ORIGINAL ARTICLE
Ranolazine Attenuates Hypoxia- and Hydrogen Peroxide-induced Increases in Sodium Channel Late Openings in Ventricular Myocytes Jihua Ma, MSc,* Yejia Song, MD,† John C. Shryock, PhD,‡ Liangkun Hu, MSc,* Weiping Wang, MD,* Xisheng Yan, MD,* Peihua Zhang, BSc,* and Luiz Belardinelli, MD‡
Abstract: Ranolazine attenuates cardiac arrhythmic activity associated with hypoxia and hydrogen peroxide (H2O2) by inhibition of late sodium current (late INa). The mechanism of ranolazine’s action on Na+ channels was investigated using whole-cell and single-channel recording from guinea pig isolated ventricular myocytes. Hypoxia increased whole-cell late INa from 20.48 6 0.02 to 23.99 6 0.07 pA/pF. Ranolazine at 3 and 9 mmol/L reduced the hypoxia-induced late INa by 16% 6 3% and 55% 6 3%, respectively. Hypoxia increased the mean open probability and open time of Na+-channel late openings from 0.016 6 0.001 to 0.064 6 0.007 milliseconds and from 0.693 6 0.043 to 1.081 6 0.098 milliseconds, respectively. Ranolazine at 3 and 9 mmol/L attenuated the hypoxia-induced increase of open probability by 19% 6 7% and 61% 6 1%, and increase of open time by 26% 6 19% and 74 6 21%, respectively. H2O2 increased the mean open probability and open time of Na+-channel late openings from 0.013 6 0.002 to 0.107 6 0.015 milliseconds and from 0.689 6 0.075 to 1.487 6 0.072 milliseconds, respectively. Ranolazine at 3 and 6 mmol/L reduced the H2O2-induced increase of mean open probability by 60% 6 7% and 95% 6 2%, and the increase of mean open time by 31% 6 21% and 82% 6 8%. In conclusion, the inhibition by ranolazine of hypoxia- and H2O2-stimulated late INa is due to reduction of both the open probability and open time of Na+-channel late openings. Key Words: ranolazine, late sodium current, hypoxia, hydrogen peroxide, cardiac myocytes (J Cardiovasc Pharmacol Ô 2014;64:60–68)
INTRODUCTION Myocardial hypoxia and excessive production of reactive oxygen species, such as superoxide anion (O22) and Received for publication December 20, 2013; accepted February 16, 2014. From the *Cardio-Electrophysiological Research Laboratory, Wuhan University of Science and Technology, Wuhan, China; †Division of Cardiovascular Medicine, University of Florida, Gainesville, FL; and ‡Gilead Sciences, Foster City, CA. Supported by Gilead Sciences. Jihua Ma, MSc, and Yejia Song, MD, have received a grant (to their institutions) from Gilead Sciences. John C. Shryock, PhD, is a consultant for and received payment from Gilead Sciences. Luiz Belardinelli, MD, is employed by and holds stock in Gilead Sciences. The other authors report no conflicts of interest. Reprints: Luiz Belardinelli, MD, Gilead Sciences, Inc., Lakeside Drive, Foster City, CA 94404 (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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hydrogen peroxide (H2O2), are contributors to cardiac ischemia-reperfusion injury.1 Both hypoxia and H2O2 have been shown to increase the late inward sodium current (late INa) in cardiac myocytes.2–6 Unlike the brief, high-amplitude, fast INa that underlies the upstroke of the cardiac action potential (AP), late INa is normally a small inward current that persists throughout the duration of the AP plateau.7 An increase of late INa due to hypoxia, H2O2, heart failure, or Na+-channel toxins such as anemone toxin II (ATX-II) can cause a significant increase of sodium influx leading to Na+ and Ca2+ overload in the heart, and is associated with increases of diastolic tension and arrhythmic activity.6,8–11 In whole-cell recordings, late INa appears as a persistent inward current, whereas in single-channel measurements, it manifests as brief channel openings during a prolonged depolarization. Ranolazine is a cardioprotective agent currently used for the treatment of chronic stable angina.12 Ranolazine preferentially inhibits late relative to peak INa with a selectivity of 9–38fold.13–15 In whole-cell recordings, ranolazine significantly reduces H2O2- and ATX-II-stimulated late INa and suppresses H2O2- and ATX-II-induced AP after depolarizations (both early and delayed) in isolated cardiac myocytes.6,16 Ranolazine also reduces Na+/Ca2+ loading and diastolic dysfunction in ischemic or H2O2-treated isolated hearts17–19 and heart tissues and/or myocytes.6,10,20 The effect of ranolazine on hypoxiainduced late INa has not been studied to date. The objective of this study was to determine the effect of ranolazine on hypoxia-induced late INa. When initial results suggested that ranolazine reduced whole-cell late INa in myocytes exposed to hypoxia, additional studies were done using single-channel recording to better understand the mechanism of ranolazine’s effect on Na+-channel function during hypoxia and in the presence of H2O2 (ie, oxidative stress). For comparison, the effects of the Na+-channel blockers lidocaine and tetrodotoxin (TTX) on late INa were also determined.
METHODS Cell Isolation All animal procedures conform to the Guide for the Care and Use of Laboratory Animals issued by the Administrative Regulation of Laboratory Animals of Hubei Province. Adult guinea pigs of either sex weighing 250–300 g were anesthetized with pentobarbital sodium (30 mg/kg, J Cardiovasc Pharmacol ä Volume 64, Number 1, July 2014
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Ranolazine Attenuates Hypoxia-induced Late Na Current
intraperitoneal) 20 minutes after an intraperitoneal injection of 2000 units of heparin. Hearts were rapidly excised and perfused retrogradely through the aorta on a Langendorff apparatus with oxygenated and pre-warmed (378C) solutions in the following order: (1) Ca2+-free solution containing (in mmol/L) NaCl 135, KCl 5.4, MgCl2 1, NaH2PO4 0.33, HEPES 10, and glucose 10, pH 7.4, for 5 minutes; (2) Ca2+-free solution containing (in g/L) collagenase type I 0.1, protease E 0.01, and albumin 0.5, for 8–10 minutes; and (3) KB solution containing (in mmol/L) KOH 70, taurine 20, glutamic acid 50, KCl 40, KH2PO4 20, MgCl2 3, EGTA 0.5, HEPES 10, and glucose 10, pH 7.4, for 5 minutes. At the end of perfusion, the ventricles were cut into small chunks and gently agitated in KB solution to free individual cells. The cell suspension was filtered through nylon mesh and stored in KB solution at 48C.
bath solutions.21 The oxygen tension of bath solutions was monitored with an ISO oxygen meter (WPI, Sarasota, FL). The PO2 of the medium in the recording chamber dropped quickly from about 13 kPa (normoxia; 100 mm Hg) to 1.33– 2.66 kPa (hypoxia; 10–20 mm Hg) in 3–5 minutes during continued perfusion of nitrogen-saturated solutions. For each group of experiments, the PO2 of bath solution was maintained constant throughout an experiment.
Chemicals Collagenase type I and CsCl were obtained from Gibco (Invitrogen, Paisley, United Kingdom). Bovine serum albumin, HEPES, and taurine were obtained from Roche (Basel, Switzerland). TTX was purchased from Hebei Fisheries Research Institute (Qinhuangdao, China). Ranolazine was
Electrophysiological Recording Myocytes were placed into a recording chamber and superfused with bath solutions as indicated below. Whole-cell or cell-attached single-channel membrane currents were recorded using an EPC-9 amplifier (Heka Electronic, Lambrecht, Pfalz, Germany) and PulseFit and TACFit software. Sodium channels were activated by 300- or 500-millisecond depolarizing pulses from a holding potential of 2100 (for whole-cell recording) or 2120 mV (for single-channel recording) to various test potentials. Late INa was determined by measuring the whole-cell or the single-channel current 200 milliseconds after the onset of a depolarizing pulse. Current signals were filtered at 2 kHz and digitized at 10 kHz. Capacitance transients and leak currents were nulled off-line. All experiments were carried out at 21 6 28C. In whole-cell experiments, the recording pipettes had a resistance of 1.5–3 MV when filled with a solution containing (in mmol/L) CsCl 120, CaCl2 1, MgCl2 5, Na2ATP 5, TEA-Cl 10, EGTA 11, and HEPES 10, pH 7.3. To record the late sodium current, cells were superfused with Tyrode solution containing (in mmol/L) NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1, NaH2PO4 0.33, and HEPES 20, pH 7.4. For recording the peak (transient) sodium current, the Tyrode solution contained (in mmol/L) NaCl 10, CsCl 130, CaCl2 1, MgCl2 2, CdCl2 0.05, HEPES 5, and glucose 5, pH 7.4. For cell-attached single-channel recordings, the recording pipettes had a resistance of 6–10 MV when filled with a pipette solution composed of (in mmol/L): NaCl 180, KCl 1.3, CaCl2 1.5, MgCl2 0.5, Na2ATP 5, CoCl2 2.0, TEACl 10, 4-AP 10, CsCl 10, HEPES 5, and glucose 5, pH 7.4. After obtaining the cell-attached configuration, the bath solution was switched from Tyrode solution to a high-potassium solution containing (in mmol/L): K-acetate 140, NaCl 5, MgCl2 2, CdCl2 0.05, HEPES 10, and glucose 25, pH 7.4, to bring the cell membrane potential close to 0 mV.
Induction of Hypoxia Hypoxia was induced and maintained by bubbling the bath solutions in flasks with 100% nitrogen. To minimize diffusion of air into bath solutions, a cover was placed over the recording chamber, and a constant flow of 100% N2 was delivered to the chamber to form a laminar layer of N2 over Ó 2014 Lippincott Williams & Wilkins
FIGURE 1. Inhibition by tetrodotoxin (TTX, 2 mmol/L) of a hypoxia-induced increase of whole-cell late INa in guinea pig isolated ventricular myocytes. (A) Superimposed current traces recorded from a myocyte sequentially superfused with normoxic bath solution (a, control), hypoxic bath solution (b, hypoxia), hypoxic bath solution containing 2 mmol/L TTX (c), or hypoxic bath solution during washout of TTX (d). Late INa was activated by a 300-millisecond voltage-clamp pulse from 2100 to 230 millivolts. (B) Summary data (mean 6 SD) for amplitudes of late INa in 6 myocytes. * and † indicate P , 0.001 versus control and hypoxia alone, respectively. www.jcvp.org |
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obtained from Gilead Sciences (Foster City, CA). All other chemicals were purchased from Sigma (St. Louis, MO).
Data Analysis Single-channel records were analyzed using TAC + TACFit (Bruxton, Seattle, WA). Channel openings were detected using half-amplitude threshold analysis. Channel open probability was calculated from the total of channel open times during 100 sweeps divided by the total sweep duration. Histograms of channel open time distribution were fitted to single exponentials using TACFit. Statistical significance between test groups was evaluated by one-way analysis of variance. The Holm–Sidak method was used when needed. All values were expressed as mean 6 SD. The number of cells/patches tested in each group is indicated by “n.” A P , 0.05 was considered to indicate a statistically significant difference.
RESULTS Effects of Ranolazine and TTX on Hypoxia-induced Whole-cell Late INa The amplitude of late INa was small and stable during 30 minutes of normoxia (ie, the duration of an experiment). During normoxia, the amplitudes of late INa elicited by 300millisecond test pulses from 2100 to 230 mV at 0, 10, 20, and 30 minutes after establishment of whole-cell configuration were 20.66 6 0.14, 20.66 6 0.16, 20.67 6 0.13, and 20.68 6 0.13 pA/pF, respectively (n = 6, P . 0.05). In comparison, inward late INa steadily increased when myocytes were exposed to hypoxic medium equilibrated with 100% N2. The value of PO2 in the myocyte chamber perfused with hypoxic medium for 20 minutes ranged from 1.33 to 2.66 kPa (10–20 mm Hg). In myocytes (n = 6) exposed to hypoxia for 20 minutes, the amplitude of late INa at 230 mV was increased from 20.51 6 0.55 (0 time, control) to 22.92 6 0.48 pA/pF (P , 0.001 vs. control; Fig. 1). Hypoxia-stimulated late INa was decreased by 2 mmol/L TTX from 22.92 6 0.48 to 20.77 6 0.53 pA/pF (n = 6, P , 0.001) (Fig. 1). The effect of TTX was reversible (Fig. 1). The high sensitivity to TTX inhibition is consistent with the identification of late current as INa. The concentration- and voltage-dependence of the effects of ranolazine on hypoxia-stimulated late INa were studied in separate series of experiments. In the concentrationdependence study, late INa was activated by depolarizing pulses from 2100 to 230 mV, and ranolazine was applied
FIGURE 2. Inhibition by ranolazine (Ran) of a hypoxia (H)induced increase of whole-cell late INa in guinea pig isolated ventricular myocyte. (A) Superimposed current traces obtained from a myocyte sequentially exposed to normoxic bath solution (a, control), hypoxic bath solution (b), hypoxic bath solution containing 3 or 9 mmol/L Ran (c and d), and hypoxic bath solution during washout of Ran (e). Late INa was activated by a 300-millisecond voltage-clamp pulse from 2100 to 230
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mV. (B) Summary data (mean 6 SD) for concentrationdependent inhibition by Ran of hypoxia-induced late INa in 8 myocytes. * and † indicate P , 0.001 versus control and hypoxia alone, respectively. (C) Current-voltage relationship for late INa recorded from 8 myocytes superfused sequentially with normoxic bath solution (control), with hypoxic bath solution in the absence (hypoxia) and presence of 9 mmol/L Ran, and with hypoxic solution after washout of Ran. Late INa was activated from a holding potential of 2100 mV to various test potentials ranging from 290 to +20 mV. Ó 2014 Lippincott Williams & Wilkins
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FIGURE 3. Ranolazine (Ran) reduced single Na+-channel late openings in ventricular myocytes exposed to hypoxia (H). (A) (a–f) Ran (1, 3, and 9 mM) concentration-dependently inhibited hypoxia-induced Na+-channel late openings in a cell-attached patch. Four current traces are shown for each treatment. The upper 3 traces are original single-channel recordings, and the bottom trace is the ensemble average current of 50–100 sweeps. Current and time scales and the voltage-clamp protocol are shown at the bottom of the panel. (B) Amplitude (left) and open-time (t, right) histograms of Na+-channel late openings in a cell-attached patch superfused with hypoxic solution in the absence and presence of 9 mmol/L Ran. Ran reduced Na+-channel open probability and mean open time in the presence of hypoxia.
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FIGURE 4. Summary data for the concentrationdependent reduction by ranolazine (Ran) of mean open probability and open time of Na+channel late openings shown in Figure 3. Bars represent average values obtained from 8 cells. * and † indicate P , 0.001 versus control and hypoxia alone, respectively.
in the bath solution (ie, through superfusate) in the presence of hypoxia. Hypoxia increased the amplitude of late INa from 20.47 6 0.01 to 23.99 6 0.07 pA/pF (n = 8, P , 0.001; Fig. 2). The addition of ranolazine to the hypoxic superfusate for 6–10 minutes concentration-dependently reduced hypoxia-stimulated late INa (Fig. 2). Ranolazine sequentially applied at concentrations of 1, 3, and 9 mmol/L significantly (n = 8, P , 0.001) attenuated hypoxia-induced late INa by 8% 6 4%, 16% 6 3%, and 55% 6 3%, respectively, from 23.99 6 0.07 to 23.69 6 0.13, 23.39 6 0.07, and 22.04 6 0.11 pA/pF (Fig. 2B). The effect of ranolazine was reversible after the drug was washed out (e in Fig. 2A). The amplitude of late INa in the presence hypoxia after washout of ranolazine was 23.78 6 0.04 pA/pF, which was not significantly different from the value
FIGURE 5. Effect of tetrodotoxin (TTX, 2 mmol/L in pipette solution) to block the time-dependent increases in mean open probability and open time of Na+-channel late openings in cell-attached patches during superfusion of myocytes (n = 7) with hypoxic bath solution. and : indicate hypoxia alone; B and 6indicate the values in the presence of TTX. Na+channel openings were activated by 500-millisecond voltageclamp pulses from 2100 to 250 mV.
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obtained before addition of ranolazine (23.99 6 0.07 pA/pF; Fig. 2B). The voltage dependence of hypoxia-induced late INa was examined in the absence and presence of 9 mmol/L ranolazine (Fig. 2C). Late INa was activated by depolarizing pulses from 2100 mV to various test potentials from 290 to +20 mV, and ranolazine was applied after a 20-minute exposure of myocytes (n = 8) to hypoxia. Inward late INa could be detected within a broad range of membrane potentials from 270 to +20 mV and was maximal at a potential of approximately 220 mV (Fig. 2C). The amplitudes of late INa at test potentials between 250 to +10 mV were markedly increased by hypoxia (Fig. 2C). The stimulatory effect of hypoxia on late INa was reversibly attenuated by ranolazine (Fig. 2C). At a test potential of 230 mV, for example, the amplitude of late INa was increased by hypoxia from 20.94 6 0.14 to 22.46 6 0.28 pA/pF (P , 0.05) and was decreased by ranolazine from 22.46 6 0.28 to 21.25 6 0.21 pA/pF (P , 0.05) in the continued presence of hypoxia. Neither hypoxia nor ranolazine altered the voltage-dependence of late INa (Fig. 2C). The effects of hypoxia and ranolazine on the peak (transient) INa were also studied. In the peak current experiments (n = 8), the concentration of sodium in the bath solution was reduced to 10 mmol/L, and the current was activated by depolarizing voltage-clamp pulses from 2100 to 230 mV. Ranolazine was sequentially applied at concentrations of 1, 3, and 9 mmol/L. The duration of ranolazine treatment at each concentration was 10 minutes. In contrast to the significant increase of late INa, a progressive decrease of the peak (transient) INa was observed in the presence of hypoxia. The decreasing process of peak INa was not altered after treatment with ranolazine. The amplitude of peak INa in the presence of normoxia, hypoxia (15 minutes), hypoxia plus 1, 3, and 9 mmol/L ranolazine, and hypoxia alone (wash out ranolazine) was 232 6 0.6, 224 6 0.6, 222 6 0.9, 221 6 0.7, 220 6 0.7, and 218 6 0.9 pA/pF, respectively. The inhibitory effect of hypoxia and the lack of effect of ranolazine on the peak INa were consistent with the result of our previous study22 and with the preferential action of ranolazine on the late INa.13–15 Ó 2014 Lippincott Williams & Wilkins
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FIGURE 6. Ranolazine (Ran, 6 mmol/L) blocked H2O2 (300 mmol/L)-induced Na+-channel late openings. (A) Single-channel recordings obtained from a cell-attached patch in the absence (control) and presence of H2O2 alone or H2O2 plus Ran. In each treatment, the upper 3 traces are original records and the bottom trace is the ensemble average current of 50–100 sweeps. Current and time scales and voltage-clamp protocol are shown at the bottom. (B) Amplitude (left) and open-time (right) histograms of Na+-channel late openings in a patch in the absence and presence of H2O2 and H2O2 plus Ran.
Effects of Ranolazine and TTX on Hypoxiastimulated Late Openings of Single Na+ Channels Single Na+-channel activity was studied using the cellattached patch-clamp configuration. Na+ channels were activated by 500-millisecond depolarizing pulses to 250 mV from a holding potential of 2120 mV. During normoxia, Na+-channel late openings were rare (a in Fig. 3A). Hypoxia markedly increased the mean open probability and open time of Na+-channel late openings (b in Fig. 3A). In a series of experiments (n = 8), a 20-minute hypoxia treatment increased the mean open probability from 0.016 6 0.001 to 0.064 6 0.007 (P , 0.001), and the mean open time from 0.693 6 0.042 to 1.081 6 0.097 milliseconds (P , 0.001) (Fig. 4). Ranolazine at 3 and 9 mmol/L significantly (P , 0.001) attenuated hypoxia-induced increases in open probability by 19% 6 7% and 61% 6 1% and in open time by 26% 6 19% and 74% 6 21%, respectively (Fig. 4). The inhibitory effect of ranolazine on hypoxia-induced Na+-channel late openings was reversed on drug washout (f in Fig. 3A and Fig. 4). In the absence of drugs, hypoxia alone (n = 7) steadily increased both the open probability and open time Ó 2014 Lippincott Williams & Wilkins
of Na+-channel late openings for up to 40 minutes (Fig. 5). When the pipette solution contained 2 mmol/L TTX (n = 7), there were no significant changes in either the open probability or the open time in the presence of hypoxia over the same 40-minute period of time (Fig. 5).
Effects of Ranolazine and Lidocaine on H2O2stimulated Late Openings of Single Na+ Channels In whole-cell experiments, ranolazine and lidocaine have been reported to inhibit H2O2-induced late INa.4,6 In this study, the effects of ranolazine and lidocaine on H2O2induced single Na+-channel late openings were investigated and compared. In these experiments, Na+ channels were activated by 500-millisecond depolarizing pulses from 2120 to 250 mV in the cell-attached patch-clamp configuration. Exposure of myocytes to H2O2 (300 mmol/L, n = 23) for 15 minutes resulted in significant (P , 0.001) increases in the mean open probability and open time of Na+-channel late openings, from 0.013 6 0.002 to 0.107 6 0.015 milliseconds and from 0.689 6 0.075 to 1.487 6 0.072 milliseconds, respectively (Figs. 6, 7). The stimulatory effect of H2O2 on www.jcvp.org |
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FIGURE 7. Summary of the effects of ranolazine (Ran, 1, 3, and 6 mmol/L) and H2O2 (300 mmol/L) on open probability and open time of Na+-channel late openings in the cellattached patch. Na+-channels were activated by 500-millisecond voltage-clamp pulses from 2120 to 250 mV. Ran concentration-dependently attenuated both H2O2-induced increases in open probability and open time. Bars represent values obtained in the presence of no drugs (control, n = 23), H2O2 alone (n = 23), and H2O2 plus 1 (n = 6), 3 (n = 7), or 6 (n = 10) mmol/L Ran, as indicated. * and † indicate P , 0.001 versus control and H2O2 alone, respectively.
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Na+-channel late openings was significantly attenuated by ranolazine in a concentration-dependent manner. Ranolazine at 3 (n = 7) and 6 (n = 10) mmol/L significantly (P , 0.001) reduced the H2O2-induced increase of mean open probability by 60% 6 7% and 95% 6 2%, and the H2O2-induced increase of mean open time by 31% 6 21% and 82% 6 8%, respectively (Figs. 6, 7). In parallel experiments with lidocaine, H2O2 (300 mmol/L, n = 12) alone increased the mean open probability and open time from 0.013 6 0.002 to 0.108 6 0.012 milliseconds and from 0.699 6 0.075 to 1.491 6 0.068 milliseconds, respectively (Figs. 8, 9). In the presence of 30 (n = 6) and 60 (n = 6) mmol/L lidocaine, the H2O2-induced increase of open probability was attenuated by 58% 6 9% and 94% 6 3%, and the increase of open time was reduced by 27% 6 8% and 85% 6 5%, respectively (Figs. 8, 9). Thus, the inhibitory effects of lidocaine at 30 and 60 mmol/L were comparable with those of ranolazine at 3 and 6 mmol/L. The effect of lidocaine to attenuate H2O2-induced late INa is consistent with previous findings.4,23 In the absence of other drugs, H2O2 (300 mmol/L) alone steadily increased the mean open probability and open time of Na+-channel late openings for up to 40 minutes (Fig. 10).
FIGURE 8. Inhibition by 60 mmol/L lidocaine (Lido) of H2O2 (300 mmol/L)-induced Na+-channel late openings. (A) Single-channel recordings obtained from a cell-attached patch. The upper 3 traces are original records, and the bottom trace is the ensemble average current of 50–100 sweeps. Current and time scales and voltage-clamp protocol are shown at the bottom. (B) Amplitude (left) and open-time (right) histograms of Na+-channel late openings in a patch in the absence and presence of H2O2 and H2O2 plus Lido.
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DISCUSSION
FIGURE 9. Summary of the effects of lidocaine (Lido, 30 and 60 mmol/L) and H2O2 (300 mmol/L) on open probability and open time of Na+-channel late openings in the cell-attached patch. Na+ channels were activated by 500-millisecond voltage-clamp pulses from 2120 to 250 mV. Bars represent values obtained in the presence of no drugs (control, n = 12), H2O2 alone (n = 12), and H2O2 plus 30 (n = 6) or 60 (n = 6) mmol/L Lido, as indicated. The inhibitory effects of Lido at 30 and 60 mmol/L were comparable to those of ranolazine at 3 and 6 mmol/L, respectively (see Fig. 7). * and † indicate P , 0.001 versus control and H2O2 alone, respectively.
When TTX (2 mmol/L, n = 7) was included in the pipette solution, the stimulatory effect of H2O2 (300 mmol/L, 30 minutes) on Na+-channel late openings was abolished (Fig. 10).
FIGURE 10. Time-course of actions of H2O2 (300 mmol/L) on Na+-channel late openings in cell-attached patches in the absence and presence of tetrodotoxin (TTX, 2 mmol/L in the pipette solution). Na+ channels were activated by 500-millisecond voltage-clamp pulses from 2120 to 250 mV. In the absence of other drugs (n = 7), H2O2 steadily increased the open probability () and open time (:). In the presence of TTX (n = 7), the actions of H2O2 on channel open probability (B) and open time (6) were completely blocked. Ó 2014 Lippincott Williams & Wilkins
The antianginal drug ranolazine was shown to inhibit hypoxia- and H2O2-induced late INa in this study of guinea pig isolated ventricular myocytes. Acute exposures of myocytes to either hypoxia (PO2, 1.33–2.66 kPa) or H2O2 caused marked increases of late INa in whole cells (Figs. 1, 2 and Song et al, 2006) and cell-attached patches (Figs. 3–10). Ranolazine inhibited hypoxia- and H2O2-stimulated late INa by reducing both the open probability and open time of Na+channel late openings (Figs. 3, 4, 6, 7). These findings are consistent with previous reports that ranolazine reduces whole-cell late INa in myocytes isolated from dog failing hearts,14 and late INa in rabbit and guinea pig ventricular myocytes treated with the late INa enhancers ATX-II or H2O2.6,10,16 The effects of hypoxia to increase late INa and to increase Na+-channel mean open time and open probability were rapid and substantial (Figs. 3–5). Significant increases in these parameters occurred within 10 minutes of the onset of superfusion with a hypoxic bathing solution (Fig. 5) or with H2O2 (Fig. 10). The mechanism by which acute hypoxia leads to changes in Na+-channel function that increase late INa is not clear. Increases of lysophosphatidylcholine, palmitoyl-Lcarnitine, and other intermediary metabolites may occur rapidly during hypoxia24 and are reported to increase late INa.25,26 Activations of protein kinase C27,28 and Ca2+/calmodulindependent protein kinase II (CaMKII) subtype dC are reported to occur during hypoxia and oxidative stress,29 and oxidation of CaMKII markedly increases its sensitivity to Ca2+, such that, CaM-independent activity of the kinase is increased.30,31 Both kinases can phosphorylate the cardiac Na+ channels and may increase late INa.32–35 In addition, redox regulation of the Na+ channels has been proposed to modulate late INa.22 Further investigation is needed to ascertain the mechanisms by which hypoxia disrupts Na+-channel function to increase late INa. Ranolazine is not reported to directly inhibit the activities of CaMKII, protein kinase C, or other protein kinases. There are no published reports of the effect of ranolazine on Na+-channel late current in the ischemic heart, because to the best of our knowledge, it is not possible to measure an ion channel current in the ischemic environment. However, ranolazine reduces Na+/Ca2+ overload, diastolic dysfunction, and arrhythmic activity caused by ischemia.18,21 The effects of several components of the ischemic environment on Na+-channel late current have been studied in the absence and presence of ranolazine. Ranolazine reduces the increases of whole-cell late INa caused by exposure of cardiac myocytes to the ischemic metabolite palmitoyl-L-carnitine,36 to acidosis,37 to reactive oxygen species,6 to activation of CaMKII,38 and in this study, to hypoxia. A limitation of this study is that we did not investigate the intracellular signaling by which hypoxia causes an increase in late INa.
CONCLUSIONS Ranolazine inhibits hypoxia- and H2O2-stimulated late INa by reducing both the open probability and open time of Na+-channel late openings. The present findings support the www.jcvp.org |
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hypothesis that ranolazine is cardioprotective during ischemia by virtue of its effects to reduce Na+-channel late openings and thereby cellular Na+ uptake in cardiac myocytes. REFERENCES 1. Zweier JL. Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury. J Biol Chem. 1988;263:1353–1357. 2. Ju YK, Saint DA, Gage PW. Hypoxia increases persistent sodium current in rat ventricular myocytes. J Physiol. 1996;497:337–347. 3. Ward CA, Giles WR. Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes. J Physiol. 1997;500:631–642. 4. Ma JH, Luo AT, Zhang PH. Effect of hydrogen peroxide on persistent sodium current in guinea pig ventricular myocytes. Acta Pharmacol Sin. 2005;26:828–834. 5. Saint DA. The role of persistent Na+ current during cardiac ischemia and hypoxia. J Cardiovasc Electrophysiol. 2006;17(suppl 1):S96–S103. 6. Song Y, Shryock JC, Wagner S, et al. Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction. J Pharmacol Exp Ther. 2006;318:214–222. 7. Kiyosue T, Arita M. Late sodium current and its contribution to action potential configuration in guinea pig ventricular myocytes. Circ Res. 1989;64:389–397. 8. Le Grand B, Vie B, Talmant JM, et al. Alleviation of contractile dysfunction in ischemic hearts by slowly inactivating Na+ current blockers. Am J Physiol. 1995;269:H533–H540. 9. Undrovinas AI, Maltsev VA, Sabbah HN. Repolarization abnormalities in cardiomyocytes of dogs with chronic heart failure: role of sustained inward current. Cell Mol Life Sci. 1999;55:494–505. 10. Sossalla S, Wagner S, Rasenack ECL, et al. Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts— role of late sodium current and intracellular ion accumulation. J Mol Cell Cardiol. 2008;45:32–43. 11. Shryock JC, Song Y, Rajamani S, et al. The arrhythmogenic consequences of increasing late INa in the cardiomyocyte. Cardiovasc Res. 2013;99: 600–611. 12. Chaitman BR. Ranolazine for the treatment of chronic angina and potential use in other cardiovascular conditions. Circulation. 2006;113:2462– 2472. 13. Fredj S, Sampson KJ, Liu H, et al. Molecular basis of ranolazine block of LQT-3 mutant sodium channels: evidence for site of action. Br J Pharmacol. 2006;148:16–24. 14. Undrovinas AI, Belardinelli L, Undrovinas NA, et al. Ranolazine improves abnormal repolarization and contraction in left ventricular myocytes of dogs with heart failure by inhibiting late sodium current. J Cardiovasc Electrophysiol. 2006;17(suppl 1):S169–S177. 15. Rajamani S, El-Bizri N, Shryock JC, et al. Use-dependent block of cardiac late Na+ current by ranolazine. Heart Rhythm. 2009;6:1625– 1631. 16. Song Y, Shryock JC, Wu L, et al. Antagonism by ranolazine of the proarrhythmic effects of increasing late INa in guinea pig ventricular myocytes. J Cardiovasc Pharmacol. 2004;44:192–199. 17. Matsumura H, Hara A, Hashizume H, et al. Protective effects of ranolazine, a novel anti-ischemic drug, on the hydrogen-peroxide-induced derangements in isolated perfused rat heart: comparison with dichloroacetate. Jpn J Pharmacol. 1998;77:31–39. 18. Fraser H, Belardinelli L, Wang L, et al. Ranolazine decreases calcium accumulation caused by ATX-II or ischemia in rat hearts. J Mol Cell Cardiol. 2006;41:1031–1038. 19. Aldakkak M, Camara AKS, Heisner JS, et al. Ranolazine reduces Ca2+ overload and oxidative stress and improves mitochondrial integrity to
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protect against ischemia reperfusion injury in isolated hearts. Pharmacol Res. 2011;64:381–392. Zhang XQ, Yamada S, Barry WH. Ranolazine inhibits an oxidative stress-induced increase in myocyte sodium and calcium loading during simulated-demand ischemia. J Cardiovasc Pharmacol. 2008;51:443– 449. Tang Q, Ma J, Zhang P, et al. Persistent sodium current and Na+/H+ exchange contributes to the augmentation of the reverse Na+/Ca2+ exchange during hypoxia or acute ischemia in ventricular myocytes. Pflügers Arch. 2012;463:513–522. Wang W, Ma J, Zhang P, et al. Redox reaction modulates transient and persistent sodium current during hypoxia in guinea pig ventricular myocytes. Pflügers Arch. 2007;454:461–475. Nilius B, Benndorf K, Markwardt F. Effects of lidocaine on single cardiac sodium channels. J Mol Cell Cardiol. 1987;19:865–874. Sobel BE, Corr PB, Robison AK, et al. Accumulation of lysophosphoglycerides with arrhythmogenic properties in ischemic myocardium. J Clin Invest. 1978;62:546–553. Undrovinas AI, Fleidervish IA, Makielski JC. Inward sodium current at resting potentials in single cardiac myocytes induced by the ischemic metabolite lysophosphatidylcholine. Circ Res. 1992;71:1231–1241. Kohlhardt M, Fichtner H, Fröbe U. Metabolites of the glycolytic pathway modulate the activity of single cardiac Na+ channels. FASEB J. 1989;3: 1963–1967. Takeishi Y, Jalili T, Ball NA, et al. Responses of cardiac protein kinase C isoforms to distinct pathological stimuli are differentially regulated. Circ Res. 1999;85:264–271. Barnett ME, Madgwick DK, Takemoto DJ. Protein kinase C as a stress sensor. Cell Signal. 2007;19:1820–1829. Peng W, Zhang Y, Zheng M, et al. Cardioprotection by CaMKII-dB is mediated by phosphorylation of heat shock factor 1 and subsequent expression of inducible heat shock protein 70. Circ Res. 2010;106: 102–110. Erickson JR, Joiner MA, Guan X, et al. A dynamic pathway for calciumindependent activation of CaMKII by methionine oxidation. Cell. 2008; 133:462–474. Palomeque J, Rueda OV, Sapia L, et al. Angiotensin II-induced oxidation stress resets the Ca2+ dependence of Ca2+-calmodulin protein kinase II and promotes a death pathway conserved across different species. Circ Res. 2009;105:1204–1212. Wagner S. Ca2+/calmodulin-dependent protein kinase II regulates cardiac Na+ channels. J Clin Invest. 2006;116:3127–3138. Hallaq H, Wang DW, Kunic JD, et al. Activation of protein kinase C alters intracellular distribution and mobility of cardiac sodium channels. Am J Physiol Heart Circ Physiol. 2012;302:H782–H789. Ma J, Luo A, Wu L, et al. Calmodulin kinase II and protein kinase C mediate the effect of increased intracellular calcium to augment late sodium current in rabbit ventricular myocytes. Am J Physiol Cell Physiol. 2012;302:C1141–C1151. Ashpole NM, Herren AW, Ginsburg KS, et al. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1. 5 gating by multiple phosphorylation sites. J Biol Chem. 2012;187: 19856–19869. Wu Y, Song Y, Belardinelli L, et al. The late Na+ current (INa) inhibitor ranolazine attenuates effects of palmitoyl-L-carnitine to increase late INa and cause ventricular diastolic dysfunction. J Pharmacol Exp Ther. 2009; 330:550–557. Sokolov S, Peters CH, Rajamani S, et al. Proton-dependent inhibition of the cardiac sodium channel NaV1.5 by ranolazine. Front Pharmacol. 2013;4:78. Sossalla S, Maurer U, Schotola H, et al. Diastolic dysfunction and arrhythmias caused by overexpression of CaMKIIvC can be reversed by inhibition of late Na+ current. Basic Res Cardiol. 2011;106:263–272.
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Ranolazine Attenuates Hypoxia- and Hydrogen Peroxide-induced Increases in Sodium Channel Late Openings in Ventricular Myocytes Jihua Ma, MSc,* Yejia Song, MD,† John C. Shryock, PhD,‡ Liangkun Hu, MSc,* Weiping Wang, MD,* Xisheng Yan, MD,* Peihua Zhang, BSc,* and Luiz Belardinelli, MD‡
Abstract: Ranolazine attenuates cardiac arrhythmic activity associated with hypoxia and hydrogen peroxide (H2O2) by inhibition of late sodium current (late INa). The mechanism of ranolazine’s action on Na+ channels was investigated using whole-cell and single-channel recording from guinea pig isolated ventricular myocytes. Hypoxia increased whole-cell late INa from 20.48 6 0.02 to 23.99 6 0.07 pA/pF. Ranolazine at 3 and 9 mmol/L reduced the hypoxia-induced late INa by 16% 6 3% and 55% 6 3%, respectively. Hypoxia increased the mean open probability and open time of Na+-channel late openings from 0.016 6 0.001 to 0.064 6 0.007 milliseconds and from 0.693 6 0.043 to 1.081 6 0.098 milliseconds, respectively. Ranolazine at 3 and 9 mmol/L attenuated the hypoxia-induced increase of open probability by 19% 6 7% and 61% 6 1%, and increase of open time by 26% 6 19% and 74 6 21%, respectively. H2O2 increased the mean open probability and open time of Na+-channel late openings from 0.013 6 0.002 to 0.107 6 0.015 milliseconds and from 0.689 6 0.075 to 1.487 6 0.072 milliseconds, respectively. Ranolazine at 3 and 6 mmol/L reduced the H2O2-induced increase of mean open probability by 60% 6 7% and 95% 6 2%, and the increase of mean open time by 31% 6 21% and 82% 6 8%. In conclusion, the inhibition by ranolazine of hypoxia- and H2O2-stimulated late INa is due to reduction of both the open probability and open time of Na+-channel late openings. Key Words: ranolazine, late sodium current, hypoxia, hydrogen peroxide, cardiac myocytes (J Cardiovasc Pharmacol Ô 2014;64:60–68)
INTRODUCTION Myocardial hypoxia and excessive production of reactive oxygen species, such as superoxide anion (O22) and Received for publication December 20, 2013; accepted February 16, 2014. From the *Cardio-Electrophysiological Research Laboratory, Wuhan University of Science and Technology, Wuhan, China; †Division of Cardiovascular Medicine, University of Florida, Gainesville, FL; and ‡Gilead Sciences, Foster City, CA. Supported by Gilead Sciences. Jihua Ma, MSc, and Yejia Song, MD, have received a grant (to their institutions) from Gilead Sciences. John C. Shryock, PhD, is a consultant for and received payment from Gilead Sciences. Luiz Belardinelli, MD, is employed by and holds stock in Gilead Sciences. The other authors report no conflicts of interest. Reprints: Luiz Belardinelli, MD, Gilead Sciences, Inc., Lakeside Drive, Foster City, CA 94404 (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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hydrogen peroxide (H2O2), are contributors to cardiac ischemia-reperfusion injury.1 Both hypoxia and H2O2 have been shown to increase the late inward sodium current (late INa) in cardiac myocytes.2–6 Unlike the brief, high-amplitude, fast INa that underlies the upstroke of the cardiac action potential (AP), late INa is normally a small inward current that persists throughout the duration of the AP plateau.7 An increase of late INa due to hypoxia, H2O2, heart failure, or Na+-channel toxins such as anemone toxin II (ATX-II) can cause a significant increase of sodium influx leading to Na+ and Ca2+ overload in the heart, and is associated with increases of diastolic tension and arrhythmic activity.6,8–11 In whole-cell recordings, late INa appears as a persistent inward current, whereas in single-channel measurements, it manifests as brief channel openings during a prolonged depolarization. Ranolazine is a cardioprotective agent currently used for the treatment of chronic stable angina.12 Ranolazine preferentially inhibits late relative to peak INa with a selectivity of 9–38fold.13–15 In whole-cell recordings, ranolazine significantly reduces H2O2- and ATX-II-stimulated late INa and suppresses H2O2- and ATX-II-induced AP after depolarizations (both early and delayed) in isolated cardiac myocytes.6,16 Ranolazine also reduces Na+/Ca2+ loading and diastolic dysfunction in ischemic or H2O2-treated isolated hearts17–19 and heart tissues and/or myocytes.6,10,20 The effect of ranolazine on hypoxiainduced late INa has not been studied to date. The objective of this study was to determine the effect of ranolazine on hypoxia-induced late INa. When initial results suggested that ranolazine reduced whole-cell late INa in myocytes exposed to hypoxia, additional studies were done using single-channel recording to better understand the mechanism of ranolazine’s effect on Na+-channel function during hypoxia and in the presence of H2O2 (ie, oxidative stress). For comparison, the effects of the Na+-channel blockers lidocaine and tetrodotoxin (TTX) on late INa were also determined.
METHODS Cell Isolation All animal procedures conform to the Guide for the Care and Use of Laboratory Animals issued by the Administrative Regulation of Laboratory Animals of Hubei Province. Adult guinea pigs of either sex weighing 250–300 g were anesthetized with pentobarbital sodium (30 mg/kg, J Cardiovasc Pharmacol ä Volume 64, Number 1, July 2014
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Ranolazine Attenuates Hypoxia-induced Late Na Current
intraperitoneal) 20 minutes after an intraperitoneal injection of 2000 units of heparin. Hearts were rapidly excised and perfused retrogradely through the aorta on a Langendorff apparatus with oxygenated and pre-warmed (378C) solutions in the following order: (1) Ca2+-free solution containing (in mmol/L) NaCl 135, KCl 5.4, MgCl2 1, NaH2PO4 0.33, HEPES 10, and glucose 10, pH 7.4, for 5 minutes; (2) Ca2+-free solution containing (in g/L) collagenase type I 0.1, protease E 0.01, and albumin 0.5, for 8–10 minutes; and (3) KB solution containing (in mmol/L) KOH 70, taurine 20, glutamic acid 50, KCl 40, KH2PO4 20, MgCl2 3, EGTA 0.5, HEPES 10, and glucose 10, pH 7.4, for 5 minutes. At the end of perfusion, the ventricles were cut into small chunks and gently agitated in KB solution to free individual cells. The cell suspension was filtered through nylon mesh and stored in KB solution at 48C.
bath solutions.21 The oxygen tension of bath solutions was monitored with an ISO oxygen meter (WPI, Sarasota, FL). The PO2 of the medium in the recording chamber dropped quickly from about 13 kPa (normoxia; 100 mm Hg) to 1.33– 2.66 kPa (hypoxia; 10–20 mm Hg) in 3–5 minutes during continued perfusion of nitrogen-saturated solutions. For each group of experiments, the PO2 of bath solution was maintained constant throughout an experiment.
Chemicals Collagenase type I and CsCl were obtained from Gibco (Invitrogen, Paisley, United Kingdom). Bovine serum albumin, HEPES, and taurine were obtained from Roche (Basel, Switzerland). TTX was purchased from Hebei Fisheries Research Institute (Qinhuangdao, China). Ranolazine was
Electrophysiological Recording Myocytes were placed into a recording chamber and superfused with bath solutions as indicated below. Whole-cell or cell-attached single-channel membrane currents were recorded using an EPC-9 amplifier (Heka Electronic, Lambrecht, Pfalz, Germany) and PulseFit and TACFit software. Sodium channels were activated by 300- or 500-millisecond depolarizing pulses from a holding potential of 2100 (for whole-cell recording) or 2120 mV (for single-channel recording) to various test potentials. Late INa was determined by measuring the whole-cell or the single-channel current 200 milliseconds after the onset of a depolarizing pulse. Current signals were filtered at 2 kHz and digitized at 10 kHz. Capacitance transients and leak currents were nulled off-line. All experiments were carried out at 21 6 28C. In whole-cell experiments, the recording pipettes had a resistance of 1.5–3 MV when filled with a solution containing (in mmol/L) CsCl 120, CaCl2 1, MgCl2 5, Na2ATP 5, TEA-Cl 10, EGTA 11, and HEPES 10, pH 7.3. To record the late sodium current, cells were superfused with Tyrode solution containing (in mmol/L) NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1, NaH2PO4 0.33, and HEPES 20, pH 7.4. For recording the peak (transient) sodium current, the Tyrode solution contained (in mmol/L) NaCl 10, CsCl 130, CaCl2 1, MgCl2 2, CdCl2 0.05, HEPES 5, and glucose 5, pH 7.4. For cell-attached single-channel recordings, the recording pipettes had a resistance of 6–10 MV when filled with a pipette solution composed of (in mmol/L): NaCl 180, KCl 1.3, CaCl2 1.5, MgCl2 0.5, Na2ATP 5, CoCl2 2.0, TEACl 10, 4-AP 10, CsCl 10, HEPES 5, and glucose 5, pH 7.4. After obtaining the cell-attached configuration, the bath solution was switched from Tyrode solution to a high-potassium solution containing (in mmol/L): K-acetate 140, NaCl 5, MgCl2 2, CdCl2 0.05, HEPES 10, and glucose 25, pH 7.4, to bring the cell membrane potential close to 0 mV.
Induction of Hypoxia Hypoxia was induced and maintained by bubbling the bath solutions in flasks with 100% nitrogen. To minimize diffusion of air into bath solutions, a cover was placed over the recording chamber, and a constant flow of 100% N2 was delivered to the chamber to form a laminar layer of N2 over Ó 2014 Lippincott Williams & Wilkins
FIGURE 1. Inhibition by tetrodotoxin (TTX, 2 mmol/L) of a hypoxia-induced increase of whole-cell late INa in guinea pig isolated ventricular myocytes. (A) Superimposed current traces recorded from a myocyte sequentially superfused with normoxic bath solution (a, control), hypoxic bath solution (b, hypoxia), hypoxic bath solution containing 2 mmol/L TTX (c), or hypoxic bath solution during washout of TTX (d). Late INa was activated by a 300-millisecond voltage-clamp pulse from 2100 to 230 millivolts. (B) Summary data (mean 6 SD) for amplitudes of late INa in 6 myocytes. * and † indicate P , 0.001 versus control and hypoxia alone, respectively. www.jcvp.org |
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obtained from Gilead Sciences (Foster City, CA). All other chemicals were purchased from Sigma (St. Louis, MO).
Data Analysis Single-channel records were analyzed using TAC + TACFit (Bruxton, Seattle, WA). Channel openings were detected using half-amplitude threshold analysis. Channel open probability was calculated from the total of channel open times during 100 sweeps divided by the total sweep duration. Histograms of channel open time distribution were fitted to single exponentials using TACFit. Statistical significance between test groups was evaluated by one-way analysis of variance. The Holm–Sidak method was used when needed. All values were expressed as mean 6 SD. The number of cells/patches tested in each group is indicated by “n.” A P , 0.05 was considered to indicate a statistically significant difference.
RESULTS Effects of Ranolazine and TTX on Hypoxia-induced Whole-cell Late INa The amplitude of late INa was small and stable during 30 minutes of normoxia (ie, the duration of an experiment). During normoxia, the amplitudes of late INa elicited by 300millisecond test pulses from 2100 to 230 mV at 0, 10, 20, and 30 minutes after establishment of whole-cell configuration were 20.66 6 0.14, 20.66 6 0.16, 20.67 6 0.13, and 20.68 6 0.13 pA/pF, respectively (n = 6, P . 0.05). In comparison, inward late INa steadily increased when myocytes were exposed to hypoxic medium equilibrated with 100% N2. The value of PO2 in the myocyte chamber perfused with hypoxic medium for 20 minutes ranged from 1.33 to 2.66 kPa (10–20 mm Hg). In myocytes (n = 6) exposed to hypoxia for 20 minutes, the amplitude of late INa at 230 mV was increased from 20.51 6 0.55 (0 time, control) to 22.92 6 0.48 pA/pF (P , 0.001 vs. control; Fig. 1). Hypoxia-stimulated late INa was decreased by 2 mmol/L TTX from 22.92 6 0.48 to 20.77 6 0.53 pA/pF (n = 6, P , 0.001) (Fig. 1). The effect of TTX was reversible (Fig. 1). The high sensitivity to TTX inhibition is consistent with the identification of late current as INa. The concentration- and voltage-dependence of the effects of ranolazine on hypoxia-stimulated late INa were studied in separate series of experiments. In the concentrationdependence study, late INa was activated by depolarizing pulses from 2100 to 230 mV, and ranolazine was applied
FIGURE 2. Inhibition by ranolazine (Ran) of a hypoxia (H)induced increase of whole-cell late INa in guinea pig isolated ventricular myocyte. (A) Superimposed current traces obtained from a myocyte sequentially exposed to normoxic bath solution (a, control), hypoxic bath solution (b), hypoxic bath solution containing 3 or 9 mmol/L Ran (c and d), and hypoxic bath solution during washout of Ran (e). Late INa was activated by a 300-millisecond voltage-clamp pulse from 2100 to 230
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mV. (B) Summary data (mean 6 SD) for concentrationdependent inhibition by Ran of hypoxia-induced late INa in 8 myocytes. * and † indicate P , 0.001 versus control and hypoxia alone, respectively. (C) Current-voltage relationship for late INa recorded from 8 myocytes superfused sequentially with normoxic bath solution (control), with hypoxic bath solution in the absence (hypoxia) and presence of 9 mmol/L Ran, and with hypoxic solution after washout of Ran. Late INa was activated from a holding potential of 2100 mV to various test potentials ranging from 290 to +20 mV. Ó 2014 Lippincott Williams & Wilkins
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Ranolazine Attenuates Hypoxia-induced Late Na Current
FIGURE 3. Ranolazine (Ran) reduced single Na+-channel late openings in ventricular myocytes exposed to hypoxia (H). (A) (a–f) Ran (1, 3, and 9 mM) concentration-dependently inhibited hypoxia-induced Na+-channel late openings in a cell-attached patch. Four current traces are shown for each treatment. The upper 3 traces are original single-channel recordings, and the bottom trace is the ensemble average current of 50–100 sweeps. Current and time scales and the voltage-clamp protocol are shown at the bottom of the panel. (B) Amplitude (left) and open-time (t, right) histograms of Na+-channel late openings in a cell-attached patch superfused with hypoxic solution in the absence and presence of 9 mmol/L Ran. Ran reduced Na+-channel open probability and mean open time in the presence of hypoxia.
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FIGURE 4. Summary data for the concentrationdependent reduction by ranolazine (Ran) of mean open probability and open time of Na+channel late openings shown in Figure 3. Bars represent average values obtained from 8 cells. * and † indicate P , 0.001 versus control and hypoxia alone, respectively.
in the bath solution (ie, through superfusate) in the presence of hypoxia. Hypoxia increased the amplitude of late INa from 20.47 6 0.01 to 23.99 6 0.07 pA/pF (n = 8, P , 0.001; Fig. 2). The addition of ranolazine to the hypoxic superfusate for 6–10 minutes concentration-dependently reduced hypoxia-stimulated late INa (Fig. 2). Ranolazine sequentially applied at concentrations of 1, 3, and 9 mmol/L significantly (n = 8, P , 0.001) attenuated hypoxia-induced late INa by 8% 6 4%, 16% 6 3%, and 55% 6 3%, respectively, from 23.99 6 0.07 to 23.69 6 0.13, 23.39 6 0.07, and 22.04 6 0.11 pA/pF (Fig. 2B). The effect of ranolazine was reversible after the drug was washed out (e in Fig. 2A). The amplitude of late INa in the presence hypoxia after washout of ranolazine was 23.78 6 0.04 pA/pF, which was not significantly different from the value
FIGURE 5. Effect of tetrodotoxin (TTX, 2 mmol/L in pipette solution) to block the time-dependent increases in mean open probability and open time of Na+-channel late openings in cell-attached patches during superfusion of myocytes (n = 7) with hypoxic bath solution. and : indicate hypoxia alone; B and 6indicate the values in the presence of TTX. Na+channel openings were activated by 500-millisecond voltageclamp pulses from 2100 to 250 mV.
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obtained before addition of ranolazine (23.99 6 0.07 pA/pF; Fig. 2B). The voltage dependence of hypoxia-induced late INa was examined in the absence and presence of 9 mmol/L ranolazine (Fig. 2C). Late INa was activated by depolarizing pulses from 2100 mV to various test potentials from 290 to +20 mV, and ranolazine was applied after a 20-minute exposure of myocytes (n = 8) to hypoxia. Inward late INa could be detected within a broad range of membrane potentials from 270 to +20 mV and was maximal at a potential of approximately 220 mV (Fig. 2C). The amplitudes of late INa at test potentials between 250 to +10 mV were markedly increased by hypoxia (Fig. 2C). The stimulatory effect of hypoxia on late INa was reversibly attenuated by ranolazine (Fig. 2C). At a test potential of 230 mV, for example, the amplitude of late INa was increased by hypoxia from 20.94 6 0.14 to 22.46 6 0.28 pA/pF (P , 0.05) and was decreased by ranolazine from 22.46 6 0.28 to 21.25 6 0.21 pA/pF (P , 0.05) in the continued presence of hypoxia. Neither hypoxia nor ranolazine altered the voltage-dependence of late INa (Fig. 2C). The effects of hypoxia and ranolazine on the peak (transient) INa were also studied. In the peak current experiments (n = 8), the concentration of sodium in the bath solution was reduced to 10 mmol/L, and the current was activated by depolarizing voltage-clamp pulses from 2100 to 230 mV. Ranolazine was sequentially applied at concentrations of 1, 3, and 9 mmol/L. The duration of ranolazine treatment at each concentration was 10 minutes. In contrast to the significant increase of late INa, a progressive decrease of the peak (transient) INa was observed in the presence of hypoxia. The decreasing process of peak INa was not altered after treatment with ranolazine. The amplitude of peak INa in the presence of normoxia, hypoxia (15 minutes), hypoxia plus 1, 3, and 9 mmol/L ranolazine, and hypoxia alone (wash out ranolazine) was 232 6 0.6, 224 6 0.6, 222 6 0.9, 221 6 0.7, 220 6 0.7, and 218 6 0.9 pA/pF, respectively. The inhibitory effect of hypoxia and the lack of effect of ranolazine on the peak INa were consistent with the result of our previous study22 and with the preferential action of ranolazine on the late INa.13–15 Ó 2014 Lippincott Williams & Wilkins
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FIGURE 6. Ranolazine (Ran, 6 mmol/L) blocked H2O2 (300 mmol/L)-induced Na+-channel late openings. (A) Single-channel recordings obtained from a cell-attached patch in the absence (control) and presence of H2O2 alone or H2O2 plus Ran. In each treatment, the upper 3 traces are original records and the bottom trace is the ensemble average current of 50–100 sweeps. Current and time scales and voltage-clamp protocol are shown at the bottom. (B) Amplitude (left) and open-time (right) histograms of Na+-channel late openings in a patch in the absence and presence of H2O2 and H2O2 plus Ran.
Effects of Ranolazine and TTX on Hypoxiastimulated Late Openings of Single Na+ Channels Single Na+-channel activity was studied using the cellattached patch-clamp configuration. Na+ channels were activated by 500-millisecond depolarizing pulses to 250 mV from a holding potential of 2120 mV. During normoxia, Na+-channel late openings were rare (a in Fig. 3A). Hypoxia markedly increased the mean open probability and open time of Na+-channel late openings (b in Fig. 3A). In a series of experiments (n = 8), a 20-minute hypoxia treatment increased the mean open probability from 0.016 6 0.001 to 0.064 6 0.007 (P , 0.001), and the mean open time from 0.693 6 0.042 to 1.081 6 0.097 milliseconds (P , 0.001) (Fig. 4). Ranolazine at 3 and 9 mmol/L significantly (P , 0.001) attenuated hypoxia-induced increases in open probability by 19% 6 7% and 61% 6 1% and in open time by 26% 6 19% and 74% 6 21%, respectively (Fig. 4). The inhibitory effect of ranolazine on hypoxia-induced Na+-channel late openings was reversed on drug washout (f in Fig. 3A and Fig. 4). In the absence of drugs, hypoxia alone (n = 7) steadily increased both the open probability and open time Ó 2014 Lippincott Williams & Wilkins
of Na+-channel late openings for up to 40 minutes (Fig. 5). When the pipette solution contained 2 mmol/L TTX (n = 7), there were no significant changes in either the open probability or the open time in the presence of hypoxia over the same 40-minute period of time (Fig. 5).
Effects of Ranolazine and Lidocaine on H2O2stimulated Late Openings of Single Na+ Channels In whole-cell experiments, ranolazine and lidocaine have been reported to inhibit H2O2-induced late INa.4,6 In this study, the effects of ranolazine and lidocaine on H2O2induced single Na+-channel late openings were investigated and compared. In these experiments, Na+ channels were activated by 500-millisecond depolarizing pulses from 2120 to 250 mV in the cell-attached patch-clamp configuration. Exposure of myocytes to H2O2 (300 mmol/L, n = 23) for 15 minutes resulted in significant (P , 0.001) increases in the mean open probability and open time of Na+-channel late openings, from 0.013 6 0.002 to 0.107 6 0.015 milliseconds and from 0.689 6 0.075 to 1.487 6 0.072 milliseconds, respectively (Figs. 6, 7). The stimulatory effect of H2O2 on www.jcvp.org |
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FIGURE 7. Summary of the effects of ranolazine (Ran, 1, 3, and 6 mmol/L) and H2O2 (300 mmol/L) on open probability and open time of Na+-channel late openings in the cellattached patch. Na+-channels were activated by 500-millisecond voltage-clamp pulses from 2120 to 250 mV. Ran concentration-dependently attenuated both H2O2-induced increases in open probability and open time. Bars represent values obtained in the presence of no drugs (control, n = 23), H2O2 alone (n = 23), and H2O2 plus 1 (n = 6), 3 (n = 7), or 6 (n = 10) mmol/L Ran, as indicated. * and † indicate P , 0.001 versus control and H2O2 alone, respectively.
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Na+-channel late openings was significantly attenuated by ranolazine in a concentration-dependent manner. Ranolazine at 3 (n = 7) and 6 (n = 10) mmol/L significantly (P , 0.001) reduced the H2O2-induced increase of mean open probability by 60% 6 7% and 95% 6 2%, and the H2O2-induced increase of mean open time by 31% 6 21% and 82% 6 8%, respectively (Figs. 6, 7). In parallel experiments with lidocaine, H2O2 (300 mmol/L, n = 12) alone increased the mean open probability and open time from 0.013 6 0.002 to 0.108 6 0.012 milliseconds and from 0.699 6 0.075 to 1.491 6 0.068 milliseconds, respectively (Figs. 8, 9). In the presence of 30 (n = 6) and 60 (n = 6) mmol/L lidocaine, the H2O2-induced increase of open probability was attenuated by 58% 6 9% and 94% 6 3%, and the increase of open time was reduced by 27% 6 8% and 85% 6 5%, respectively (Figs. 8, 9). Thus, the inhibitory effects of lidocaine at 30 and 60 mmol/L were comparable with those of ranolazine at 3 and 6 mmol/L. The effect of lidocaine to attenuate H2O2-induced late INa is consistent with previous findings.4,23 In the absence of other drugs, H2O2 (300 mmol/L) alone steadily increased the mean open probability and open time of Na+-channel late openings for up to 40 minutes (Fig. 10).
FIGURE 8. Inhibition by 60 mmol/L lidocaine (Lido) of H2O2 (300 mmol/L)-induced Na+-channel late openings. (A) Single-channel recordings obtained from a cell-attached patch. The upper 3 traces are original records, and the bottom trace is the ensemble average current of 50–100 sweeps. Current and time scales and voltage-clamp protocol are shown at the bottom. (B) Amplitude (left) and open-time (right) histograms of Na+-channel late openings in a patch in the absence and presence of H2O2 and H2O2 plus Lido.
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J Cardiovasc Pharmacol ä Volume 64, Number 1, July 2014
Ranolazine Attenuates Hypoxia-induced Late Na Current
DISCUSSION
FIGURE 9. Summary of the effects of lidocaine (Lido, 30 and 60 mmol/L) and H2O2 (300 mmol/L) on open probability and open time of Na+-channel late openings in the cell-attached patch. Na+ channels were activated by 500-millisecond voltage-clamp pulses from 2120 to 250 mV. Bars represent values obtained in the presence of no drugs (control, n = 12), H2O2 alone (n = 12), and H2O2 plus 30 (n = 6) or 60 (n = 6) mmol/L Lido, as indicated. The inhibitory effects of Lido at 30 and 60 mmol/L were comparable to those of ranolazine at 3 and 6 mmol/L, respectively (see Fig. 7). * and † indicate P , 0.001 versus control and H2O2 alone, respectively.
When TTX (2 mmol/L, n = 7) was included in the pipette solution, the stimulatory effect of H2O2 (300 mmol/L, 30 minutes) on Na+-channel late openings was abolished (Fig. 10).
FIGURE 10. Time-course of actions of H2O2 (300 mmol/L) on Na+-channel late openings in cell-attached patches in the absence and presence of tetrodotoxin (TTX, 2 mmol/L in the pipette solution). Na+ channels were activated by 500-millisecond voltage-clamp pulses from 2120 to 250 mV. In the absence of other drugs (n = 7), H2O2 steadily increased the open probability () and open time (:). In the presence of TTX (n = 7), the actions of H2O2 on channel open probability (B) and open time (6) were completely blocked. Ó 2014 Lippincott Williams & Wilkins
The antianginal drug ranolazine was shown to inhibit hypoxia- and H2O2-induced late INa in this study of guinea pig isolated ventricular myocytes. Acute exposures of myocytes to either hypoxia (PO2, 1.33–2.66 kPa) or H2O2 caused marked increases of late INa in whole cells (Figs. 1, 2 and Song et al, 2006) and cell-attached patches (Figs. 3–10). Ranolazine inhibited hypoxia- and H2O2-stimulated late INa by reducing both the open probability and open time of Na+channel late openings (Figs. 3, 4, 6, 7). These findings are consistent with previous reports that ranolazine reduces whole-cell late INa in myocytes isolated from dog failing hearts,14 and late INa in rabbit and guinea pig ventricular myocytes treated with the late INa enhancers ATX-II or H2O2.6,10,16 The effects of hypoxia to increase late INa and to increase Na+-channel mean open time and open probability were rapid and substantial (Figs. 3–5). Significant increases in these parameters occurred within 10 minutes of the onset of superfusion with a hypoxic bathing solution (Fig. 5) or with H2O2 (Fig. 10). The mechanism by which acute hypoxia leads to changes in Na+-channel function that increase late INa is not clear. Increases of lysophosphatidylcholine, palmitoyl-Lcarnitine, and other intermediary metabolites may occur rapidly during hypoxia24 and are reported to increase late INa.25,26 Activations of protein kinase C27,28 and Ca2+/calmodulindependent protein kinase II (CaMKII) subtype dC are reported to occur during hypoxia and oxidative stress,29 and oxidation of CaMKII markedly increases its sensitivity to Ca2+, such that, CaM-independent activity of the kinase is increased.30,31 Both kinases can phosphorylate the cardiac Na+ channels and may increase late INa.32–35 In addition, redox regulation of the Na+ channels has been proposed to modulate late INa.22 Further investigation is needed to ascertain the mechanisms by which hypoxia disrupts Na+-channel function to increase late INa. Ranolazine is not reported to directly inhibit the activities of CaMKII, protein kinase C, or other protein kinases. There are no published reports of the effect of ranolazine on Na+-channel late current in the ischemic heart, because to the best of our knowledge, it is not possible to measure an ion channel current in the ischemic environment. However, ranolazine reduces Na+/Ca2+ overload, diastolic dysfunction, and arrhythmic activity caused by ischemia.18,21 The effects of several components of the ischemic environment on Na+-channel late current have been studied in the absence and presence of ranolazine. Ranolazine reduces the increases of whole-cell late INa caused by exposure of cardiac myocytes to the ischemic metabolite palmitoyl-L-carnitine,36 to acidosis,37 to reactive oxygen species,6 to activation of CaMKII,38 and in this study, to hypoxia. A limitation of this study is that we did not investigate the intracellular signaling by which hypoxia causes an increase in late INa.
CONCLUSIONS Ranolazine inhibits hypoxia- and H2O2-stimulated late INa by reducing both the open probability and open time of Na+-channel late openings. The present findings support the www.jcvp.org |
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hypothesis that ranolazine is cardioprotective during ischemia by virtue of its effects to reduce Na+-channel late openings and thereby cellular Na+ uptake in cardiac myocytes. REFERENCES 1. Zweier JL. Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury. J Biol Chem. 1988;263:1353–1357. 2. Ju YK, Saint DA, Gage PW. Hypoxia increases persistent sodium current in rat ventricular myocytes. J Physiol. 1996;497:337–347. 3. Ward CA, Giles WR. Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes. J Physiol. 1997;500:631–642. 4. Ma JH, Luo AT, Zhang PH. Effect of hydrogen peroxide on persistent sodium current in guinea pig ventricular myocytes. Acta Pharmacol Sin. 2005;26:828–834. 5. Saint DA. The role of persistent Na+ current during cardiac ischemia and hypoxia. J Cardiovasc Electrophysiol. 2006;17(suppl 1):S96–S103. 6. Song Y, Shryock JC, Wagner S, et al. Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction. J Pharmacol Exp Ther. 2006;318:214–222. 7. Kiyosue T, Arita M. Late sodium current and its contribution to action potential configuration in guinea pig ventricular myocytes. Circ Res. 1989;64:389–397. 8. Le Grand B, Vie B, Talmant JM, et al. Alleviation of contractile dysfunction in ischemic hearts by slowly inactivating Na+ current blockers. Am J Physiol. 1995;269:H533–H540. 9. Undrovinas AI, Maltsev VA, Sabbah HN. Repolarization abnormalities in cardiomyocytes of dogs with chronic heart failure: role of sustained inward current. Cell Mol Life Sci. 1999;55:494–505. 10. Sossalla S, Wagner S, Rasenack ECL, et al. Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts— role of late sodium current and intracellular ion accumulation. J Mol Cell Cardiol. 2008;45:32–43. 11. Shryock JC, Song Y, Rajamani S, et al. The arrhythmogenic consequences of increasing late INa in the cardiomyocyte. Cardiovasc Res. 2013;99: 600–611. 12. Chaitman BR. Ranolazine for the treatment of chronic angina and potential use in other cardiovascular conditions. Circulation. 2006;113:2462– 2472. 13. Fredj S, Sampson KJ, Liu H, et al. Molecular basis of ranolazine block of LQT-3 mutant sodium channels: evidence for site of action. Br J Pharmacol. 2006;148:16–24. 14. Undrovinas AI, Belardinelli L, Undrovinas NA, et al. Ranolazine improves abnormal repolarization and contraction in left ventricular myocytes of dogs with heart failure by inhibiting late sodium current. J Cardiovasc Electrophysiol. 2006;17(suppl 1):S169–S177. 15. Rajamani S, El-Bizri N, Shryock JC, et al. Use-dependent block of cardiac late Na+ current by ranolazine. Heart Rhythm. 2009;6:1625– 1631. 16. Song Y, Shryock JC, Wu L, et al. Antagonism by ranolazine of the proarrhythmic effects of increasing late INa in guinea pig ventricular myocytes. J Cardiovasc Pharmacol. 2004;44:192–199. 17. Matsumura H, Hara A, Hashizume H, et al. Protective effects of ranolazine, a novel anti-ischemic drug, on the hydrogen-peroxide-induced derangements in isolated perfused rat heart: comparison with dichloroacetate. Jpn J Pharmacol. 1998;77:31–39. 18. Fraser H, Belardinelli L, Wang L, et al. Ranolazine decreases calcium accumulation caused by ATX-II or ischemia in rat hearts. J Mol Cell Cardiol. 2006;41:1031–1038. 19. Aldakkak M, Camara AKS, Heisner JS, et al. Ranolazine reduces Ca2+ overload and oxidative stress and improves mitochondrial integrity to
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