Jihua Ma

Late Sodium Current Contributes to the Reverse Rate-Dependent Effect of IKr Inhibition on Ventricular Repolarization

Background— The reverse rate dependence (RRD) of actions of IKr-blocking drugs to increase the action potential duration (APD) and beat-to-beat variability of repolarization (BVR) of APD is proarrhythmic. We determined whether inhibition of endogenous, physiological late Na+ current (late INa) attenuates the RRD and proarrhythmic effect of IKr inhibition. Methods and Results— Duration of the monophasic APD (MAPD) was measured from female rabbit hearts paced at cycle lengths from 400 to 2000 milliseconds, and BVR was calculated. In the absence of a drug, duration of monophasic action potential at 90% completion of repolarization (MAPD90) and BVR increased as the cycle length was increased from 400 to 2000 milliseconds (n=36 and 26; P<0.01). Both E-4031 (20 nmol/L) and d-sotalol (10 &mgr;mol/L) increased MAPD90 and BVR at all stimulation rates, and the increase was greater at slower than at faster pacing rates (n=19, 11, 12 and 7, respectively; P<0.01). Tetrodotoxin (1 &mgr;mol/L) and ranolazine significantly attenuated the RRD of MAPD90, reduced BVR (P<0.01), and abolished torsade de pointes in hearts treated with either 20 nmol/L E-4031 or 10 &mgr;mol/L d-sotalol. Endogenous late INa in cardiomyocytes stimulated at cycle lengths from 500 to 4000 milliseconds was greater at slower than at faster stimulation rates, and rapidly decreased during the first several beats at faster but not at slower rates (n=8; P<0.01). In a computational model, simulated RRD of APD caused by E-4031 and d-sotalol was attenuated when late INa was inhibited. Conclusion— Endogenous late INa contributes to the RRD of IKr inhibitor–induced increases in APD and BVR and to bradycardia-related ventricular arrhythmias.

Effect of hydrogen peroxide on persistent sodium current in guinea pig ventricular myocytes

AbstractAim:To study the effect of hydrogen peroxide (H2O2) on persistent sodium current (INa.P) in guinea pig ventricular myocytes.Methods:The whole-cell, cell-attached, and inside-out patch-clamp techniques were applied on isolated ventricular myocytes from guinea pig.Results:H2O2 (0.1 mmol/L, 0.5 mmol/L and 1.0 mmol/L) increased the amplitude of whole-cell INa.P in a concentration-dependent manner, and glutathione (GSH 1 mmol/L) reversed the increased INa.P. H2O2 (1 mmol/L) increased persistent sodium channel activity in cell-attached and inside out patches. The mean open probability was increased from control values of 0.015±0.004 and 0.012±0.003 to 0.106±0.011 and 0.136±0.010, respectively (P<0.01 vs control). They were then decreased to 0.039±0.024 and 0.027±0.006, respectively, after the addition of 1 mmol/L GSH (P<0.01 vs H2O2). The time when open probability began to increase and reached a maximum was shorter in inside out patches than that in cell-attached patches (4.8±1.0 min vs 11.5±3.9 min, P<0.01; 9.6±1.6 min vs 18.7±4.7 min, P<0.01).Conclusion:H2O2 increased the INa.P of guinea pig ventricular myocytes in a concentration-dependent manner, possibly by directly oxidating the cell membrane.

Calmodulin kinase II and protein kinase C mediate the effect of increased intracellular calcium to augment late sodium current in rabbit ventricular myocytes

An increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) augments late sodium current (I(Na.L)) in cardiomyocytes. This study tests the hypothesis that both Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) mediate the effect of increased [Ca(2+)](i) to increase I(Na.L). Whole cell and open cell-attached patch clamp techniques were used to record I(Na.L) in rabbit ventricular myocytes dialyzed with solutions containing various concentrations of [Ca(2+)](i). Dialysis of cells with [Ca(2+)](i) from 0.1 to 0.3, 0.6, and 1.0 μM increased I(Na.L) in a concentration-dependent manner from 0.221 ± 0.038 to 0.554 ± 0.045 pA/pF (n = 10, P < 0.01) and was associated with an increase in mean Na(+) channel open probability and prolongation of channel mean open-time (n = 7, P < 0.01). In the presence of 0.6 μM [Ca(2+)](i), KN-93 (10 μM) and bisindolylmaleimide (BIM, 2 μM) decreased I(Na.L) by 45.2 and 54.8%, respectively. The effects of KN-93 and autocamtide-2-related inhibitory peptide II (2 μM) were not different. A combination of KN-93 and BIM completely reversed the increase in I(Na.L) as well as the Ca(2+)-induced changes in Na(+) channel mean open probability and mean open-time induced by 0.6 μM [Ca(2+)](i). Phorbol myristoyl acetate increased I(Na.L) in myocytes dialyzed with 0.1 μM [Ca(2+)](i); the effect was abolished by Gö-6976. In summary, both CaMKII and PKC are involved in [Ca(2+)](i)-mediated augmentation of I(Na.L) in ventricular myocytes. Inhibition of CaMKII and/or PKC pathways may be a therapeutic target to reduce myocardial dysfunction and cardiac arrhythmias caused by calcium overload.

The effect of ligustrazine on L-type calcium current, calcium transient and contractility in rabbit ventricular myocytes.

ETHNOPHARMACOLOGICAL RELEVANCE Ligustrazine, the biologically active ingredient isolated from a popular Chinese medicinal plant, Ligusticum chuanxiong Hort. (Umbelliferae), has been used effectively to treat ischemic heart diseases, cerebrovascular and thrombotic vascular diseases since the 1970s. MATERIALS AND METHODS At present, the effect of ligustrazine on L-type calcium current (I(Ca-L)) of ventricular myocytes remains controversial. In this study, we use the whole-cell patch-clamp techniques and video-based edge detection and dual excitation fluorescence photomultiplier systems to study the effects of ligustrazine on I(Ca-L), and calcium transient and contractility in rabbit ventricular myocytes in the absence and presence of isoprenaline (ISO). RESULTS Ligustrazine (5 μM) in low concentration did not affect I(Ca-L) (P>0.05), higher concentrations of this drug (10, 20, 40, 80 μM) inhibited I(Ca-L) in a concentration-dependent manner and reduced I(Ca-L) by 9.6 ± 2.9%, 21.0 ± 4.3%, 33.9 ± 4.3%, and 51.6 ± 7.3%, respectively. Under normal conditions, ligustrazine (40 μΜ) reduced baseline of fura-2 fluorescence intensities (FFI, 340/380 ratio), namely diastolic calcium concentration, changes in FFI (ΔFFI, 340/380 ratio) and maximal velocity of Ca(2+) rise and decay (340/380 ratio/ms) by 6.3%, 26.1%, 25.2%, and 26.5%, and decreased sarcomere peak shorting (PS) and maximal velocity of shorting and relengthening by 36.4%, 31.9%, and 25.0%, respectively. Similarly, ligustrazine (40 μM) reduced baseline FFI, ΔFFI, and maximal velocity of Ca(2+) rise and decay by 14.1%, 51.1%, 35.2%, and 41.1%, and reduced sarcomere PS and maximal velocity of shorting and relengthening by 38.6%, 50.0% and 39.1%, respectively, in the presence of ISO. CONCLUSIONS Ligustrazine not only significantly inhibits I(Ca-L) in a concentration-dependent manner but also suppressed calcium transient and contraction in the absence and presence of ISO.

Resveratrol attenuates the Na(+)-dependent intracellular Ca(2+) overload by inhibiting H(2)O(2)-induced increase in late sodium current in ventricular myocytes.

Background/Aims Resveratrol has been demonstrated to be protective in the cardiovascular system. The aim of this study was to assess the effects of resveratrol on hydrogen peroxide (H2O2)-induced increase in late sodium current (I Na.L) which augmented the reverse Na+-Ca2+ exchanger current (I NCX), and the diastolic intracellular Ca2+ concentration in ventricular myocytes. Methods I Na.L, I NCX, L-type Ca2+ current (I Ca.L) and intracellular Ca2+ properties were determined using whole-cell patch-clamp techniques and dual-excitation fluorescence photomultiplier system (IonOptix), respectively, in rabbit ventricular myocytes. Results Resveratrol (10, 20, 40 and 80 µM) decreased I Na.L in myocytes both in the absence and presence of H2O2 (300 µM) in a concentration dependent manner. Ranolazine (3–9 µM) and tetrodotoxin (TTX, 4 µM), I Na.L inhibitors, decreased I Na.L in cardiomyocytes in the presence of 300 µM H2O2. H2O2 (300 µM) increased the reverse I NCX and this increase was significantly attenuated by either 20 µM resveratrol or 4 µM ranolazine or 4 µM TTX. In addition, 10 µM resveratrol and 2 µM TTX significantly depressed the increase by 150 µM H2O2 of the diastolic intracellular Ca2+ fura-2 fluorescence intensity (FFI), fura-fluorescence intensity change (△FFI), maximal velocity of intracellular Ca2+ transient rise and decay. As expected, 2 µM TTX had no effect on I Ca.L. Conclusion Resveratrol protects the cardiomyocytes by inhibiting the H2O2-induced augmentation of I Na.L.and may contribute to the reduction of ischemia-induced lethal arrhythmias.

Verapamil blocks HERG channel by the helix residue Y652 and F656 in the S6 transmembrane domain

AbstractAim:The objectives of this study were to investigate the inhibitory action of verapamil on wild-type(WT) and mutation HERG K+ channel current (IHERG), and to determine whether mutations in the S6 region are important for the inhibition of IHERG by verapamil.Methods:HERG channels (WT, Y652A, and F656A) were expressed in oocytes of Xenopus laevis and studied using the 2-electrode voltage-clamp technique.Results:WT HERG is blocked in a concentration-dependent manner by verapamil (half-maximal inhibition concentration [IC50;[equals;5.1 μmol/L), and the steady state activation and inactivation parameters are shifted to more negative values. However, mutation to Ala of Y652 and F656 located on the S6 domain produced 16-fold and 20-fold increases in IC50 for IHERG blockade, respectively. Simultaneously, the steady state activation and inactivation parameters for Y652A are also shifted to more negative values in the presence of the blockers.Conclusion:Verapamil preferentially binds to and blocks open HERG channels. Tyr-652 and Phe-656, 2 aromatic amino-acid residues in the inner (S6) helix, are critical in the verapamil-binding site.

Sodium Channel Gating Modes During Redox Reaction

Background/Aims: Many studies have confirmed that persistent sodium current (I_NaP) is altered during a redox reaction, but little attention has been paid to transient sodium current (I_NaT) and its correlation with I_NaP during the redox reaction. The aim of the study was to investigate the effect of the redox states on the correlation between I_NaT and I_NaP in cardiomyocytes. Methods: I_NaT and I_NaP were recorded using whole-cell and cell-attached patch-clamp techniques in guinea pig ventricular myocytes. Results: In whole-cell recordings, dithiothreitol (DTT, 1 mM) simultaneously increased I_NaT and decreased I_NaP. Hydrogen peroxide (H₂O₂, 0.3 mM) increased I_NaP and decreased I_NaT in a time-dependent manner, which were reversed by DTT (1 mM). In cell-attached recordings, the increasing of I_NaP and decreasing of I_NaT induced by H₂O₂ (0.3 mM) were similarly recovered by DTT (1 mM). H₂O₂ (0.3 mM) prolonged the action potential (AP) duration of ventricular papillary cells whereas decreased the AP amplitude and maximum rate of depolarization (V_max) in a time-dependent manner, which were reversed by DTT (1 mM). Conclusion: These results indicate that the redox states could modulate the sodium channel gating modes in guinea pig ventricular myocytes.

Modulation of KATP currents in rat ventricular myocytes by hypoxia and a redox reaction

AbstractAim:The present study investigated the possible regulatory mechanisms of redox agents and hypoxia on the KATP current (IKATP) in acutely isolated rat ventricular myocytes.Methods:Single-channel and whole-cell patch-clamp techniques were used to record the KATP current (IKATP) in acutely isolated rat ventricular myocytes.Results:Oxidized glutathione (GSSG, 1 mmol/L) increased the IKATP, while reduced glutathione (GSH, 1 mmol/L) could reverse the increased IKATP during normoxia. To further corroborate the effect of the redox agent on the KATP channel, we employed the redox couple DTT (1 mmol/L)/H2O2 (0.3, 0.6, and 1 mmol/L) and repeated the previous processes, which produced results similar to the previous redox couple GSH/GSSG during normoxia. H2O2 increased the IKATP in a concentration dependent manner, which was reversed by DTT (1 mmol/L). In addition, our results have shown that 15 min of hypoxia increased the IKATP, while GSH (1 mmol/L) could reverse the increased IKATP. Furthermore, in order to study the signaling pathways of the IKATP augmented by hypoxia and the redox agent, we applied a protein kinase C(PKC) inhibitor bisindolylmaleimide VI (BIM), a protein kinase G(PKG) inhibitor KT5823, a protein kinase A (PKA) inhibitor H-89, and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-62 and KN-93. The results indicated that BIM, KT5823, KN-62, and KN-93, but not H-89, inhibited the IKATP augmented by hypoxia and GSSG; in addition, these results suggest that the effects of both GSSG and hypoxia on KATP channels involve the activation of the PKC, PKG, and CaMK II pathways, but not the PKA pathway.Conclusion:The present study provides electrophysiological evidence that hypoxia and the oxidizing reaction are closely related to the modulation of IKATP.

Larger late sodium current density as well as greater sensitivities to ATX II and ranolazine in rabbit left atrial than left ventricular myocytes

An increase of cardiac late sodium current (INa.L) is arrhythmogenic in atrial and ventricular tissues, but the densities of INa.L and thus the potential relative contributions of this current to sodium ion (Na(+)) influx and arrhythmogenesis in atria and ventricles are unclear. In this study, whole-cell and cell-attached patch-clamp techniques were used to measure INa.L in rabbit left atrial and ventricular myocytes under identical conditions. The density of INa.L was 67% greater in left atrial (0.50 ± 0.09 pA/pF, n = 20) than in left ventricular cells (0.30 ± 0.07 pA/pF, n = 27, P < 0.01) when elicited by step pulses from -120 to -20 mV at a rate of 0.2 Hz. Similar results were obtained using step pulses from -90 to -20 mV. Anemone toxin II (ATX II) increased INa.L with an EC50 value of 14 ± 2 nM and a Hill slope of 1.4 ± 0.1 (n = 9) in atrial myocytes and with an EC50 of 21 ± 5 nM and a Hill slope of 1.2 ± 0.1 (n = 12) in ventricular myocytes. Na(+) channel open probability (but not mean open time) was greater in atrial than in ventricular cells in the absence and presence of ATX II. The INa.L inhibitor ranolazine (3, 6, and 9 μM) reduced INa.L more in atrial than ventricular myocytes in the presence of 40 nM ATX II. In summary, rabbit left atrial myocytes have a greater density of INa.L and higher sensitivities to ATX II and ranolazine than rabbit left ventricular myocytes.

Sophocarpine Attenuates the Na+-dependent Ca2+ Overload Induced by anemonia Sulcata Toxin—increased Late Sodium Current in Rabbit Ventricular Myocytes

Abstract: Many studies indicate that an increase in late sodium current (INa.L) of cardiomyocytes causes intracellular Na+ overload and subsequently raises the reverse Na+/Ca2+ exchanger current (INCX), ultimately resulting in intracellular Ca2+ overload. Therefore, using drugs to inhibit the increased INa.L under various pathological conditions can lower intracellular Ca2+ overload. This study was intended to explore the effect of sophocarpine (SOP) on the increase in INa.L, INCX, calcium transient and contraction in rabbit ventricular myocytes induced by Anemonia sulcata toxin II (ATX II), an opener of sodium channel, with the application of whole-cell patch-clamp techniques, the video-based motion edge detection system, and the intracellular calcium concentration determination system. The results indicate that tetrodotoxin (TTX, 4 &mgr;M ) obviously decreased INa.L and INCX enlarged by ATX II (30 nM), and SOP (20, 40, and 80 &mgr;M) also inhibited both the parameters concentration dependently in rabbit ventricular myocytes. However, transient sodium current remained unaffected by the above-mentioned concentrations of ATX II, TTX, and SOP. In addition, SOP also reversed diastolic calcium concentration, calcium transient amplitude, and ventricular muscle contractility augmented by ATX II. Its effects were similar to those of TTX, a specific inhibitor of the sodium channel. In conclusion, SOP inhibits INa.L, INCX, diastolic Ca2+ concentration, and contractility in rabbit ventricular myocytes, which suggests that relief of intracellular Ca2+ overload through inhibiting INa.L is likely to become a new therapeutic mechanism of SOP against arrhythmia and myocyte damage associated with intracellular Ca2+ overload.