Circulation Journal ORIGINAL ARTICLE Circ J 2020; 84: 1931 – 1940 doi: 10.1253/circj.CJ-20-0288 Arrhythmia/Electrophysiology Binge Alcohol Exposure Triggers Atrial Fibrillation Through T-Type Ca2+ Channel Upregulation via Protein Kinase C (PKC) / Glycogen Synthesis Kinase 3β (GSK3β) / Nuclear Factor of Activated T-Cells (NFAT) Signaling ― An Experimental Account of Holiday Heart Syndrome ― Yan Wang, MD, PhD; Masaki Morishima, PhD; Dan Li; Naohiko Takahashi, MD, PhD; Tetsunori Saikawa, MD, PhD; Stanley Nattel, MD, PhD; Katsushige Ono, MD, PhD Background: The association between binge alcohol ingestion and atrial fibrillation (AF), often termed “holiday heart syndrome”, has long been recognized. However, the underlying cellular and molecular mechanisms are unknown. Methods and Results: An experimental model of binge alcohol-induced AF was developed to elucidate the mechanisms linking acute ethanol exposure to changes in ion channel transcription and AF susceptibility. AF-susceptibility during transesophageal electrical stimulation was enhanced 8 h after, but not immediately or 24 h after, acute alcohol intake. T-type calcium channel (TCC) blockade and calcineurin inhibition diminished the AF-promoting effect of ethanol. Long-term (8–24 h) exposure to ethanol augmented TCC isoform-expression (Cav3.1 and Cav3.2) and currents in cardiomyocytes, accompanied by upregulation of the transcription factors, Csx/Nkx2.5 and nuclear factor of activated T-cells (NFAT), in the nucleus, and of phospho-glycogen synthesis kinase 3β (GSK3β) in the cytosol. Inhibition of protein kinase C (PKC) during the 7- to 8-h period following ethanol exposure attenuated susceptibility to AF, whereas acute exposure did not. GSK3β inhibition itself upregulated TCC expression and increased AF susceptibility. Conclusions: The present study results suggest a crucial role for TCC upregulation in the AF substrate following binge alcohol- drinking, resulting from ethanol-induced PKC-activation that hyperphosphorylates GSK3β to cause enhanced calcineurin-NFAT-Csx/ Nkx2.5 signaling. These observations elucidate for the first time the potential mechanisms underlying the clinically well-recognized, but mechanistically enigmatic, “holiday heart syndrome”. Key Words: Alcohol; Atrial fibrillation; Electrophysiology; Ion channels trial fibrillation (AF) is the most commonly sus- tained cardiac arrhythmia. Although certain risk Editorial p 1909 factors, such as age, hypertension, serum metabolites A 1–3 6 and heart failure are well-established, the causes and within 24 h. Several potential mechanisms have been pos- mechanisms remain unknown in many patients. There is tulated by which an alcoholic binge could cause arrhyth- conflicting evidence regarding an association between mias. Alcohol-induced increases in plasma-free fatty acids long-term alcohol consumption and the risk of AF.4 In and catecholamines are thought to be arrhythmogenic,7 as contrast, an association between binge alcohol-drinking is the principal metabolite of alcohol, acetaldehyde.8 How- and AF, often termed “holiday heart syndrome”, has been ever, the precise electrophysiological mechanisms that connect widely recognized.5 Holiday heart syndrome refers to cardiac acute ethanol consumption to AF are largely unknown. arrhythmias, particularly AF, that occur after an alcoholic Pulmonary veins (PVs) are important foci for initiation binge in individuals showing no other evidence of heart of paroxysmal AF and are also associated with AF main- disease, which usually convert to normal sinus rhythm tenance.9 PVs contain cardiomyocytes with electrical activ- Received March 31, 2020; revised manuscript received July 20, 2020; accepted August 7, 2020; J-STAGE Advance Publication released online October 7, 2020 Time for primary review: 20 days Department of Pathophysiology (Y.W., M.M., D.L., K.O.), Department of Clinical Examination and Diagnostics (Y.W., N.T., T.S., K.O.), Oita University School of Medicine, Yufu, Japan; Montreal Heart Institute Research Center, University of Montreal, Montreal (S.N.), Canada The first two authors contributed equally to this study (Y.W., M.M.). Mailing address: Katsushige Ono, MD, PhD, Department of Pathophysiology, Oita University School of Medicine, 1-1 Idaigaoka, Hasama, Yufu 879-5593, Japan. E-mail: [email protected] All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] ISSN-1346-9843 Circulation Journal Vol.84, November 2020 1932 WANG Y et al. Figure 1. Effect of ethanol on heart rate and atrial fibrillation (AF) inducibility. Changes in heart rate over time after (A) drinking water (20 mL) or binge drinking of ethanol (20%, 20 mL), and (B) injection of saline (10 mL) or ethanol (20%, 10 mL) into the abdominal cavity, are shown. Examples of the electrocardiogram (ECG) record for vehicle (water or saline; ○) and ethanol (●) 8 h after the procedure are shown in the insets. Number of animals are indicated in parentheses. Examples of the ECG lead II deflec- tion from a rat 8 h after receiving (C) a saline injection, (D) an ethanol injection, (E) an ethanol injection with a short-term (1 h) application of kurtoxin (0.1 mg/kg), (F) an ethanol injection with a long-term (7 h) application of a PKC inhibitor, chelerythrine (5 mg/kg), or (G) an injection of a GSK3β inhibitor, BIO (0.1 μg/kg). The duration of AF was designated as the period from the end of electrical stimulation to the appearance of the first P wave (▼). Magnification of the ECGs for 1 s duration are shown in the insets (C,D). (H) Incidence of AF episodes in the different groups and (I) mean AF duration in rats treated with saline (10 mL) or with ethanol (20%, 10 mL) alone or cotreated with kurtoxin (0.1 mg/kg), mibefradil (5 mg/kg), cyclosporine (50 mg/kg), BIO (0.1 μg/kg), SB216763 (0.5 μg/kg), chelerythrine (5 mg/kg) or atenolol (5 mg/kg) for the durations indicated in parentheses. Each rat (n=10) received 10 sets of electrical stimulations with an interval of 10 min. *P<0.01 vs. vehicle, #P<0.01 vs. ethanol (8 h). Circulation Journal Vol.84, November 2020 Binge Alcohol-Induced AF Mechanism 1933 Figure 2. Changes in action potentials (APs) induced by ethanol applied in vivo and in vitro. (A) Representative APs recorded from pulmonary vein (PV) cardiomyocytes isolated from an adult rat 8 h after injection of saline (vehicle, 10 mL) or 20% ethanol (10 mL) into the abdominal cavity. (B) Representative APs recorded from neonatal cardiomyocytes cultured without ethanol (vehicle) or with ethanol (0.1%) for 24 h. (C–E) AP parameters of PV and neonatal cardiomyocytes (Neo) with or without ethanol procedures: AP cycle (C), V˙max at −50 mV (D), and the maximum diastolic potential (MDP) (E). (F) Group data of the beating rate of cardiomyocytes with (gray) or without (white, vehicle) ethanol exposure for 0, 1, 3, 6, 12, and 24 h. *P<0.01 vs. vehicle. #P<0.05 vs. vehicle. Number of cells (C–F) are indicated; each animal heart was assessed by using 10 different preparations. ity, which have been suggested to function as subsidiary animal model of “holiday heart syndrome”; (2) to use the pacemakers and to induce atrial arrhythmias.10 In addi- model to clarify the molecular links between acute ethanol tion, PV cardiomyocytes have distinct electrophysiological consumption and subsequent transient AF; and (3) to characteristics and may possess arrhythmogenic activity establish the role, if any, of TCC dysregulation in experi- through several mechanisms.11 mental “holiday heart syndrome”. The T-type Ca2+ channel (TCC) current (ICa.T) is present in nodal cells, Purkinje fibers, and PV cardiomyocytes, but Methods is less important in normal atrial and ventricular myo- cytes.12 Cardiac pacemaker cells possess a greater density A detailed description of the experimental methods is pro- of ICa.T than non-pacemaker cells, and ICa.T contributes to vided in Supplementary File 1. the genesis of automaticity.12 In contrast, in pathological conditions like hypertrophy, myocardial infarction, and Animal ECG Recordings and Electrophysiological heart failure, TCC is re-expressed in ventricular myo- Measurements cytes.13 TCC blockade may prevent the development of a The experimental protocol was approved in advance by the substrate for AF.14 Moreover, Chen et al demonstrated Ethics Review Committee for Animal Experimentation of that TCC blockade decreases spontaneous activity, sup- Oita University School of Medicine (Approval number presses delayed afterdepolarizations, and inhibits transient C004003). Detailed profiles and procedures for ECG, inward currents in PV cardiomyocytes, indicating a role development of an animal model for ethanol-induced AF, 15 for ICa.T in PV arrhythmogenesis. It has thus been sug- and patch clamp recordings were described in previous gested that TCC is correlated with the onset and/or the studies.17,18 Briefly, we applied 10 mL ethanol (20%) for maintenance of AF.16 Regarding the pathophysiological injections and 20 mL (20%) for the alcohol binge. Rats signals associated with the T-type Ca2+ channel, the role of voluntarily drank this amount of ethanol with ease after a the cardiac transcription factor, Csx/Nkx2.5, in positive 24- to 30-h water-fasting period. AF inducibility was assessed regulation of the Cav3.2-TCC has recently been pro- after a burst of transesophageal atrial electrical pacing posed.17 As a working hypothesis, we assumed that actions (70 V, 15 s, 33.3 Hz). For short-term ECG recording with a of TCCs may form part of the mechanism by which binge burst of transesophageal atrial pacing, a 400 series PowerLab alcohol consumption leads to AF. In addition, we consid- with Chart v4 software (ADInstruments, Bella Vista, NSW, ered that a sub-acute transient action of ethanol, rather Australia) connected with a BIO amp (ADInstruments) than an immediate or long-term effect, may create a sub- was used. strate favoring AF occurrence via modulation of TCC. The purpose of the present study was: (1) to create an Circulation Journal Vol.84, November 2020 1934 WANG Y et al.