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1 818 Exp Physiol 12.7 (217) pp Research Paper Research Paper Modulation of late sodium current by Ca 2+ calmodulin-dependent protein kinase II, protein kinase C and Ca 2+ during hypoxia in rabbit ventricular myocytes Chen Fu 1,2,JieHao 1,MengliuZeng 1, Yejia Song 3, Wanzhen Jiang 1, Peihua Zhang 1, Antao Luo 1, Zhenzhen Cao 1, Luiz Belardinelli 4 and Jihua Ma 1 1 Cardio-Electrophysiological Research Laboratory, Medical College of Wuhan University of Science and Technology, Wuhan, China 2 Department of Cardiovascular Medicine, Tianyou Hospital, affiliated to Wuhan University of Science and Technology, Wuhan, China 3 Division of Cardiovascular Medicine, University of Florida College of Medicine, Gainesville, FL, USA 4 Department of Biology, Gilead Sciences, Fremont, CA, USA Edited by: Ming Lei Experimental Physiology New Findings What is the central question of this study? -induced increase in late sodium current (I Na,L ) is associated with conditions causing cellular Ca 2+ overload and contributes to arrhythmogenesis in the ventricular myocardium. The I Na,L is an important drug target. We investigated intracellular signal transduction pathways involved in modulation of I Na,L during hypoxia. What is the main finding and its importance? caused increases in I Na,L,reverseNa + Ca 2+ exchange current and diastolic [Ca 2+ ], which were attenuated by inhibitors of Ca 2+ calmodulin-dependent protein kinase II (CaMKII) and protein kinase C and by a Ca 2+ chelator. The findings suggest that CaMKII, protein kinase C and Ca 2+ all participate in mediation of the effect of hypoxia to increase I Na,L. leads to augmentation of the late sodium current (I Na,L )andcellularna + loading, increased reverse Na + Ca 2+ exchange current (reverse I NCX ) and intracellular Ca 2+ loading in rabbit ventricular myocytes. The purpose of this study was to determine the intracellular signal transduction pathways involved in the modulation of I Na,L during hypoxia in ventricular myocytes. Whole-cell and cell-attached patch-clamp techniques were used to record I Na,L, and the whole-cell mode was also used to record reverse I NCX and to study intercellular signal transduction mechanisms that mediate the increased I Na,L. Dual excitation fluorescence photomultiplier systems were used to record the calcium transient in ventricular myocytes. caused increases of I Na,L and reverse I NCX. These increases were attenuated by KN-93 (an inhibitor of Ca 2+ calmodulin-dependent protein kinase II), bisindolylmaleimide VI (BIM; an inhibitor of protein kinase C) and BAPTA AM (a Ca 2+ chelator). KN-93, BIM and BAPTA AM had no effect on I Na,L in normoxia. In studies of KN-93, hypoxia alone increased the density of I Na,L from.31 ±.2 to.66 ±.3 pa pf 1 (n = 6, P <.1 versus control) and the C.Fu,J.HaoandM.Zengcontributedequallytothiswork. DOI: /EP8599 C 217 The Authors. Experimental Physiology C 217 The Physiological Society

2 Exp Physiol 12.7 (217) pp Modulation of late sodium current during hypoxia 819 density of reverse I NCX from 1.2 ±.6 to 1.91 ±.2 pa pf 1 (n = 7, P <.1 versus control) in rabbit ventricular myocytes. In the presence of 1 µm KN-93, the densities of I Na,L and reverse I NCX during hypoxia were significantly attenuated to.44 ±.3 (n = 6, P <.1 versus hypoxia) and 1.36 ±.15 pa pf 1 (n = 7, P <.1 versus hypoxia), respectively. In studies of BIM, hypoxia increased I Na,L from.3 ±.3 to.6 ±.3 pa pf 1 (n = 6, P <.1 versus control) and reverse I NCX from.91 ±.1 to 1.71 ±.27 pa pf 1 (n = 6, P <.1 versus control).inthepresenceof1µm BIM, the densities of I Na,L and reverse I NCX during hypoxia were significantly attenuated to.48 ±.2 (n = 6, P <.1 versus hypoxia) and 1.33 ±.21 pa pf 1 (n = 6, P <.1 versus hypoxia), respectively. In studies of BAPTA AM, hypoxia increased I Na,L from.26 ±.4 to.63 ±.5 pa pf 1 (n = 6, P <.1 versus control) and reverse I NCX from.86 ±.9 to 1.68 ±.35 pa pf 1 (n = 6, P <.1 versus control). The effects of hypoxia on I Na,L and reverse I NCX were significantly attenuated in the presence of 1 mm BAPTAAMto.39 ±.2 (n = 6, P <.1 versus hypoxia) and 1.12 ±.27 pa pf 1 (n = 6, P <.1 versus hypoxia), respectively. Results of single-channel studies showed that hypoxia apparently increased the mean open probability and mean open time of sodium channels. These effects were inhibited by either 1 µm KN-93 or 1 mm BAPTA AM. The suppressant effects of drug interventions were reversed upon washout. In addition, KN-93, BIM and BAPTA AM also reversed the hypoxia-enhanced diastolic Ca 2+ concentration and the attenuated amplitude of the [Ca 2+ ] i transient, maximal velocities of Ca 2+ increase and Ca 2+ decay. In summary, the findings suggest that Ca 2+ calmodulin-dependent protein kinase II, protein kinase C and Ca 2+ all participate in mediation of the effect of hypoxia to increase I Na,L. (Received 3 August 216; accepted after revision 13 April 217; first published online 24 April 217) Corresponding author J. Ma: Cardio-Electrophysiological Research Laboratory, Medical College of Wuhan University of Science and Technology, Wuhan 4381, China. mjhua@wust.edu.cn Introduction In patch-clamp experiments using cardiac myocytes, a persistent and small-amplitude sodium current, known as the late sodium current (I Na,L ), is recorded after the transient sodium current (which is responsible for the rapid upstroke of the action potential) has peaked and returned to baseline (Carmeliet, 1987; Sakmann et al. 2; Huang et al. 21). The I Na,L is increased by toxins, such as anemone toxin II and veratridine, that delay and/or destabilize the Na + channel inactivated state (Fekete et al. 29; Luoet al. 214; Rizzettoet al. 215). The amplitude of I Na,L is increased during hypoxia and is associated with conditions that cause cellular Ca 2+ overload (Haigney et al. 1994; Xiao & Allen, 1999; Hammarström & Gage, 22). Augmentation of I Na,L may increase the intracellular Na + concentration ([Na + ] i ) and thereby potentiate reverse Na + Ca 2+ exchanger activity, increasing Ca 2+ influx and causing cytoplasmic calcium overload (Jansen et al. 24; Maier & Hasenfuss, 26; Williams et al. 27). The I Na,L contributes to the shaping and duration of the action potential in ventricular myocytes (Sakmann et al. 2), and results of computer modelling studies indicate that even a slight increase of I Na,L may cause action potential prolongation that can lead to the occurrence of life-threatening arrhythmias (Antzelevitch, 2; Sakmann et al. 2; Gima & Rudy, 22). Therefore, an abnormally increased I Na,L is considered to be an important drug target for suppression of arrhythmias and irreversible cell damage associated with Ca 2+ overload (Zaza, 211; Aguilar-Shardonofsky et al. 212). A variety of pathological factors, including heart failure, acute myocardial ischaemia and congenital Na + channelopathies (long QT syndrome type 3), may lead to an increase in I Na,L in ventricular myocytes (Haigney et al. 1994; Ju et al. 1996; Hammarström & Gage, 22; Luo et al. 27; Wang et al. 27, 214; Zheng et al. 29; Tang et al. 212). Results of in vitro studies indicate that hypoxia increases I Na,L ; however, the cellular mechanism(s) by which hypoxia causes I Na,L to increase are poorly understood. A positive feedback circuit between an increase of cytoplasmic Ca 2+ and I Na,L appears to exist in ventricular myocytes. This positive feedback is furthermore reported to involve increases of the activities of protein kinase C (PKC) and Ca 2+ calmodulin-dependent protein kinase II (CaMKII; Maltsev & Undrovinas, 28; Maltsev et al. 28), both of which are activated by Ca 2+ (Ma et al. 212). We hypothesized that the effect of hypoxia to increase I Na,L might also be mediated by [Ca 2+ ] i, PKC and CaMKII in ventricular myocytes. To test this hypothesis, acutely isolated rabbit ventricular myocytes were subjected C 217 The Authors. Experimental Physiology C 217 The Physiological Society

3 82 C. Fu and others Exp Physiol 12.7 (217) pp to hypoxia. Whole-cell and cell-attached patch-clamp techniques were used to record I Na,L and reverse Na + Ca 2+ exchange current (reverse I NCX ) in the absence and presence of inhibitors of CaMKII and PKC and the Ca 2+ chelator BAPTA AM, respectively. Methods Ethical approval All of the experimental ventricular myocytes were obtained from adult New Zealand rabbits (White breed, either sex, age 7 ± 1months,bodyweight2±.5 kg). They were purchased and housed (with feeding ad libitum) by the Institutional Animal Care and Use Committee of Wuhan University of Science and Technology. The use of animals in this investigation conformed to the Guide for the Care and Use of Laboratory Animals (Hubei Province, China), the Guidance for Ethical Treatment of Laboratory Animals (the Ministry of Science and Technology of China, 26) and the Operation of the Animals (Scientific Procedures) Act 1986 passed in 1986 by EU Directive 86/69/EEC (updated and replaced by EU Directive 21/63/EU) and was approved by the Institutional Ethics Committee of Wuhan University of Science and Technology (approval no. MD-medical science 21323). Isolation of rabbit ventricular myocytes Single ventricular myocytes were isolated enzymatically. Heparin (2 IU) was injected into the marginal ear vein, followed 1 min later by inducted of general anaesthesia with urethane (4 mg kg 1 I.V.) and xylazine (7.5 mg kg 1 I.M.) urethane and xylazine were purchased from Sigma Chemical (Saint Louis, MO, USA). After corneal reflexes and reflexes in response to a painful stimulus had disappeared, hearts were quickly excised (<3 s) and perfused retrogradely (using a Langendorff apparatus) with Ca 2+ -free Tyrode solution for 5 min to wash out residual blood from the ventricular chambers. Hearts were subsequently digested by perfusion with an enzyme-containing Ca 2+ -free Tyrode solution (.1 g l 1 collagenase type I and.5 g l 1 bovine serum albumin) for 4 5 min. The perfusate was then switched to Kraftbrühe (KB) solution for 5 min. All solutions were bubbled with 1% O 2 and maintained at a temperature of 37 C. The left ventricle was excised and cut into small chunks that were gently agitated in KB solution. The resulting cell suspension was filtered through nylon and stored in KB solution at room temperature until use in experiments. Solutions and drugs The Tyrode solution contained (mm): 135 NaCl, 5.4 KCl, 1 MgCl 2,1.8CaCl 2,.33NaH 2 PO 4,1Hepesand1glucose (ph adjusted to 7.4 with NaOH). The Ca 2+ -free Tyrode solution was normal Tyrode solution without Ca 2+.The KB solution contained (mm): 7 KOH, 4 KCl, 3 MgCl 2, 2 KH 2 PO 4,.5 EGTA, 5 L-glutamic acid, 2 taurine, 1 Hepes and 1 glucose (ph adjusted to 7.4 with KOH). To record I Na,L, the extracellular solution contained (mm): 135 NaCl, 5.4 CsCl, 1.8 CaCl 2, 1 MgCl 2,1Hepes and 1 glucose (ph adjusted to 7.4 with NaOH). The pipette solution contained (mm): 12 CsCl, 1 CaCl 2,5 Na 2 ATP, 5 MgCl 2, 1 TEA-Cl, 1 EGTA and 1 Hepes (ph adjusted to 7.4 with CsOH). In addition, 1 μm nifedipine was used to block L-type Ca 2+ channels. To record I NCX, the extracellular solution contained (mm): 14 NaCl, 2 CsCl, 2 CaCl 2, 2 MgCl 2,1Hepes and 1 glucose (ph adjusted to 7.4 with NaOH). The pipette solution contained (mm): 12 CsOH, 2 NaCl, 1 CaCl 2, 5 MgATP, 5 aspartic acid, 3 MgCl 2, 2 EGTA and 1 Hepes (ph adjusted to 7.4 with CsOH). In addition, 1 μm nifedipine, 2 μm ouabain and 1 μm BaCl 2 were used to block L-type Ca 2+ channels, the Na + K + pump and K + channels, respectively. The I NCX was measured as the Ni 2+ -sensitive current that was blocked by 5 μm NiCl 2. The pipette solution used in experiments to record single-channel currents contained (mm): 18 NaCl, 1.3 KCl, 1.5 CaCl 2,.5 MgCl 2,5Na 2 ATP, 3 CoCl 2, 1 TEA-Cl, 1 4-aminopyridine, 1 CsCl, 5 glucose and 5 Hepes (ph adjusted to 7.4 with NaOH). The cells were first superfused with Tyrode solution. After obtaining the cell-attached configuration, the cell membrane potential (RP) was brought close to mv by superfusion of the cell with a high-potassium extracellular solution containing (mm): 14 potassium aspartate, 5 NaCl, 4 MgCl 2,25 glucose and 1 Hepes (ph adjusted to 7.4 with KOH). For intracellular Ca 2+ transient recordings, the bath solution contained the following (mm): 135 NaCl, 4 KCl, 1.8 CaCl 2, 1 MgCl 2, 1 Hepes and 1 glucose (ph adjusted to 7.4 with NaOH). Taurine, bovine serum albumin (BSA) and Hepes were products from Roche Corporation. Collagenase type I was obtained from Gibco. All other chemicals were purchased from Sigma. KN-93 and bisindolylmaleimide VI (BIM) were both dissolved in DMSO. Each of the stock solutions was diluted in the appropriate buffer to the required concentration immediately before use. Induction of hypoxia Ventricular myocytes were transferred into a custom-made perfusion chamber. Acute hypoxia was produced by bubbling the extracellular perfusate (normal extracellular solution for current recording, without glucose) with 1% N 2 gas for at least 5 min before experimentation. Bubbling with N 2 caused no change in the ph of the perfusate. A constant flow of 1% N 2 from a catheter into a radially arrayed slot was used to create a stable C 217 The Authors. Experimental Physiology C 217 The Physiological Society

4 Exp Physiol 12.7 (217) pp Modulation of late sodium current during hypoxia 821 laminar layer of nitrogen above the bath solution in the recording chamber, in order to prevent O 2 in the air from diffusing into the bath solution. In general, bath P O2 was reduced to 1.33 kpa (1 mmhg) within 3 5 min (Tang et al. 212). Whole-cell current recording All experiments were performed at room temperature (25 ± 2 C). The whole-cell patch-clamp technique was used to record I Na,L and I NCX in isolated ventricular myocytes in normoxic conditions (control), during hypoxia and during exposure to drug in the presence of hypoxia. To avoid contamination of either current by hypoxia-induced ATP-dependent potassium current, 5mM ATP was added to the pipette solution (Yan et al. 29). Patch microelectrodes were pulled with a two-stage puller (PP-83; Narishige Group, Tokyo, Japan). Pipette resistances were in the range of 1 2 M. After forming the whole-cell clamp, I Na,L was recorded using the following two depolarizing pulse protocols: (i) a single 3 ms voltage step from a holding potential (HP) of 9 mv to 2 mv; and (ii) a series of 3 ms pulses at a rate of.5 Hz from a HP of 9 mv to a test potential between 8 and +6mV,in1mVincrements.The former was the single-pulse protocol, and the latter was the I V relationship protocol. For I NCX recording, currents were elicited using a ramp pulse from a HP of 4 mv to +6 mv for 3 ms, followed by a ramp to 12 mv over a period of 2 ms (i.e. at 9 mv s 1 ) before returning to 4 mv. Capacitance and series resistances were adjusted to obtain a minimal contribution of the capacitive transients. A 6 8% compensation of the series resistance was usually achieved without ringing. Currents were filtered at 2 khz, digitized at 1 khz and stored on a computer hard disk for analysis. Single-channel current recording Single sodium channel currents were recorded using the cell-attached configuration of the patch-clamp technique. The shanks of pipettes with resistances of 6 1 M were coated with Sylgard, and the tips were then heat polished. This procedure decreased the electrode capacitance from 6 8 to <1 pf and significantly reduced background noise, without affecting the gigaseal resistance (>4 G ). Currents were activated with a 5 ms voltage pulse from a HP of 1 mv to a test potential of 5 mv. Other settings were the same as those used for whole-cell recording. Measurement of intracellular Ca 2+ fluorescence Ventricular myocytes were loaded with fura-2 AM (.5 μm) for 3 min at 25 C in the dark. Fluorescence measurements were made with a dual-excitation fluorescence photomultiplier system (Ionoptix). Ventricular myocytes were imaged through an Olympus IX-7 Fluor 4 oil objective. Cell contractions were elicited by field stimulations at a frequency of.5 Hz, 3 ms duration, using a pair of platinum wires placed on opposite sides of the chamber connected to an FHC stimulator (Brunswick, NE, USA). The polarity of the stimulating electrodes was reversed frequently to avoid possible build-up of electrolyte byproducts. Cells were exposed to light emitted by a 75 W lamp and passed through either a 34 or a 38 nm filter (bandwidths were 4 ± 15 nm) while being field stimulated. Fluorescence emissions were detected between 48 and 52 nm by a photomultiplier tube after first illuminating the cells at 34 nm for.5 s then at 38 nm for the duration of the recording protocol (333 Hz sampling rate). Quantitative changes in intracellular Ca 2+ concentration were inferred from the ratio of the fura-fluorescence intensity (FFI) at both wavelengths. Intracellular Ca 2+ fluorescence measurements were assessed using the following indices: diastolic intracellular Ca 2+ concentration (diastolic FFI; 34/38 ratio), amplitude of intracellular Ca 2+ transient (34/38 ratio), maximal rate of intracellular Ca 2+ rise and Ca 2+ decay (34/38 ratio). Data analysis and statistics Whole-cell currents were analysed using FitMaster (HEkA Electronic, Lambrecht, Pfalz, Germany). Current density was calculated by dividing the current amplitude by the cell capacitance. In general, the amplitude of I Na,L was measured 2 ms after the onset of a depolarizing pulse to avoid contamination of I Na,L with the transient sodium current. The I V relationship for I Na,L was plotted using data from the ramp pulse protocol (see Whole-cell current recording section). For analysis of I NCX, currents measured at +5 and 1 mv were used to determine the magnitudes of reverse and positive I NCX, respectively (Blaustein & Lederer, 1999; Tang et al. 212). Single-channel recordings were analysed using TAC+TACFit (X4..9; Bruxton, Seattle, WA, USA). Capacitance transients and leakage currents were nullified by off-line subtracting fits of average blunt traces. Channel openings at times >5 ms following the onset of a depolarizing pulse were used for analysis of persistent Na + channel activity. Openings and closures were identified by the half-height criterion. Open probability was calculated from the total open times of at least 1 sweeps divided by the total sweep duration. Histograms of channel open time distribution were fitted to single exponentials using TACFit. For all data, the Kolmogorov Smirnov statistic was used to confirm that they conformed to a normal distribution, followed by Student s paired t test to compare the C 217 The Authors. Experimental Physiology C 217 The Physiological Society

5 822 C. Fu and others Exp Physiol 12.7 (217) pp Table 1. The average current densities of late sodium current at 2 mv before and after addition of 1 µmol l 1 bisindolylmaleimide VI (BIM) Membrane potential (mv) current density (pa pf 1 ) Current density in presence of 1 μmol l 1 BIM (pa pf 1 ) Mean ± SD.3156 ± ±.134 P >.5 (n = 7) versus control conditions. Table 3. The average current densities of late sodium current at 2 mv potential before and after addition 1 mmol l 1 BAPTA AM Membrane potential (mv) current density (pa pf 1 ) Current density in presence of 1 mmol l 1 BAPTA (pa pf 1 ) Mean ± SD.3267 ± ±.284 P >.5 (n = 6) versus control conditions. Table 2. The average current densities of late sodium current at 2 mv before and after addition of 1 µmol l 1 KN-93 Membrane potential (mv) current density (pa pf 1 ) Current density in presence of 1 μmol l 1 KN-93 (pa pf 1 ) Mean ± SD.3292 ± ±.48 P >.5 (n = 7) versus control conditions. differences before and after a treatment factor of one cell. Furthermore, one-way ANOVA followed by Bonferroni s multiple comparison was also used to test the statistical significance of difference. All values are expressed as mean ± SD, and the number of cells (n) ineachgroupis given. A value of P <.5 was considered to be statistically significant. Figures were plotted using Origin (V7.; OriginLab, Northampton, MA, USA). Data were analysed using Microsoft Excel and SPSS (V17.; IBM Co., USA). Results Effect of KN-93, BIM and BAPTA AM on I Na,L during normoxia In order to detect the effects of the CaMKII and PKC inhibitors and the Ca 2+ chelator on I Na,L in ventricular myocytes in normoxic conditions, we recorded the I Na,L at apotentialof 2 mv before and after adding 1 μmol l 1 BIM, 1 μmol l 1 KN-93 or 1 mmol l 1 BAPTA in normoxia (Tables 1 3). The average current densities of I Na,L recorded in control (normoxic) conditions in three groups of experiments were.32 ±.2,.33 ±.4 and.33 ±.3 pa pf 1 at 2 mv. After adding 1 μmol l 1 BIM, 1 μmol l 1 KN-93 or 1 mmol l 1 BAPTA, respectively, the densities of I Na,L in myocytes were.32 ±.1,.34 ±.4 and.32±.3 pa pf 1 (BIM group, n = 7; KN-93 group, n = 7; BAPTA group, n = 6; all P >.5 versus control). There was no significant difference compared with control conditions. Effect of hypoxia to increase I Na,L The effects of hypoxia on I Na,L in the absence and presence of drug were measured in three series of experiments, respectively, to investigate the roles of CaMKII, PKC and Ca 2+ in mediating the action of hypoxia (Figs 1 3). The density of I Na,L was augmented during hypoxia in all three experimental series (Figs 1A,2A and 3A). Analyses of the I Na,L I V curves for all experiments are presented and plotted in Figs 1B,2B and 3B. was associated with an increased density of I Na,L at all voltages tested (Figs 1B, 2B and 3B). The I V data indicate that the density of I Na,L was greatest at 2 mv during both normoxia and hypoxia. As seen in Figs 1C, 2C and 3C, the densities of current recorded in control (normoxic) conditions in the three series of experiments were.31 ±.2,.3 ±.3 and.26 ±.4 pa pf 1 at 2 mv. The densities of I Na,L in myocytes exposed to 1 min of hypoxia were significantly greater than control:.66 ±.3,.6 ±.3 and.63 ±.5 pa pf 1, respectively (Figs 1 3, each n = 6, all P <.1 versus control). Effects of KN-93, BIM and BAPTA AM to attenuate the hypoxia-induced increase of I Na,L To investigate the intracellular signal transduction pathways involved in mediation of the hypoxia-induced increase of I Na,L, the effects of antagonists of CaMKII (1 μm KN-93) and PKC (1 μm BIM) and a Ca 2+ -chelating C 217 The Authors. Experimental Physiology C 217 The Physiological Society

6 Exp Physiol 12.7 (217) pp Modulation of late sodium current during hypoxia 823 agent (1 mm BAPTA AM) were investigated. Each of the three treatments resulted in a significant reduction of I Na,L in myocytes exposed to hypoxia (Figs 1 3). The CaMKII inhibitor KN-93 significantly reduced I Na,L recorded at 2 mv in myocytes exposed to hypoxia from.66 ±.3 to.44 ±.3 pa pf 1 (Fig. 1B and C; n = 6, P <.1 versus hypoxia alone). The PKC blocker BIM reduced I Na,L from.63 ±.5 to.48 ±.2 pa pf 1 (Fig. 2B and C; n = 6, P <.1 versus hypoxia). The Ca 2+ -chelating agent BAPTA AM (1 mm) reduced I Na,L from.63 ±.5 to.39 ±.2 pa pf 1 (Fig. 3B and C; n = 6, P <.1 versus hypoxia). The data in Figs 1B, 2B and 3B show the values of I Na,L recorded at different depolarizing test voltages. The data suggest that a hypoxia-induced increase of I Na,L can be suppressed by blocking either CaMKII or PKC or by chelation of cytoplasmic Ca 2+. Effect of hypoxia to increase I NCX in the absence and presence of BIM, KN-93 or BAPTA AM Exposures of myocytes to hypoxia for 1 min caused a significant increase (all three, P <.1 versus control) of reverse-mode I NCX [i.e. Na + exit and Ca 2+ entry to A +KN-93 I Na,L (pa/pf) B mv 6 mv 4mV 3 mv 2 mv mv 6 mv 4 mv 3 mv 2 mv ms 6 mv 9 mv +KN C mv 6 mv 4 mv 3 mv 2 mv (KN-93) 3ms 2mV 9mV Figure 1. Effect of hypoxia to increase the late sodium current (I Na,L ) in the absence and presence of the Ca 2+ calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 (1 µm) A, records of currents recorded from individual myocytes exposed to normoxia (control), hypoxia or hypoxia plus KN-93. B, summary of data for I V relationships for I Na,L recorded during normoxia, hypoxia or hypoxia plus KN-93 (n = 6 for all points). C, representative records of I Na,L recorded at 2 mv from single myocytes exposed to normoxia, hypoxia or hypoxia in the presence of KN-93. Values are means ± SD, n = 6 cells per group; P <.1 versus control; P <.1, P <.5 versus hypoxia. C 217 The Authors. Experimental Physiology C 217 The Physiological Society

7 824 C. Fu and others Exp Physiol 12.7 (217) pp the cell, recorded in this study as an outward, positive (greater than zero) membrane current at +5 mv; Fig. 4]. These results are consistent with previous findings that hypoxia causes cellular Na + loading that facilitates an increase of reverse-mode I NCX.Inmyocytesexposedto hypoxia, BIM (1 μm), KN-93 (1 μm) or BAPTA AM (1 mm) each significantly reduced reverse-mode (but not forward-mode) I NCX (respectively: n = 6, P <.1 versus hypoxia; n = 7, P <.1 versus hypoxia; n = 6, P <.1 versus hypoxia; Fig. 4). Effect of hypoxia on single Na + channel activity in the absence and presence of either KN-93 or BAPTA AM To confirm and extend the results of the whole-cell current experiments, we recorded single Na + channel currents using the cell-attached patch-clamp technique in two series of experiments (one with KN-93 and one with BAPTA AM). As expected, single Na + channel openings and bursts of openings both increased from control (normoxia) after 1 min of hypoxia (Fig. 5B versusa A +BIM mv 6 mv 4 mv 3 mv 2 mv mv 6 mv 4 mv 3 mv 2 mv mv 6 mv 4 mv 3 mv 2 mv B. I Na,L (pa/pf) ms 6 mv 9 mv +BIM C ms 2 mv 9 mv (BIM) Figure 2. Effect of hypoxia to increase I Na,L in the absence and presence of the protein kinase C (PKC) antagonist bisindolylmaleimide VI (BIM; 1 µm) A, records of currents recorded from individual myocytes exposed to normoxia (control), hypoxia or hypoxia plus 1 μm BIM. B, summary of data for I V relationships for I Na,L recorded during normoxia, hypoxia and hypoxia plus BIM (n = 6 for all points). C, representative records of I Na,L recorded at 2 mv from single myocytes exposed to normoxia, hypoxia or hypoxia in the presence of BIM. Values are means ± SD, n = 6 cells per group; P <.1 versus control; P <.1, P <.5 versus hypoxia. C 217 The Authors. Experimental Physiology C 217 The Physiological Society

8 Exp Physiol 12.7 (217) pp Modulation of late sodium current during hypoxia 825 and Fig. 6B versusa). Na + channel mean open time and mean open probability also increased during exposure of myocytes to hypoxia (Figs 7 and 8). Application of either 1 μm KN-93 (Fig. 5C) or1mmbapta AM (Fig. 6C) reduced the frequency of Na + channel openings during hypoxia. The effects of KN-93 and BAPTA AM were reversible, as the frequency of Na + channel openings during hypoxia again increased when these agents were washed out of the perfusion chamber (Figs 5D and 6D). During hypoxia, Na + channelmeanopenprobabilityand mean open time increased from control (normoxia) values of.38 ±.2 and.728 ±.79 ms, respectively, to.126 ±.13 and 1.72 ±.243 ms (Fig. 6; n = 6, both P <.1 versus control). After addition of 1 μm KN-93 to the perfusate, these values significantly declined to.86 ±.13 and ±.19 ms, respectively (n = 6, both P <.1 versus hypoxia), and after washout of KN-93, the values increased again to.118 ±.12 and ±.193 ms (n = 6, both P <.1 versus 1 μm KN-93; Fig. 7). A +BAPTA mv 6 mv 4 mv 3 mv 2 mv 2 7 mv 6 mv 4 mv 3 mv 2 mv 2 7 mv 6 mv 4 mv 3 mv 2 mv B C I Na,L (pa/pf) ms 6 mv 9 mv +BAPTA (BAPTA) 3 ms 2 mv 9 mv Figure 3. Effect of hypoxia to increase I Na,L in the absence and presence of the Ca 2+ -chelating agent BAPTA AM (1 mm) A, records of currents recorded from individual myocytes exposed to normoxia (control), hypoxia or hypoxia plus BAPTA AM. B, summary of data for I V relationships for I Na,L recorded during normoxia, hypoxia and hypoxia plus BAPTA AM (n = 6 for all points). C, representative records of I Na,L recorded at 2 mv from single myocytes exposed to normoxia, hypoxia or hypoxia in the presence of BAPTA AM. Values are means ± SD, n = 6 cells per group; P <.1 versus control; P <.1 versus hypoxia. C 217 The Authors. Experimental Physiology C 217 The Physiological Society

9 826 C. Fu and others Exp Physiol 12.7 (217) pp A B C /ramp (pa) a () 6 mv 4 mv 4 mv 2 s 12 mv b (+BIM) /ramp (pa) c () c-d d (+BIM+NiCl 2) 1 6 mv 4 mv 4 mv 2 s 12 mv a-d b-d /NCX (pa/pf) / NCX (+5 mv) / NCX ( 1 mv) D / ramp (pa) a () 6 mv 4 mv b (+KN-93) 4 mv 12 mv c () 2 s E /ramp (pa) a-d c-d 6 mv 4 mv b-d 2 s 4 mv 12 mv F /NCX (pa/pf) BIM / NCX (+5 mv) / NCX ( 1 mv) 1 d (+KN-93+NiCl 2) KN-93 G H I /ramp (pa) 3 6 mv a () 4 mv 4 mv 2 s 12 mv 2 b (+BATPA) 1 c () /ramp (pa) mv 4 mv 4 mv 2 s 12 mv a-d b-d c-d /NCX (pa/pf) / NCX (+5 mv) / NCX ( 1 mv) 1 d (+BATPA+NiCl 2) BAPTA Figure 4. Effect of hypoxia to increase reverse-mode Na + Ca 2+ exchange current (reverse I NCX )inthe absence and presence of BIM, KN-93 or BAPTA AM C 217 The Authors. Experimental Physiology C 217 The Physiological Society

10 Exp Physiol 12.7 (217) pp Modulation of late sodium current during hypoxia 827 A, D and G, rriginal I V relationships obtained using a ramp voltage pulse stimulation protocol and recorded from myocytes exposed to normoxia (control, trace c), hypoxia (trace a), then hypoxia plus 1 μm BIM (A, trace b), hypoxia plus 1 μm KN-93 (D) or hypoxia plus 1 mm BAPTA AM (G), and finally, 5mM NiCl 2 in the continued presence of hypoxia and test agent (trace d). B, E and H, plotsofi NCX as a function of voltage. Using the data shown in A, D and G, theni 2+ -sensitive current (i.e. I NCX )was calculated by subtracting the data in trace d from the data in traces a, b and c. C, F and I, bar graphs of summary data for mean current densities of I NCX at potentials of +5 mv (reverse mode, open bars) and 1 mv (forward mode, filled bars). Values are means ± SD, n = 6, 7 and 6 cells in each group, respectively. P <.1 versus control; P <.1 versus hypoxia alone. In the series of experiments with BAPTA AM, sodium channelmeanopenprobabilityandmeanopentime increased from control values of.32 ±.2 and.76 ±.13 ms, respectively, to.115 ±.16 and 1.76 ±.325 ms (Fig. 8; n = 6, P <.1 versus control). In the presence of 1 mm BAPTA AM, these values decreased to.61±.6 and.982±.19 ms after 1 min (Fig. 8; n = 6, P <.1 versus hypoxia), and after washout of BAPTA AM, the values increased to.118 ±.12 and ±.193 ms (Fig. 8; n = 6, P <.1 versus 1mM BAPTA AM). Effects of KN-93, BIM and BAPTA AM on the diastolic calcium transient induced by hypoxia In order to reflect the changes of [Ca 2+ ] i directly, intracellular Ca 2+ fluorescence measurements were conducted during normoxia, hypoxia and hypoxia plus one of the drugs (KN-93, BIM or BAPTA AM). As shown in Fig. 9A F, the diastolic FFI was enhanced, whereas the amplitude of the Ca 2+ transient and maximal velocities of Ca 2+ increase and Ca 2+ decay were significantly attenuated after 1 min of hypoxia (n = 7cellspergroup,allP <.1 versus control). In the continued presence of hypoxia, KN-93 (1 μm), BIM (1 μm) andbaptaam(1mm) significantly reversed these effects induced by hypoxia (n = 7cellspergroup,allP <.1 versus hypoxia; Fig. 9). Discussion The major findings in this study were as follows: (i) late sodium current (I Na,L ) in rabbit isolated ventricular myocytes was significantly increased by exposure of these cells to 1 min of hypoxia (P O2 = 1.33 kpa, equivalent to 1 mmhg; Figs 1 3); (ii) for sodium channel late openings (i.e. openings >5 ms after the onset of a depolarizing pulse), mean open probability and mean open time were significantly increased during hypoxia, relative to normoxia (Figs 5 8); (iii) the effects of hypoxia to increase I Na,L,sodiumchannelmeanopenprobability and channel mean open time were significantly attenuated by the CaMKII inhibitor KN-93 (1 μm)andbybaptaam (1 mm), a chelator of cytoplasmic Ca 2+ (Figs 5 8); the PKC inhibitor BIM also significantly attenuated the effect of hypoxia to increase I Na,L (Fig. 2), but they had no effect on I Na,L during normoxia; and (iv) the reverse I NCX was also increased in myocytes exposed to hypoxia; the effect of hypoxia was significantly attenuated by KN-93, BIM or BAPTA AM (Figs 4 and 5). The diastolic [Ca 2+ ] i was increased, but the amplitude of [Ca 2+ ] i transient and the maximal velocities of Ca 2+ increase and Ca 2+ decay were attenuated when these myocytes were exposing to hypoxia, which could be reversed by KN-93 (1 μm), BIM (1 mm) and BAPTA AM (1 mm), respectively (Fig. 9). The findings suggest that the effect of hypoxia to increase the amplitudes of I Na,L,reverseI NCX and diastolic [Ca 2+ ] i in myocytes is mediated by signalling events involving PKC, CaMKII and Ca 2+. These events occur either in sequence or in concert to act on the cell membrane sodium channel and to increase the intracellular Na + concentration, thereby increasing the concentration gradient for extrusion of cytoplasmic Na + via Na + Ca 2+ exchange. ProteinkinaseChasbeenshowntophosphorylate serine and threonine residues in the sodium channel inactivation gate, leading to destabilization of channel inactivation and increased I Na,L (West et al. 1991; Qu et al. 1996; Murray et al. 1997). The Ca 2+ -dependent or conventional PKC comprises four major isoforms (α, β1, β2 andγ; Farhadiet al. 26). The activity of PKCα was found to be significantly increased in failing hearts (Song et al. 215). The CaMKII is also reported to phosphorylate sodium channels and to increase I Na,L (Braun & Schulman, 1995; Maier & Hasenfuss, 26), and Ca 2+ activates CaMKII. It is thus not surprising that in the present study, BIM, KN-93 and BAPTA AM each acted to attenuate the effect of hypoxia to increase I Na,L. We observed that reverse-mode I NCX and I Na,L were both augmented in the presence of hypoxia and that application of 1 μm KN-93, 1 μm BIM or 1 mm BAPTA AM attenuated the effects of hypoxia on both currents. The finding is consistent with previous reports demonstrating that an increase of I Na,L can cause [Na + ] i to increase, leading to an increase of reverse-mode I NCX and Ca 2+ overload (Hammarström & Gage, 22; Kong et al. 212; Qian et al. 212; Wang et al. 214). Results of recent studies indicate that increases of [Ca 2+ ] i and I Na,L in ventricular myocytes act in a positive but pathological feedback system, involving PKC and CaMII, to create electrical and mechanical dysfunction (Maltsev & Undrovinas, 28; Maltsev et al. 28; Ma et al. 212). Qian et al. (212) reported that 4 μm tetrodotoxin blocked the effect C 217 The Authors. Experimental Physiology C 217 The Physiological Society

11 828 C. Fu and others Exp Physiol 12.7 (217) pp of 3 μm H 2 O 2 to increase I Na,L and reverse-mode I NCX, further supporting the hypothesis that sodium channel I Na,L can cause cellular Na + loading that increases reverse-mode I NCX and Ca 2+ loading. Our experimental results indicated that KN-93, BIM and BAPTA AM could attenuate the increases of I Na,L by hypoxia, but these drugs had no influence on I Na,L in normoxic conditions. These results mean that the enlargement of I Na,L by hypoxia was as a result of the activations of CaMKII and PKC at this time. We inferred that hypoxia elevated the activity of PKC and CaMKII in rabbit myocardial cell cytoplasm, which would trigger a series of chain reactions to expedite extracellular Ca 2+ influx and elevate diastolic [Ca 2+ ] i. At the same time, we also considered that the excessive activation of two enzymes was an important step of the chain reactions. To block them would ultimately prevent cytoplasmic Ca 2+ overload during hypoxia. In addition, we found that the increases of I Na,L, I NCX and diastolic [Ca 2+ ] i by hypoxia were not blocked entirely with KN-93, BIM or BAPTA AM. This might indicate that multiple mechanisms or mechanisms not explored in this study might contribute to increases of I Na,L, I NCX and diastolic [Ca 2+ ] i during hypoxia. For example, hypoxia decreases formation of ATP (Cross et al. 199) and leads to inhibition of the Na + K + pump and enhancement of Na + A B 1 min C +1 µm KN-93 D KN-93 Washed out 2 pa 1 ms 1 mv 5 ms 5 mv Figure 5. Effect of hypoxia on Na + channel activity in the absence and presence of KN-93 Channel activity (current) in a cell-attached patch was recorded from 5 to 5 ms after the onset of a voltage step to 5 mv from a holding potential of 1 mv. A D, representative traces recorded during normoxia (A, control), after 1 min of hypoxia (B), during perfusion with 1 μm KN-93 in the continued presence of hypoxia (C) and after washout of KN-93 (D, hypoxia alone). C 217 The Authors. Experimental Physiology C 217 The Physiological Society

12 Exp Physiol 12.7 (217) pp Modulation of late sodium current during hypoxia 829 entry into the cell via H + Na + exchange (Tang et al. 212). We speculate that above-mentioned mechanisms may also be an initial factor for hypoxia increasing I Na,L.Thatisto say, inhibition of the Na + K + pump and enhancement of Na + entry into the cell via H + Na + exchange can cause [Na + ] i to increase, leading to an increase of reverse-mode I NCX and Ca 2+ increase during hypoxia; then CaMKII and PKC activated by intracellular high Ca 2+ increased I Na,L in ventricular myocytes. A limitation of our experimental method is that changes in extracellular ion concentrations, CO 2 and lactate are mitigated by constant superfusion of myocytes with bathing solution. Additional studies of in vivo preparations are therefore needed to confirm or extend our findings. Our research design is mainly conducted at the mammalian cellular level. Owing to the experimental limitations conditions, we used ventricular myocytes of Adult New Zealand rabbits. More study and analysis are required, especially in different species (especially humans) and at the organ level. In summary, hypoxia augmented the amplitude of I Na,L and the mean open probability and mean open time of A B 1 min C +1mM BAPTA D BAPTA washed out 4 pa 1 ms 1 mv 5 ms 5 mv Figure 6. Effect of hypoxia on sodium channel activity in the absence and presence of BAPTA AM Channel activity (current) in a cell-attached patch was recorded from 5 to 5 ms after the onset of a voltage step to 5 mv from a holding potential of 1 mv. A D, representative traces recorded during normoxia (A, control), after 1 min of hypoxia (B), during perfusion with 1 mm BAPTA AM in the continued presence of hypoxia (C) and after washout of BAPTA AM (D, hypoxia alone). C 217 The Authors. Experimental Physiology C 217 The Physiological Society

13 83 C. Fu and others Exp Physiol 12.7 (217) pp A B C D min +1µM KN KN-93 washed out Amplitude (pa) Amplitude (pa) Amplitude (pa) Amplitude (pa) E F G H 1 min +1 µm KN KN-93 washed out τ=.728 τ=1.72 τ=1.292 τ= log Duration (s) log Duration (s) log Duration (s) log Duration (s) I.2 J Mean open probability KN-93 KN µm washed out Mean open time (ms) KN-93 KN µm washed out Figure 7. Effect of hypoxia in the absence and presence of KN-93 on sodium channel mean open probability and mean open time Data are from experiments (n = 6 for all treatments) such as those shown in Fig. 5; 1 individual current traces were analysed for calculation of data shown in each panel. A D, all-point histograms of current amplitude. E H, histograms of channel mean open times. I and J, summary of data from all experiments (n = 6). P <.1 versus control; P <.1 versus hypoxia; + P <.1 versus KN-93. C 217 The Authors. Experimental Physiology C 217 The Physiological Society

14 Exp Physiol 12.7 (217) pp Modulation of late sodium current during hypoxia 831 A B C D 75 1 min +1 mm BAPTA BAPTA washed out Amplitude (pa) Amplitude (pa) Amplitude (pa) Amplitude (pa) E F G H 1 min +1 mm BAPTA BAPTA washed out τ= τ= τ= τ= log Duration (s) log Duration (s) log Duration (s) log Duration (s) I.2 J Mean open probability Mean open time (ms) BAPTA BAPTA. BAPTA BAPTA 1 mm washed out 1 mm washed out Figure 8. Effect of hypoxia in the absence and presence of BAPTA AM on sodium channel mean open probability and mean open time Data are from experiments (n = 6 for all treatments) such as those shown in Fig. 7; 1 individual current traces were analysed for calculation of data shown in each panel. A D, all-point histograms of current amplitude. E H, histograms of channel mean open times. I and J, summary of data from all experiments (n = 6). P <.1 versus control; P <.1 versus hypoxia; + P <.1 versus BAPTA AM. C 217 The Authors. Experimental Physiology C 217 The Physiological Society

15 Amplitude of [Ca 2+ ] i transient (F 34/F38) 832 C. Fu and others Exp Physiol 12.7 (217) pp A +1 µm KN-93 [Ca 2+ ] i (F 34 /F 38 ) [Ca 2+ ] i (F 34 /F 38 ) [Ca 2+ ] i (F 34 /F 38 ) B C µm BIM +1 mm BAPTA 1. D Diastolic [Ca 2+ ] i (F 34 /F 38 ) Maximal rate of [Ca 2+ ] i rise (ratio/ms) Maximal rate of [Ca 2+ ] i decay (ratio/ms) μm KN-93 E Diastolic [Ca 2+ ] i (F 34 /F 38 ) & Amplitude of [Ca 2+ ] i transient (F 34/F38) & Maximal rate of [Ca 2+ ] i rise (ratio/ms) & Maximal rate of [Ca 2+ ] i decay (ratio/ms) μm BIM & F Diastolic [Ca 2+ ] i (F 34 /F 38 ) $ Amplitude of [Ca 2+ ] i transient (F34 /F 38) $ $ Maximal rate of [Ca 2+ ] i rise (ratio/ms) Maximal rate of [Ca 2+] i decay (ratio/ms) mm BAPTA $ Figure 9. Effects of KN-93, BIM and BAPTA AM on the diastolic calcium transient induced by hypoxia A C, representative recordings of [Ca 2+ ] i transients in the absence (control) and presence of hypoxia before and after exposure to KN-93 (1 μm), BIM (1 μm) or BAPTA AM (1 mm), respectively. D F, summary data for representative parameters of diastolic [Ca 2+ ] i, amplitude of [Ca 2+ ] i transients, maximal rate of [Ca 2+ ] i rise and [Ca 2+ ] i decay. The results are expressed as the mean ± SD (n = 7 cells in each group). P <.1, hypoxia versus control; P <.1, 1 μm KN-93 versus hypoxia; & P <.1, 1 μm BIM versus hypoxia; $ P <.5 1 mm BAPTA versus hypoxia. C 217 The Authors. Experimental Physiology C 217 The Physiological Society

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