Histone Deacetylase Inhibition Reduces Pulmonary Vein ...

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Sep 21, 2014 - Baigalmaa Lkhagva a, Shih-Lin Chang b, Yao-Chang Chen c, Yu-Hsun Kao a ... Cindy Tzu-Hsuan Chiu f, Shih-Ann Chen b, Yi-Jen Chen a,e,⁎.
International Journal of Cardiology 177 (2014) 982–989

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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard

Histone Deacetylase Inhibition Reduces Pulmonary Vein Arrhythmogenesis through Calcium Regulation Baigalmaa Lkhagva a, Shih-Lin Chang b, Yao-Chang Chen c, Yu-Hsun Kao a,d, Yung-Kuo Lin e, Cindy Tzu-Hsuan Chiu f, Shih-Ann Chen b, Yi-Jen Chen a,e,⁎ a

Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan Division of Cardiology and Cardiovascular Research Center, Taipei Veterans General Hospital, Taipei, Taiwan Department of Biomedical Engineering, National Defense Medical Center, Taipei, Taiwan d Department of Medical Education and Research, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan e Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan f Department of Anatomy and Cell Biology, McGill University, Montreal, Canada b c

a r t i c l e

i n f o

Article history: Received 31 May 2014 Received in revised form 21 September 2014 Accepted 28 September 2014 Available online 5 October 2014 Keywords: Atrial fibrillation Histone deacetylase inhibition Pulmonary vein Calcium homeostasis

a b s t r a c t Pulmonary veins (PVs) play a critical role in the pathophysiology of atrial fibrillation (AF). Histone deacetylases (HDACs) are vital to calcium homeostasis and AF genesis. However, the electrophysiological effects of HDAC inhibition were unclear. This study evaluated whether HDAC inhibition can regulate PV electrical activity through calcium modulation. Whole-cell patch-clamp, confocal microscopic with fluorescence, and Western blot were used to evaluate electrophysiological characteristics and Ca2+ dynamics in isolated rabbit PV cardiomyocytes with and without MPT0E014 (a pan HDAC inhibitor), MS-275 (HDAC1 and 3 inhibitor), and MC-1568 (HDAC4 and 6 inhibitor) for 5 ~ 8 h. Atrial electrical activity and induced-AF (rapid atrial pacing and acetylcholine infusion) were measured in rabbits with and without MPT0E014 (10 mg/kg treated for 5 hours) in vivo. MPT0E014 (1 μM)-treated PV cardiomyocytes (n = 12) had slower beating rates (2.1 ± 0.2 vs. 2.8 ± 0.1 Hz, p b 0.05) than control PV cardiomyocytes. However, control (n = 11) and MPT0E014 (1 μM)-treated (n = 12) SAN cardiomyocytes had similar beating rates (3.2 ± 0.2 vs. 2.9 ± 0.3 Hz). MS-275-treated PV cardiomyocytes (n = 12, 2.3 ± 0.2 Hz), but not MC-1568-treated PV cardiomyocytes (n = 14, 3.1 ± 0.3 Hz) had slower beating rates than control PV cardiomocytes. MPT0E014-treated PV cardiomyocytes (n = 14) had a lower frequency (2.4 ± 0.6 vs. 0.3 ± 0.1 spark/mm/s, p b 0.05) of Ca2+ sparks than control PV (n = 17) cardiomyocytes. As compared to control, MPT0E014-treated PV cardiomyocytes had reduced Ca2+ transient amplitudes, sodium-calcium exchanger currents, and ryanodine receptor expressions. Moreover, MPT0E014-treated rabbits had less AF and shorter AF duration than control rabbits. In conclusions, HDAC inhibition reduced PV arrhythmogenesis and AF inducibility with modulation on calcium homeostasis. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Abbreviations: Ac-H3, acetyl-Histone H3; AF, atrial fibrillation; AP, action potential; APD50, action potential duration at 50% repolarization; APD90, action potential duration at 90% repolarization; Ca2 +i, intracellular calcium; CaMKII, Calmodulin-dependent protein kinase II; HCN4, hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; HDACs, histone deacetylases; ICa-L, L-type calcium current; LA, left atrium; NCX, sodium calcium exchanger; PLB, phospholamban; PV, pulmonary veins; RyR, ryanodine receptor; SAN, sinoatrial node; SERCA, sarcoendoplasmic reticulum Ca2 +-ATPase; SR, sarcoplasmic reticulum. ⁎ Corresponding author at: Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, 111 Hsin-Lung Road, Sec. 3, Taipei 110, Taiwan. Tel./fax: +886 2 27390500. E-mail address: [email protected] (Y.-J. Chen).

http://dx.doi.org/10.1016/j.ijcard.2014.09.175 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia seen in clinical practice, and contributes to morbidity and mortality in the general population [1]. However, the pathophysiology underlying AF is not fully elucidated, and the current treatment of AF is unsatisfactory [2,3]. Histone deacetylases (HDACs), which are epigenetic regulators, play critical roles in altering gene expressions during the progression of remodeling processes of cardiovascular diseases [4]. Reversible acetylation of histone and non-histone nuclear and cytosolic proteins was implicated in multiple cellular regulatory processes of cardiovascular diseases [5]. HDAC inhibitors can reduce cardiac hypertrophy and fibrosis [4,6–8]. Our previous study has shown that a novel pan HDAC inhibitor (3-(1-Benzenesulfonyl-1H-indol-5-yl)-N-

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hydroxyacrylamide, MPT0E014) can improve heart failure with modulation on calcium regulation proteins [6]. Moreover, HDAC inhibitors were also shown to reduce AF induction in genetically modified mice with increased HDAC activity [9]. Activation of HDAC6 can enhance the occurrence of AF with contractile dysfunction, and AF patients had increased HDAC6 activities [10]. These findings suggest the potential role of inhibiting HDACs to treat AF. However, knowledge of the electrophysiological effects and calcium regulatory mechanisms of HDAC inhibition is limited. Pulmonary veins (PVs), are the most important ectopic foci for AF initiation [11,12]. PV cardiomyocytes contain distinctive electrophysiological characteristics with abnormally triggered activities [12,13]. PV cardiomyocytes have increased sarcoplasmic reticulum (SR) Ca2 + stores and Ca2+ sparks [13], which suggests that calcium dysregulation may lead to PV arrhythmogenesis. Enhanced calmodulin-dependent protein kinase II (CaMKII) and the phosphorylated ryanodine receptor (RyR) can enhance calcium leak which produces calcium sparks and generates triggered activity [14]. In addition, increased sodium calcium exchanger (NCX) currents and CaMKII-hyperphosphorylated RyR were demonstrated in AF patients [15]. Modulating PV calcium homeostasis controls PV electrical activity, which may reduce the risk of AF [16–18]. In contrast, sinoatrial node (SAN) dysfunction may enhance the occurrences of AF [19]. HDACs and HDAC inhibitors can modulate expressions of calcium regulatory proteins [20,21]. Therefore, the purpose of this study was to examine whether HDAC inhibition can modulate PV or SAN electrical activity through calcium modulation.

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2. Materials and methods 2.1. Isolation of PV and SAN cardiomyocytes The investigation was approved by a local ethics review board and was conducted in accordance with the US National Institute of Health’s Guide for the Care and Use of Laboratory Animals. Male rabbits (1.5 ~ 2 kg) were intravenously injected with sodium pentobarbital (100 mg⁄kg) and heparin (2500 units). Single cardiomyocytes from rabbit PVs and SANs were enzymatically dissociated by a previously described procedure [22]. Cardiomyocytes with spontaneous activity were identified by the presence of constant beating during perfusion with Tyrode’s solution. PV or SAN cardiomyocytes were incubated with or without (control) MPT0E014 (HDAC1, 2, 3, 10 ≥ 90% inhibition; HDAC6, 8 ≥ 70% inhibition, and HDAC4, 5, 7, 9 ≤ 50% inhibition at 1 μM, synthesized in-house, Supplemental Figure) for 5-8 hours. We also studied MS-275 (more inhibition on HDAC1 than HDAC3 at 1 μM [23], Cayman Chemical, Michigan, USA) and MC-1568 (more inhibition on HDAC4 than HDAC6 at 1 μM [24], Tocris Bioscience, Bristol, UK) in PV cardiomyocytes to clarify whether more specific HDAC inhibition may have electrophysiological effects.

2.2. Electrophysiological and pharmacological studies A whole-cell patch-clamp analysis was performed on single isolated PV or SAN cardiomyocytes using an Axopatch 1D amplifier (Axon Instruments, CA, USA) at 35 ± 1 °C. Borosilicate glass electrodes (o.d., 1.8 mm) were used, with tip resistances of 3 ~ 5 MΩ. Before the formation of the membrane-pipette seal, the tip potentials were zeroed in Tyrode’s solution. The junction potentials measured from differences between the bath and pipette (9 mV) solutions were corrected for action potential (AP) recordings. APs were recorded in the current-clamp mode. Ionic currents of myocytes were measured in the voltage-clamp mode [22]. A small hyperpolarizing step from a holding potential of -50 mV to a test potential of -55 mV for 80 ms was delivered at the beginning of each experiment. The area under the capacitive current was divided by the applied voltage

Fig. 1. Action potentials of control and MPT0E014-treated sinoatrial node (SAN) and pulmonary vein (PV) cardiomyocytes with spontaneous activity. (A) Tracings and average data of control (n = 15) and MPT0E014-treated (0.1 and 1 μM), (n = 12) PV cardiomyocytes with spontaneous activities. (B). Tracings and average data of control (n = 11) and MPT0E014-treated (0.1 μM, n = 15, and 1 μM, n = 12) SAN cardiomyocytes. *p b 0.05 versus control.

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step to obtain the total cell capacitance. Normally, 60% ~ 80% series resistance was electronically compensated for. AP measurements began 5 min after cell rupture. Micropipettes were filled with a solution containing (in mM) CsCl 130, MgCl2 1, Mg2ATP 5, HEPES 10, EGTA 10, NaGTP 0.1, and Na2 phosphocreatine 5 (at pH 7.2 adjusted with CsOH) for the L-type calcium current (ICa-L); containing (in mM) NaCl 20, CsCl 110, MgCl2 0.4, CaCl2 1.75, TEACl 20, BAPTA 5, glucose 5, Mg2ATP 5, and HEPES 10 (at pH 7.25 adjusted with CsOH) for the NCX current; and containing (in mM) KCl 20, K aspartate 110, MgCl2 1, Mg2ATP 5, HEPES 10, EGTA 0.5, LiGTP 0.1, and Na2 phosphocreatine 5 (at pH 7.2 adjusted with KOH) for the AP. The ICa-L was measured as an inward current during depolarization from a holding potential of -50 mV to the test potentials which ranged from -40 to +60 mV in 10-mV steps for 300 ms at a frequency of 0.1 Hz by means of a ruptured patch-clamp. The NaCl and KCl in the external solution were respectively replaced by TEACl and CsCl. The NCX current was elicited by depolarizing pulses between -100 and + 100 mV from a holding potential of -40 mV for 300 ms at a frequency of 0.1 Hz. Amplitudes of the NCX current were measured as 10-mM nickel-sensitive currents. The external solution consisted of (in mM) NaCl 140, CaCl2 2, MgCl2 1, HEPES 5, and glucose 10 at pH 7.4 and contained strophanthidin (10 μM), nitrendipine (10 μM), and niflumic acid (100 μM). After achieving a steady-state Ca2+ transients with the repeated pulses (1 Hz for 5 sec), the SR Ca2+ content was measured by integrating the NCX current after rapidly adding 20 mM of caffeine to cells within 0.5 sec at rest with the membrane potential clamped to -40 mV. The time integral of the NCX current was converted to amoles of Ca2+ released from the SR. 2.3. Measurement of intracellular calcium and Ca2+ spark imaging As described previously [25], PV cardiomyocytes were loaded with fluorescent Ca2+ (10 μM) fluo-3/AM for 30 min at room temperature. Excess extracellular dye was removed

Fig. 2. Effects of MS-275 and MC-1568 on pulmonary vein (PV) cardiomyocytes with spontaneous activity. Tracings and average data that MS-275 (1 μM)-treated (n = 12) PV cardiomyocytes had a slower beating rate than MC-1568 (1 μM)-treated (n = 14) or control PV cardiomyocytes.*p b 0.05 versus control.

by changing the bath solution, and intracellular hydrolysis of fluo-3/AM occurred at 35 ± 1 °C after 30 min. Fluo-3 fluorescence was excited with a 488-nm line of an argon ion laser. The emission was recorded at N 515 nm. Cells were repetitively scanned at 3-ms intervals for a total duration of 6 s. Fluorescence imaging was performed with a laser scanning confocal microscope (Zeiss LSM 510, Carl Zeiss, Jena, Germany) and an inverted microscope (Axiovert 100). The fluorescent signals were corrected for variations in the dye concentration by normalizing the fluorescence (F) against the baseline fluorescence (F0) to obtain reliable information about transient [Ca2+]i changes from baseline values (F/F0) and to exclude variations in the fluorescence intensity with different volumes of injected dye. [Ca2+]i transients, and peak systolic and diastolic [Ca2+]i were measured during a 2-Hz field stimulation with 10-ms twice-threshold strength square-wave pulses. As described previously [25], Ca2+ sparks were detected through the line scan mode along a line parallel to the longitudinal axis of single PV cardiomyocytes, while avoiding nuclei. Each line was composed of 512 pixels. Ca2+ sparks were detected as an increase in the signal mass (of b 6 μm). Ca2+ sparks were analyzed using SparkMaster, and validated by the authors using criteria of the signal mass. Calcium spark frequencies were expressed as the number of observed sparks (per second and per mm of scanned distance) and the incidences as the percentage of cells exhibiting Ca2+ sparks in the diastolic phase of PV cardiomyocytes with spontaneous activity.

2.4. Western blot analysis Control and MPT0E014-treated PV cardiomyocytes were centrifuged and lysed using radio-immunoprecipitation buffer. The protein concentration was determined with a Bio-Rad protein assay reagent (Hercules, CA, USA). For the analysis, equal amounts of protein from each sample were separated using 4% ~ 16% Tris-acetate polyacrylamide gradient gel electrophoresis. After electrophoresis, protein samples were transferred onto equilibrated polyvinylidene difluoride membranes (Amersham Biosciences, Buckinghamshire, UK). Blots were probed with primary antibodies against RyR type 2 (RyR2; Affinity BioReagent, Golden, CO, USA), SERCA2a (Santa Cruz Biotechnology,

Fig. 3. Intracellular calcium (Ca2+i) and sarcoplasmic reticulum (SR) calcium stores in control and MPT0E014-treated pulmonary vein (PV) cardiomyocytes. (A) Tracings and average data of Ca2+i transients from control (n = 27) and MPT0E014-treated (n = 19) PV cardiomyocytes. (B) Tracings and average data of caffeine-induced sodium-calcium exchanger (NCX) currents from control (n = 14) and MPT0E014-treated (n = 11) PV cardiomyocytes. *p b 0.05 versus control.

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Fig. 4. Ca2+ sparks in control and MPT0E014-treated pulmonary vein (PV) cardiomyocytes. (A) Examples of line scans for detecting Ca2+ sparks in control and MPT0E014-treated PV cardiomyocytes. (B) Incidences and frequencies of Ca2+ sparks in control (n = 17) and MPT0E014-treated (n = 14) cardiomyocytes. *p b 0.05, **p b 0.01 versus control.

Fig. 5. Ion channel and acetylated proteins in control and MPT0E014-treated pulmonary vein (PV) cardiomyocytes. MPT0E014-treated PVs (n = 7) had a higher expression of acetyl histone H3 (Ac-H3), and lower expressions of the sodium-calcium exchanger (NCX) and ryanodine receptor (RyR) than control PVs (n = 7). Ca2+/calmodulin-dependent protein kinase-II (CaMKII), Cav1.2, sarcoendoplasmic reticulum Ca2+-ATPase (SERCA2a), hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4), phospholamban (PLB), and Ser16 phosphorylated phospholamban (p-PLB) levels were similar between control (n = 7) and MPT0E014-treated PVs (n = 7). *p b 0.05 versus control.

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Santa Cruz, CA, USA), NCX (Swant, Bellinzona, Switzerland), Cav1.2 (ICa-L subunit; Alomone Labs, Jerusalem, Israel), phospholamban (PLB; Thermo, Rockford, IL, USA), Ser16-phosphorylated PLB (p-PLB; Badrilla, Leeds, UK), HCN4 and acetyl-Histone H3 (Ac-H3) (Millipore, Billerica, MA, USA), CaMKII (Abcam, Cambridge, UK), and α-actin (Sigma-Aldrich, St. Louis, MO, USA). Blots reacted with primary antibodies were followed by horseradish peroxidase-conjugated secondary antibodies. Bound antibodies were detected with an enhanced chemiluminescence (ECL) detection system (ECL Plus Kit, Millipore) and analyzed with AlphaEaseFC software (Alpha Innotech, San Leandro, CA, USA). Targeted bands were normalized to α-actin to confirm equal protein loading. 2.5. Effects of MPT0E014 on LA electrophysiology and AF inducibility in vivo To study the in vivo effects of MPT0E014 on atrial electrophysiology and AF inducibility, MPT0E014 (10 mg/kg in 2 ml 30% DMSO) or vehicle was administrated in rabbits intraperitoneally. Rabbits were anesthetized and intubated with an endotracheal tube and mechanically ventilated after drug administration for 5 hours. A median sternotomy was performed, and the chest wall was removed to expose the heart structure. As described previously [16,26], the surface ECG and intra-cardiac electrograms were recorded continuously using a computer-based digital amplifier/recorder system (Lab System ™ PRO EP Recording System, Bard, MA, USA) with a sampling rate of 2 KHz. Monophasic action potentials were recorded during the sinus rhythm from the epicardium by placing a mapping catheter at the LA appendage. The monophasic action potentials were filtered from 0.05 to 500 Hz. The signals were digitized at 1 kHz to 16-bit resolution and exported from the recorder (Bard Pro, Billerica, Massachusetts) for analysis using custom PC software written in Lab view (National Instruments, Austin, Texas). The signals with an unstable baseline, noise, or artifact were excluded. The effective refractory period was measured (2 times the diastolic threshold, 2-ms pulse width and mean of 3 determinations) at basic cycle lengths of 200 ms with a train of 7 basic (S1) stimuli followed by a premature (S2) stimulus with 2-ms decrements by using the monophasic action potentials pacing catheters. The effective refractory period was defined as the longest S1-S2 interval that failed to produce an AP. The 50% and 90% of AP duration (APD90 and APD50) were measured from the steepest deflection of the slope of phase 0 of the monophasic action potentials to the time at which 90% and 50% were repolarized. The induction of AF performed by rapid atrial pacing with the cycle length of 60-70 ms (4× threshold current for 10 s, 20 attempts) from the LA appendage using a 2-mm-tip mapping catheter (electrode spacing: 2-2-2 mm, St. Jude Medical, MN, USA) under continuous acetylcholine infusion (1 μM) at least 30 minutes. The inducibility of AF was measured as the percentage of AF occurrence (N1 s) from rapid atrial pacing in each rabbit. 2.6. Statistical analysis All quantitative data are expressed as the mean ± standard error (SEM). Differences between control or HDAC inhibitor-treated groups were compared using an unpaired t-test or Mann-Whitney rank-sum test depending on the outcome of the normality test. Nominal variables were compared by a Chi-square analysis with a Yates correction or Fisher’s exact test. A value of p b 0.05 was considered statistically significant.

Fig. 6. Effects of MPT0E014 on the L-type calcium current (ICa_L) in pulmonary vein (PV) cardiomyocytes. Examples of tracings and I-V relationships of ICa-L from control (n = 6) and MPT0E014-treated (n = 8) cardiomyocytes.

treated PV cardiomyocytes had similar expressions of CaMKII, Cav1.2, SERCA2, HCN4, PLB, and phosphorylated PLB.

3. Results 3.3. Effects of MPT0E014 on ionic currents in PV cardiomyocytes 3.1. Effects of MPT0E014 on PV APs, electrical activity, and calcium homeostasis As shown in Fig. 1, MPT0E014-treated PV cardiomyocytes had a slower beating rate than control PV cardiomyocytes (2.1 ± 0.2 vs. 2.8 ± 0.1 Hz, p b 0.05) at the concentration of 1 μM, but not at 0.1 μM (Fig. 1A). However, control and MPT0E014-treated (0.1 and 1 μM) SAN cardiomyocytes had similar beating rates (3.2 ± 0.2 vs. 2.9 ± 0.3 Hz) (Fig. 1B). Moreover, MS-275(1 μM)-treated PV cardiomyocytes had a slower beating rate (n = 12, 2.3 ± 0.2 Hz) than control or MC-1568 (1 μM)-treated PV cardiomyocytes (n = 14, 3.1 ± 0.3 Hz) (Fig. 2). MPT0E014-treated PV cardiomyocytes had smaller Ca2+i transient amplitudes than control PV cardiomyocytes (Fig. 3A). Control and MPT0E014-treated PV cardiomyocytes had similar SR Ca2 + contents (Fig. 3B). Moreover, as shown in Fig. 4, MPT0E014-treated PV cardiomyocytes had a lower incidence (29% vs. 82%, p b 0.01) and frequency of Ca2+ sparks (2.4 ± 0.6 vs. 0.3 ± 0.1 spark/mm/s, p b 0.05) than control PV cardiomyocytes. 3.2. Effects of MPT0E014 on calcium-handling proteins MPT0E014-treated PV cardiomyocytes had significantly higher expression of Ac-H3 and lower expressions of RyR2 and NCX1 than control PV cardiomyocytes (Fig. 5). However, control and MPT0E014-

As shown in Fig. 6, control and MPT0E014-treated PV cardiomyocytes had similar ICa-L current densities. However, MPT0E014treated PV cardiomyocytes had lower NCX currents than control cardiomyocytes by the reverse mode (Fig. 7). 3.4. Effects of MPT0E014 on LA electrophysiology and AF inducibility in vivo Control and MPT0E014-treated rabbits had similar heart rate, PR interval and QT interval (Fig. 8). Control and MPT0E014-treated LAs had similar APD90 and APD50, but MPT0E014-treated LA had a longer effective refractory period (69 ± 1 vs. 65 ± 1 ms, p b 0.05) than control LA. Moreover, MPT0E014-treated rabbits had a lower AF inducibility (6 ± 2 vs. 38 ± 3 %, p b 0.005) and shorter AF duration (2.1 ± 0.5 vs. 10.1 ± 0.2 sec, p b 0.005) than control rabbits. 4. Discussion In the present study, for the first time, we showed that an HDAC inhibitor may contain anti-AF potential through modulating calcium regulation in PV cardiomyocytes. Increased HDAC activity can produce atrial fibrosis and atrial arrhythmic susceptibility in transgenic mice. Although pharmacologic inhibition of HDAC suppressed atrial fibrosis and atrial arrhythmia [9], the effects of HDAC inhibition on ion channels, calcium homeostasis, and arrhythmogenesis were not assessed [9,27].

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Fig. 7. Effects of MPT0E014 on sodium-calcium exchanger (NCX) currents in pulmonary vein (PV) cardiomyocytes. Current tracings and I-V relationships of nickel-sensitive NCX in control (n = 6) and MPT0E014-treated (n = 7) cardiomyocytes. *p b 0.05, ***p b 0.005 versus control.

We found that MPT0E014 at the concentration of 1 μM can increase Ac-H3 and reduce spontaneous activity in PV cardiomyocytes. Those findings suggest that HDAC inhibition can modulate electrical activity in PV cardiomyocytes. In contrast, MPT0E014 did not change pacemaker activity in SAN cardiomyocytes. Since SAN and PV electrical activities play a critical role in the genesis of AF [28]. The inhibitory effects of MPT0E014 on PV but not on SAN, suggests that this pan-HDAC inhibitor may have anti-AF potential. MPT0E014 at 1 μM inhibits more class I HDACs than class II HDACs. This study found that only MS-275 (the rather specific HDAC1 inhibitor), but not MC-1568 (the rather specific HDAC 4 inhibitor) can reduce PV spontaneous activity. Therefore, HDAC1 inhibition may contribute to the effects of MPT0E014 on PV arrhytmogenesis at least in part.

The effects of MPT0E014 on PV arrhythmogenesis may be caused by its decreases in NCX currents and calcium transients, since calcium homeostasis plays a critical role in the genesis of PV electrical activity. The decrease in Ca2 + transients by MPT0E014 in the face of similar ICa-L and SR Ca2+ contents is expected to arise from effects of reduced RyR expression by MPT0E014, which would result in smaller calcium transients at similar SR calcium stores. Ca2 + sparks are generated by calcium leaks due to calcium overload or RyR dysfunction [13,25]. We found significantly decreased Ca2+ sparks in PV cardiomyocytes treated with MPT0E014, which may have been caused by decreased expression of the RyR2 protein in those cardiomyocytes. Previous studies have shown an increased PV arrhythmogenesis in rabbit AF models [25,29]. Since Ca2+ sparks play an important role in PV electrical activity and also significantly contribute to the arrhythmogenesis of AF and/or HF

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Fig. 8. Effects of MPT0E014 on left atrial (LA) electrophysiology and atrial fibrillations (AFs) inducibility in rabbits. (A) The examples and average data of lead II ECG and LA intracardiac monophasic action potential in control (n = 5) and MPT0E014-treated (n = 5) rabbits. Control and MPT0E014-treated rabbits had a similar ECG parameter and LA monophasic action potential morphology. (B) The examples and average data of rapid atrial pacing (RAP) and acetylcholine infusion on AF induction in control (n = 5) and MPT0E014-treated (n = 5) rabbits. As compared to those in control rabbits (upper panel), RAP with acetylcholine infusion did not induce AF (middle panel), or generate AF with a shorter period (lower panel) in MPT0E014treated rabbits. *p b 0.05, ***p b 0.005 versus control.

[25], our findings suggest the antiarrhythmic potential of HDAC inhibition. Moreover, HDAC inhibition can ameliorate HF and reduce cardiac fibrosis and hypertrophy, which would also decrease the risk of AF [6, 9,30]. HDAC inhibitors have been shown to reduce AF induction and the occurrence of AF by inhibiting cellular Ca2 + handling, reducing atrial fibrosis and improving contractile dysfunction [9,10]. In this study, we found that MPT0E014 can suppress AF episodes during acetylcholine infusion and rapid atrial pacing in rabbits, which support the anti-AF potential of HDAC inhibitors. This effect may partially arise from suppression of PV arrhythmogenesis or prolongation of LA effective refractory period by MPT0E014. In addition, HDAC inhibitors also can decrease oxidative stress [31], inflammation [7,32] and renin– angiotensin system activity [6,33], which may further reduce AF occurrence through upstream therapy [34].

A previous study showed that over-stimulated NCX1 gene expression was inhibited in a dose-dependent manner by the HDAC inhibitor, trichostatin A, through decreasing deacetylation of Nkx2.5 and binding of p300 to the NCX1 promoter [21]. NCX plays important roles in the genesis of PV electrical activity and calcium regulation. Therefore, the decrease in NCX by MPT0E014 supposedly reduces PV arrhythmogenesis. More recently, Lu et al. presented other evidence that activation of CaMKIIδB led to an increase in NCX1 expression via class IIa HDAC/MEF2-dependent signaling in adult cardiomyocytes [35]. In that study, they demonstrated that the increase in CaMKIIδB was accompanied by translocation of HDAC4 from nucleus to the cytosol, and its activity increased MEF2 transcriptional activity by targeting HDAC4, which could affect transcriptional activation of the NCX1 exchanger [35].

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There are some potential limitations. In order to maintain cell viability, we treated PV or SAN cardiomyocytes with HDAC inhibitors for a rather short period. The long-term effects of HDAC inhibition on cardiomyocytes are not clear. HF has been shown to increase PV arrhythmogenesis [12]. However, this study evaluated the HDAC inhibitors on cardiomyocytes from healthy rabbits. Our previous study showed that MPT0E014 can reduce animal HF [6]. It is not clear whether HDAC inhibition may have different effects in the pathological cardiomyocytes. In conclusion, HDAC inhibition can reduce PV arrhythmogenesis and AF inducibility with modulation on calcium homeostasis. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2014.09.175. Disclosures The authors declare no conflict of interest. Acknowledgements The present work was supported by grants from Taipei Medical University, Wan Fang Hospital (103swf-10, 103-wf-eva-02 and 102wf-eva-01), and the Ministry of Science and Technology, Taiwan (MOST 100-2628-B-038-001-MY4, 102-2628-B-038-002-MY3, and 102-2325-B-010-005). References [1] Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Borden WB, et al. Heart disease and stroke statistics-2013 update: a report from the American Heart Association. Circulation 2013;127:e6-245. [2] Dobrev D, Carlsson L, Nattel S. Novel molecular targets for atrial fibrillation therapy. Nat Rev Drug Discov 2012;11:275–91. [3] Dobrev D, Nattel S. New antiarrhythmic drugs for treatment of atrial fibrillation. Lancet 2010;375:1212–23. [4] McKinsey TA. Therapeutic potential for HDAC inhibitors in the heart. Annu Rev Pharmacol Toxicol 2012;52:303–19. [5] Haberland M, Montgomery RL, Olson EN. The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet 2009;10:32–42. [6] Kao YH, Liou JP, Chung CC, Lien GS, Kuo CC, Chen SA, et al. Histone deacetylase inhibition improved cardiac functions with direct antifibrotic activity in heart failure. Int J Cardiol 2013;168:4178–83. [7] McKinsey TA. Targeting inflammation in heart failure with histone deacetylase inhibitors. Mol Med 2011;17:434–41. [8] Tao H, Shi KH, Yang JJ, Huang C, Zhan HY, Li J. Histone deacetylases in cardiac fibrosis: Current perspectives for therapy. Cell Signal 2013;26:521–7. [9] Liu F, Levin MD, Petrenko NB, Lu MM, Wang T, Yuan LJ, et al. Histone-deacetylase inhibition reverses atrial arrhythmia inducibility and fibrosis in cardiac hypertrophy independent of angiotensin. J Mol Cell Cardiol 2008;45:715–23. [10] Zhang D, Wu CT, Qi X, Meijering RA, Hoogstra-Berends F, Tadevosyan A, et al. Activation of Histone Deacetylase-6 Induces Contractile Dysfunction Through Derailment of alpha-Tubulin Proteostasis in Experimental and Human Atrial Fibrillation. Circulation 2014;129:346–58. [11] Chen YJ, Chen SA. Electrophysiology of Pulmonary Veins. J Cardiovasc Electrophysiol 2006;17:220–4. [12] Nattel S. Paroxysmal atrial fibrillation and pulmonary veins: relationships between clinical forms and automatic versus re-entrant mechanisms. Can J Cardiol 2013;29:1147–9. [13] Chang SH, Chen YC, Chiang SJ, Higa S, Cheng CC, Chen YJ, et al. Increased Ca(2+) sparks and sarcoplasmic reticulum Ca(2+) stores potentially determine the spontaneous activity of pulmonary vein cardiomyocytes. Life Sci 2008;83:284–92.

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