner pore cavity was critical for the effects of E2 on the hERG channel. Furthermore, E2 enhanced both hERG suppression and QTC prolongation by its blocker, E4031. The lack of effects of testosterone at its physiological concentrations on both of hERG currents and E4031‐sensitivity of the hERG channel implicates the critical role of aromatic centroid present in E2 but not in testosterone. Our data indicate that E2 acutely affects the hERG channel gating and the E4031‐induced QTC prolongation, and may provide a novel mechanism for the higher susceptibility to drug‐induced arrhythmia in women.

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.

Ginsenoside Re, a major ingredient of Panax ginseng, protects the heart against ischemia–reperfusion injury by shortening action potential duration (APD) and thereby prohibiting influx of excessive Ca2+. Ginsenoside Re enhances the slowly activating component of the delayed rectifier K+ current (IKs) and suppresses the L‐type Ca2+ current (ICa,L), which may account for APD shortening. We used perforated configuration of patch‐clamp technique to define the mechanism of enhancement of IKs and suppression of ICa,L by ginsenoside Re in guinea‐pig ventricular myocytes. S‐Methylisothiourea (SMT, 1 μM), an inhibitor of nitric oxide (NO) synthase (NOS), and N‐acetyl‐L‐cystein (LNAC, 1 mM), an NO scavenger, inhibited IKs enhancement. Application of an NO donor, sodium nitroprusside (SNP, 1 mM), enhanced IKs with a magnitude similar to that by a maximum dose (20 μM) of ginseonside Re, and subsequent application of ginsenoside Re failed to enhance IKs. Conversely, after IKs had been enhanced by ginsenoside Re (20 μM), subsequently applied SNP failed to further enhance IKs. An inhibitor of guanylate cyclase, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 10 μM), barely suppressed IKs enhancement, while a thiol‐alkylating reagent, N‐ethylmaleimide (NEM, 0.5 mM), clearly suppressed it. A reducing reagent, di‐thiothreitol (DTT, 5 mM), reversed both ginsenoside Re‐ and SNP‐induced IKs enhancement. ICa,L suppression by ginsenoside Re (3 μM) was abolished by SMT (1 μM) or LNAC (1 mM). NEM (0.5 mM) did not suppress ICa,L inhibition and DTT (5 mM) did not reverse ICa,L inhibition, whereas in the presence of ODQ (10 μM), ginsenoside Re (3 μM) failed to suppress ICa,L. These results indicate that ginsenoside Re‐induced IKs enhancement and ICa,L suppression involve NO actions. Direct S‐nitrosylation of channel protein appears to be the main mechanism for IKs enhancement, while a cGMP‐dependent pathway is responsible for ICa,L inhibition.

Role of Nitric Oxide in Ca2+ Sensitivity of the Slowly Activating Delayed Rectifier K+ Current in Cardiac Myocytes

Sarcolemmal Ca2+ entry is a vital step for contraction of cardiomyocytes, but Ca2+ overload is harmful and may trigger arrhythmias and/or apoptosis. To maintain the amount of Ca2+ entry within an appropriate range, cardiomyocytes have feedback systems that tightly regulate ion channel activities in response to the changes in intracellular Ca2+ concentration ([Ca2+]i), thereby regulating Ca2+ entry. In guinea pig ventricular myocytes, Ca2+ ionophore, A23187, induced suppression of the L-type Ca2+ currents (ICa,L) and enhancement of the slowly activating delayed rectifier K+ currents (IKs). At a low stimulation rate, ICa,L suppression and IKs enhancement contributed to the A23187-induced APD shortening with a similar magnitude, whereas at a high stimulation rate, IKs enhancement dominantly contributed to APD shortening. IKs enhancement induced by A23187 was attributable to actions of nitric oxide (NO), because they were inhibited by an inhibitor of NO synthase (NOS) and by a NO scavenger. A23187-induced alterations of APD and IKs were strongly suppressed by a NOS3 inhibitor, but barely affected by a NOS1 inhibitor, suggesting that NOS3 was responsible for NO release in this phenomenon. Inhibition of calmodulin (CaM), but not Akt, blocked the enhancement of IKs by A23187. Thus, CaM-dependent NOS3 activation confers the selective Ca2+-sensitivity on IKs. Ca2+-induced IKs enhancement and resultant APD shortening potentially act as a physiological regulatory mechanism of Ca2+ recycling, because they were observed at a physiological range of [Ca2+]i in cardiac myocytes and are induced by physiologically relevant Ca2+ loading, such as digitalis application and rise in extracellular Ca2+ concentration.

Ginsenoside Re, a main phytosterol of Panax ginseng, activates cardiac potassium channels via a nongenomic pathway of sex hormones.

Ginseng root is one of the most popular herbs throughout the world and is believed to be a panacea and to promote longevity. It has been used as a medicine to protect against cardiac ischemia, a major cause of death in the West. We have previously demonstrated that ginsenoside Re, a main phytosterol of Panax ginseng, inhibits Ca2+ accumulation in mitochondria during cardiac ischemia/reperfusion, which is attributable to nitric oxide (NO)-induced Ca2+ channel inhibition and K+ channel activation in cardiac myocytes. In this study, we provide compelling evidence that ginsenoside Re activates endothelial NO synthase (eNOS) to release NO, resulting in activation of the slowly activating delayed rectifier K+ current. The eNOS activation occurs via a nongenomic pathway of each of androgen receptor, estrogen receptor-α, and progesterone receptor, in which c-Src, phosphoinositide 3-kinase, Akt, and eNOS are sequentially activated. However, ginsenoside Re does not stimulate proliferation of androgen-responsive LNCaP cells and estrogen-responsive MCF-7 cells, implying that ginsenoside Re does not activate a genomic pathway of sex hormone receptors. Fluorescence resonance energy transfer experiments with a probe, SCCoR (single cell coactivator recruitment), indicate that the lack of genomic action is attributable to failure of coactivator recruitment. Thus, ginsenoside Re acts as a specific agonist for the nongenomic pathway of sex steroid receptors, and NO released from activated eNOS underlies cardiac K+ channel activation and protection against ischemia-reperfusion injury.

Electrophysiological effects of ginseng and ginsenoside Re in guinea pig ventricular myocytes.

Panax ginseng is a folk medicine with various cardiovascular actions; however, its underlying mechanisms of action are not well known. In the present study, we examined the effects of ginseng and its main component, ginsenoside Re, on action potentials and membrane currents recorded from isolated guinea pig ventricular myocytes with the whole-cell patch clamp technique. Ginseng (1 mg/ml) shortened the action potential duration in a rate-dependent manner. Ginseng depressed the L-type Ca2+ current (I(Ca-L)) in a mode of both tonic block and use-dependent block, and enhanced the slowly activating component of the delayed rectifier K+ current (I(Ks)). Ginsenoside Re 3 microM exhibited similar electrophysiological effects to those of 1 mg/ml ginseng, but of slightly smaller magnitude. Inhibition of I(Ca,L) and enhancement of I(Ks) by ginsenoside Re appear to be one of the main electrophysiological actions of ginseng in the heart, although contributions from other ingredients should be considered.

Involvement of local anesthetic binding sites on IVS6 of sodium channels in fast and slow inactivation

Local anesthetics (LAs) block Na(+) channels with a higher affinity for the fast or slow inactivated state of the channel. Their binding to the channel may stabilize fast inactivation or induce slow inactivation. We examined the role of the LA binding sites on domain IV, S6 (IVS6) of Na(+) channels in fast and slow inactivation by studying the gating properties of the mutants on IVS6 affecting LA binding. Mutation of the putative LA binding site, F1579C, inhibited fast and slow inactivation. Mutations of another putative LA binding site, Y1586C, and IVS6 residue involved in LA access and binding, I1575C, both enhanced fast and slow inactivation. None of the mutations affected channel activation. These results suggest that the LA binding site on IVS6 is involved in slow inactivation as well as fast inactivation, and these two gatings are coupled at the binding site.