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 ―

Abstract

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 (Ca_v3.1 and Ca_v3.2) 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”.

Atrial fibrillation (AF) is the most commonly sustained cardiac arrhythmia. Although certain risk factors, such as age, hypertension, serum metabolites and heart failure are well-established,^1–3 the causes and mechanisms remain unknown in many patients. There is conflicting evidence regarding an association between long-term alcohol consumption and the risk of AF.⁴ In contrast, an association between binge alcohol-drinking and AF, often termed “holiday heart syndrome”, has been widely recognized.⁵ Holiday heart syndrome refers to cardiac arrhythmias, particularly AF, that occur after an alcoholic binge in individuals showing no other evidence of heart disease, which usually convert to normal sinus rhythm within 24 h.⁶ Several potential mechanisms have been postulated by which an alcoholic binge could cause arrhythmias. Alcohol-induced increases in plasma-free fatty acids and catecholamines are thought to be arrhythmogenic,⁷ as is the principal metabolite of alcohol, acetaldehyde.⁸ However, the precise electrophysiological mechanisms that connect acute ethanol consumption to AF are largely unknown.

Editorial p 1909

Pulmonary veins (PVs) are important foci for initiation of paroxysmal AF and are also associated with AF maintenance.⁹ PVs contain cardiomyocytes with electrical activity, which have been suggested to function as subsidiary pacemakers and to induce atrial arrhythmias.¹⁰ In addition, PV cardiomyocytes have distinct electrophysiological characteristics and may possess arrhythmogenic activity through several mechanisms.¹¹

The T-type Ca²⁺ channel (TCC) current (I_Ca.T) is present in nodal cells, Purkinje fibers, and PV cardiomyocytes, but is less important in normal atrial and ventricular myocytes.¹² Cardiac pacemaker cells possess a greater density of I_Ca.T than non-pacemaker cells, and I_Ca.T contributes to the genesis of automaticity.¹² In contrast, in pathological conditions like hypertrophy, myocardial infarction, and heart failure, TCC is re-expressed in ventricular myocytes.¹³ TCC blockade may prevent the development of a substrate for AF.¹⁴ Moreover, Chen et al demonstrated that TCC blockade decreases spontaneous activity, suppresses delayed afterdepolarizations, and inhibits transient inward currents in PV cardiomyocytes, indicating a role for I_Ca.T in PV arrhythmogenesis.¹⁵ It has thus been suggested that TCC is correlated with the onset and/or the maintenance of AF.¹⁶ Regarding the pathophysiological signals associated with the T-type Ca²⁺ channel, the role of the cardiac transcription factor, Csx/Nkx2.5, in positive regulation of the Cav3.2-TCC has recently been proposed.¹⁷ As a working hypothesis, we assumed that actions of TCCs may form part of the mechanism by which binge alcohol consumption leads to AF. In addition, we considered that a sub-acute transient action of ethanol, rather than an immediate or long-term effect, may create a substrate favoring AF occurrence via modulation of TCC. The purpose of the present study was: (1) to create an animal model of “holiday heart syndrome”; (2) to use the model to clarify the molecular links between acute ethanol consumption and subsequent transient AF; and (3) to establish the role, if any, of TCC dysregulation in experimental “holiday heart syndrome”.

Methods

A detailed description of the experimental methods is provided in Supplementary File 1.

Animal ECG Recordings and Electrophysiological Measurements

The experimental protocol was approved in advance by the Ethics Review Committee for Animal Experimentation of Oita University School of Medicine (Approval number C004003). Detailed profiles and procedures for ECG, development of an animal model for ethanol-induced AF, and patch clamp recordings were described in previous studies.^17,18 Briefly, we applied 10 mL ethanol (20%) for injections and 20 mL (20%) for the alcohol binge. Rats voluntarily drank this amount of ethanol with ease after a 24- to 30-h water-fasting period. AF inducibility was assessed after a burst of transesophageal atrial electrical pacing (70 V, 15 s, 33.3 Hz). For short-term ECG recording with a burst of transesophageal atrial pacing, a 400 series PowerLab with Chart v4 software (ADInstruments, Bella Vista, NSW, Australia) connected with a BIO amp (ADInstruments) was used.

Cardiomyocyte Isolation and Culture

Neonatal cardiomyocytes were prepared from 2- to 3-day-old Wistar rat heart ventricles, as previously described.¹⁷ PV cardiomyocytes were prepared from adult Wistar rats (8 weeks old) using a procedure modified from that of Chen et al.¹⁵

Measurement of Protein Kinase C (PKC) Activation

Membranous PKC activity was measured using the StressXpress PKC kinase activity assay kit (EKS-420A; Stressgen Bioreagents Corp., Victoria, British Columbia, Canada).

Western Blot Analysis

Western blot analysis was performed according to a standard method.¹⁹ Total protein of the nucleus and the cytosol was extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce, Rockford, IL, USA) and was quantified using the BCA Protein Assay Kit (Pierce).

Transfection of siRNA and the Luciferase Activity Assay

siRNAs directed against rat Nkx2 (Nkx siRNA, pools of 3 target-specific 27 mer siRNAs) and the scrambled negative control siRNA (Control siRNA) were purchased from ORIGENE (SR512716; OriGene Technologies, MD, USA). Transfection was performed in OPTI-MEM (Invitrogen).

Statistical Analysis

Data are expressed as mean±SEM. Between group and among group comparisons were conducted using one-way ANOVA with Scheffe’s test, respectively. P<0.05 was considered significant.

Results

Ethanol-Induced Increase of Heart Rate and Cellular Automaticity

Possible actions of ethanol on heart rate change and arrhythmogenicity were examined over 24 h by using telemetric ECG recordings from adult rats that drank ethanol (20%, 20 mL) or distilled water (20 mL), as well as from rats that were injected with ethanol (20%, 10 mL) or saline (10 mL) via the abdominal cavity (Figure 1). Heart rate increased after either binge drinking or injection of ethanol, and reached its maximum 8 h later (Figure 1A,B). The increase in heart rate might not be a direct effect of ethanol or serum catecholamines, because the maximum ethanol and noradrenalin (NA) concentration in serum reached a peak 2 h after the ethanol binge (Supplementary Figure 1A). Importantly, ethanol concentration decreased significantly by 8 h (n=4–5), which did not correspond to the time course of heart rate change (Supplementary Figure 1C). Cardiac autonomic nerve function was not directly related to heart-rate change, but was based on heart rate variability in relation to serum concentrations of NA (Supplementary Figure 1B,D,E). We then assessed the induction of AF by transesophageal atrial burst pacing in rats with or without ethanol binging. After rapid transesophageal electrical stimulation, ethanol consumption increased the probability of AF development by as much as 92±4%; AF was not substantially induced in animals that were injected with saline (Figure 1C–I). AF was not induced immediately after ethanol injection (1 h), but rather predominantly 8 h later; the probability of AF induction returned towards baseline 24 h after ethanol injection. The incidence and the duration of AF caused by ethanol was strongly reduced by kurtoxin, a potent selective inhibitor of TCCs (Figure 1E), suggesting that TCCs mediate AF promotion by binge ethanol. Pretreatment with the calcineurin (CN) inhibitor, cyclosporine, or the PKC inhibitor, chelerythrine, for 7 h significantly reduced the incidence and duration of AF following ethanol administration, although acute application (1 h) of cyclosporine or chelerythrine had no effect (Figure 1H,I). Although the β-adrenergic blocking agent, atenolol (5 mg/kg), significantly reduced heart rate from 431±23 bpm to 367±20 bpm (P<0.01) 1 h after intraperitoneal application, it failed to suppress ethanol-related AF (Figure 1H,I; Supplementary Figure 2C). Intriguingly, glycogen synthesis kinase 3β (GSK3β) inhibition by BIO or SB216763 greatly increased AF susceptibility and AF duration (Figure 1G–I and Supplementary Figure 2E), suggesting PKC and GSK3β as potential signaling molecules associating binge alcohol drinking with AF.

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 deflection 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).

Increased excitability of PV cardiomyocytes due to ethanol activity has been postulated as a possible trigger for AF induction. We performed in vitro electrophysiological experiments using spontaneously beating PV cardiomyocytes from adult rats. Examples of action potentials (APs) recorded from PV cardiomyocytes isolated from a rat 8 h after injection of saline or ethanol are shown in Figure 2A. PV automatic rate increased approximately 3-fold in PV cardiomyocytes obtained from ethanol-injected rats (Figure 2C). In order to obtain an in vitro model, we studied neonatal cardiomyocytes kept in primary culture with or without 0.1% ethanol for up to 24 h (Figure 2B). The spontaneous beating rate was 88±13 bpm in the absence of ethanol (vehicle), and was 126±19 bpm in the presence of ethanol (Figure 2C). Although the upstroke velocity at a membrane potential of −50 mV (V̇@−50 mV) was markedly increased, the maximum diastolic potential (MDP) was unchanged by ethanol both in adult PV cardiomyocytes and in neonatal cardiomyocytes (Figure 2D,E). The beating rate of neonatal cardiomyocytes was significantly increased by 0.1% ethanol after incubation for 6–24 h (Figure 2F). These results indicate that ethanol increases beating rates and upstroke velocity of APs at potentials comparable to the pacemaker range several hours following ethanol drinking in vivo, and at comparable intervals following ethanol treatment in vitro.

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), V̇max 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.

Ethanol Increases TCC Current and mRNA Expression

As kurtoxin strongly decreased the effect of ethanol on AF induction (Figure 1E,H,I), actions of ethanol on TCC currents were suspected. The I_Ca.T was significantly increased in PV myocytes 8 h after ethanol injection, although I_Ca.L was unchanged (Figure 3). The increase in I_Ca.T nearly completely reversed to baseline values in PV cardiomyocytes 24 h after an ethanol binge (Supplementary Figure 3). Consistent with the results in PV cardiomyocytes, ethanol treatment (0.1%, 24 h) also increased I_Ca.T but not I_Ca.L, I_h, or I_Kr in neonatal cardiomyocytes (Supplementary Figure 4). To investigate the effects of ethanol on Ca²⁺ channel isoform expression, we used quantitative polymerase chain-reaction (qPCR) to analyze the mRNA expression of Ca_v1.2 and Ca_v1.3, representing LCC isoforms, and Ca_v3.1 and Ca_v3.2, representing TCC isoforms. The Ca_v1.2- and Ca_v1.3-LCCs were expressed in PVs at lower levels than other cardiomyocytes (Figure 4A-a,b). Neither the mRNA expression of Ca_v1.2 or Ca_v1.3 was changed in atrial, ventricular or PV cardiomyocytes 8 h after an ethanol binge in adult rats (Figure 4A-a,b), nor in neonatal myocytes cultured with ethanol (0.001–0.5%) for up to 24 h (Figure 4B-a,b; Figure 4C-a,b). In contrast, Ca_v3.1- and Ca_v3.2-TCC isoforms were highly expressed in PV cardiomyocytes, and were upregulated 8 h after an ethanol binge (Figure 4A-c,d). The upregulation of Ca_v3.1 and Ca_v3.2 by ethanol in neonatal cardiomyocytes occurred at the concentration of 0.1% or higher (Figure 4B-d), and it was in a time-dependent manner (Figure 4C-c,d). Unlike the transient upregulation of Ca_v3.1- and Ca_v3.2-TCC in rats after an acute ethanol binge, the upregulation of these channels was sustained for more than 24 h when cardiomyocytes were exposed continuously to ethanol in vitro (Figure 4C-c,d).

Figure 3.

Long-term actions of ethanol on L-type (LCC) and T-type Ca²⁺ channel (TCC) currents in PV cardiomyocytes. (A–C) Examples of current traces recorded in PV cardiomyocytes of adult rats at 8 h after a water or an ethanol binge. (A) I_Ca obtained from a V_HP of −100 mV; (I_Ca.L+I_Ca.T). (B) I_Ca obtained from a V_HP of −40 mV in the same patch; (I_Ca.L). (C) I_Ca obtained by subtraction of the traces in (B) from the traces in (A), which represents I_Ca.T. (D,E) Current (I)-voltage (V) relationship of the group data for I_Ca.L (D) and I_Ca.T (E) from 8 cells out of 8 animals in each group. *P<0.01 vs. saline.

Figure 4.

Long-term actions of ethanol on L-type (LCC) and T-type Ca²⁺ channel (TCC) expression in adult and neonatal cardiomyocytes. (A) The mRNA expression of Ca_v1.2 (A-a), Ca_v1.3 (A-b), Ca_v3.1 (A-c) and Ca_v3.2 (A-d) in atrial, ventricular and pulmonary vein (PV) cardiomyocytes of adult rats 8 h after drinking 20 mL of water (white bars) or 20 mL of 20% ethanol (gray bars). Data were normalized to Ca_v1.2 mRNA expression in atrial cardiomyocytes isolated from water-drinking rats, which was designated as 100. (B) Changes in Ca_v1.2 (B-a), Ca_v1.3 (B-b), Ca_v3.1 (B-c) and Ca_v3.2 (B-d) mRNA expression in neonatal cardiomyocytes treated with vehicle or ethanol (0.01%, 0.1% and 0.5%) in the culture medium for 24 h. (C) Time-dependent changes in Ca_v1.2 (C-a), Ca_v1.3 (C-b), Ca_v3.1 (C-c) and Ca_v3.2 (C-d) mRNA in neonatal myocytes treated with 0.1% ethanol in the culture medium for 0–24 h. Representative polymerase chain reaction (PCR) products are shown in the insets with the reference gene, GAPDH (below). *P<0.01 vs. vehicle (0% ethanol) (A,B) or specific time vs. 0 h (C).

As TCC upregulation occurred several hours after ethanol application, a genomic mechanism was postulated. To investigate the potential signaling mechanisms and considering the suppressant effect of chelerythrine on ethanol-related AF, we examined PKC activity and the involvement of transcription factors that regulate cardiac ion channel expression. PKC activity in cardiomyocytes was significantly enhanced when the cells were exposed to ethanol at a concentration of 0.1% or higher (Figure 5A). Under the same conditions, the mRNA levels of the transcription factors, Csx/Nkx2.5, known to regulate Ca_v3.2 transcription, and GATA4 (but not those of CREB, NFAT or NRSF), were increased by ethanol (Figure 5B). NFAT is an important target of GSK3β kinase activity in the heart. Using a protein expression assay, we found that the levels of Csx/Nkx2.5 and NFAT in the nuclear fraction were more than doubled when cardiomyocytes were exposed to ethanol. These increases were accompanied by an increase in phosphorylated (deactivated) GSK3β (pGSK), a substrate of PKC, in the cytosol (Figure 5C,D). To confirm that ethanol modifies transcription of the TCC gene, we cultured cardiomyocytes in the presence of actinomycin D (0.01 μmol/L), an inhibitor of transcription, with or without 0.1% ethanol. Actinomycin D in the culture medium prevented ethanol-induced increases in I_Ca.T and TCC-subunit expression; actinomycin D-induced downregulation of the expression of Csx/Nkx2.5 mRNA by 40.2% was unaffected by ethanol addition (Supplementary Figure 5).

Figure 5.

Effect of ethanol on transcription factors and signaling molecules. (A) Protein kinase C (PKC) activity in cardiomyocytes with or without 24 h treatment with culture medium containing 0.01%, 0.1% and 1% ethanol (n=8). (B) Changes in mRNA expression of the transcription factors, Csx/Nkx2.5, GATA4, CREB, NFATc4 and NRSF in myocytes treated with or without 0.1% ethanol for 24 h. Expression of each mRNA without ethanol treatment was assigned as 100% (n=8). (C) Examples of Csx/Nkx2.5, NFAT, GSK3β, phosphorylated GSK3β (p-GSK3β) and GAPDH protein expression in the nucleus and cytosol of myocytes treated with or without 0.1% ethanol for 24 h. (D) Semi-quantitative assessment of protein expression levels of Csx/Nkx2.5, NFAT, GSK3β, and phosphorylated p-GSK3β determined based on the density of the blotted bands exemplified in panel (C) (n=5). The protein expression of each sample was normalized to that in the nucleus of cells treated with vehicle. (E) A luciferase assay after transient transfection of cardiomyocytes with TOPFLASH and renilla; cells were treated with (gray bars) or without 0.1% ethanol for 24 h (open bars) in the presence or absence of PKC inhibitors (2 μmol/L chelerythrine, 5 nmol/L Gö 6976, 15–50 nmol 3-IYIAP), a Ca²⁺ chelator (50 μmol/L BAPTA-AM) or GSK3β inhibitors (20 nmol/L BIO, 10 μmol/L SB216763) (n=8). *P<0.01 vs. vehicle.

To additionally test whether ethanol-mediated upregulation of TCC is specifically due to inhibition of GSK-3β by PKC-mediated phosphorylation, ethanol-treated cardiomyocytes were analyzed by TOPFLASH (a reporter plasmid containing multiple copies of wild-type Tcf-binding sites) reporter assay with or without PKC inhibitors (chelerythrine, Gö 6976, 3-IYIAP), and the results were compared to the effects of the GSK-3β inhibitors, BIO and SB216763 (Figure 5E). Ethanol treatment enhanced TOPFLASH reporter activity, an action which depended upon intracellular Ca²⁺ and PKC actions.

Effect of PKC-Dependent Signals on TCC Transcription

To further examine downstream target molecules that might mediate the effects of ethanol on TCC expression, we added the PKC inhibitor, chelerythrine, and/or the Ca²⁺ chelator, BAPTA-AM, to the culture medium for 24 h, with or without ethanol. The ethanol-induced increase in I_Ca.T and in the mRNA expression of TCCs was completely eliminated in the presence of BAPTA (Figure 6A). Chelerythrine also abolished the effect of ethanol on I_Ca.T as well as on Ca_v3.1- and Ca_v3.2-mRNA expression (Figure 6A). To determine which [Ca²⁺]_i-dependent isoform of PKC affects TCC transcription, we examined the effect of ethanol in the absence or presence of several PKC isoform-specific inhibitors: the PKCα inhibitors, Gö 6976 and Ro-32-0432, and the PKCβ inhibitor, 3-IYIAP. The effect of ethanol on Ca_v3.1 was abolished by Gö 6976 or Ro-32-0432, whereas its effect on Ca_v3.2 was unaffected (Figure 6D,E). When a low dose of 3-IYIAP was used, which inactivates PKCβII, the levels of both Ca_v3.1 and Ca_v3.2 were increased by ethanol. In contrast, when a high concentration of 3-IYIAP was used, which blocks the activity of both PKCβI and PKCβII, the upregulation of Ca_v3.2 by ethanol was completely abolished. Finally, to confirm the participation of GSK3β in the ethanol signaling pathway, we examined the effects of ethanol in the absence or presence of BIO (20 nmol/L), a GSK3β inhibitor. The expression of Ca_v3.2-mRNA was strongly increased by BIO alone, and ethanol was unable to further increase the Ca_v3.2 level (Figure 6E,F). Ethanol regulation of Ca_v3.2 and Csx/Nkx2.5 expression in the presence of specific PKC inhibitors or a GSK3β inhibitor were strikingly similar (Figure 6E,F). Finally, to confirm our findings implicating Csx/Nkx2.5, short interfering RNAs for Nkx2.5 (Nkx siRNA) were applied along with ethanol treatment (Figure 7, Supplementary Figure 6). Nkx siRNA abolished the upregulation of Cav3.2 by ethanol as well as by PMA (Figure 7A,B). Nkx siRNA also reduced ethanol- and PMA-dependent increases in beating rates of cardiomyocytes (Figure 7C). These results confirm that activation of Csx/Nkx2.5 action by ethanol results in TCC current increases that enhance cardiomyocyte excitability.

Figure 6.

Effects of protein kinase C (PKC), cellular Ca²⁺ and GSK3β on I_Ca.T, Ca_v3 and Csx/Nkx2.5 expression. (A) Examples of current traces of I_Ca.T recorded from saline-treated (white triangles) or ethanol-treated (filled triangles) neonatal cardiomyocytes cultured in normal medium for 24 h, with 2 μmol/L chelerythrine (Chele, a PKC inhibitor), with 50 μmol/L BAPTA-AM (BAPTA, a Ca²⁺ chelator), or with chelerythrine plus BAPTA-AM. (B,C) changes in Ca_v3.1 mRNA (B) and Ca_v3.2 mRNA (C) induced by treatment with 0.1% ethanol for 24 h, with or without chelerythrine or BAPTA-AM. Changes in the mRNA expression of (D) Ca_v3.1, (E) Ca_v3.2, and (F) Csx/Nkx2.5 induced by 0.1% ethanol treatment for 24 h with or without PKC inhibitors; the PKCα inhibitors Gö 6976 (5 nmol/L) or Ro-32-0432 (30 nmol/L), the PKCβI inhibitor, 3-IYIAP (15 nmol/L), or the PKCβI/II inhibitor, 3-IYIAP (50 nmol/L), or with or without the GSK3β inhibitor, BIO (20 nmol/L). *P<0.01 vs. control (as indicated). ^#P<0.01 vs. control (ethanol (–), PKC inhibitor (−) or BIO (−)). Representative polymerase chain reaction (PCR) products are shown in the insets with the reference gene, GAPDH (below).

Figure 7.

Regulation of Cav3.2 by a transcription factor, Csx/Nkx2.5, and its modulation by siRNA-Csx/Nkx2.5 (Nkx siRNA) in cardiomyocytes. (A) Representative immunostaining of cardiomyocytes for Cav3.2 and DAPI incorporation with ethanol (EtOH) or a PKC activator, PMA (1 nmol/L) for 24 h. (B) Knockdown of Cav3.2-mRNA by Nkx siRNA. (C) Mean spontaneous beating rate of cardiomyocytes after application of EtOH (0.1%) or PMA (1 nmol/L) for 24 h. Significant reduction of the beating rate was observed in Nkx siRNA-transfected myocytes with or without EtOH/PMA. Data are expressed as mean±SE. *P<0.05 as indicated. Nkx siRNA corresponds to Nkx (B) siRNA in Supplementary Figure 6.

Discussion

Holiday Heart Syndrome and the Actions of Ethanol

Clinicians have long recognized the temporal association of episodic alcohol use with the onset of AF,²⁰ but the mechanisms have remained elusive. The present study demonstrated that acute ethanol exposure triggers transiently enhanced AF susceptibility in an animal model. Our detailed studies implicated TCC upregulation mediated by a PKC/GSK3β/NFAT/Csx pathway in the molecular pathophysiology of ethanol binge-related AF.

TCC Upregulation as a Possible Arrhythmogenic Substrate

Although the pathophysiological actions of TCC in cardiac muscle are still controversial, recent investigations revealed that TCC current is larger in PVs than in other cardiac regions.¹⁵ In addition, the TCC blocker suppressed tachycardia-induced atrial remodeling in experimental animals.²¹ Previous studies indicate that PV cardiomyocytes are arrhythmogenic through the generation of triggered activity.²² Furthermore, TCCs were highly expressed in PV cardiomyocytes and contributed to their pacemaker activity and triggered activity.^15,23 Ethanol acutely inhibits Ca²⁺ channels.²⁴ However, here we noted that ethanol is a transcriptional modulator of TCCs in PVs and neonatal cardiomyocytes. Transient upregulation of TCCs following an experimental ethanol binge, along with suppression of ethanol-induced AF promotion by a TCC blocker, suggest that TCCs are involved in the initiation of ethanol-related AF in our model.

Downstream Signal Transduction

Ethanol has been shown to interact with membrane-associated phospholipid substrates involved in signal transduction pathways.²⁵ Ethanol activates phospholipase C and triggers associated cellular signaling responses, including the formation of inositol-1,4,5-triphosphate and diacylglycerol, leading to the stimulation of PKC.²⁶ PKC is involved in a variety of pathophysiological cell signaling systems, including AF.²⁷ Based on the structural and functional properties of the kinase regulatory domain, enzymes of the PKC family are divided into 3 groups: conventional (cPKC), novel (nPKC) and atypical (aPKC) isoforms.²⁸ As the non-specific PKC inhibitor, chelerythrine, and the membrane-permeable Ca²⁺-chelator, BAPTA-AM, completely abolished the effects of ethanol on Ca_v3-mRNA and I_Ca,T, we speculated that cPKC was involved in the regulation of Ca_v3 expression. Numerous studies indicate that PKC can phosphorylate and inhibit GSK3β, which regulates a wide variety of cardiac transcription factors.²⁹ For example, GSK3β inhibits endothelin-1 (ET1)-induced hypertrophy in neonatal cardiomyocytes, and prevents NFAT transcriptional activation by retaining NFAT in the cytosol or by delaying ET1-induced nuclear importation of NFAT.³⁰ Because nuclear localization of NFAT is accelerated when it is dephosphorylated by CN, NFAT phosphorylation by GSK3β should counteract dephosphorylation by CN.³¹ In transgenic mice, overexpression of S9A (a mutant form of GSK3β, which cannot be phosphorylated at Ser 9 and is constitutively active), significantly decreased the nuclear localization of NFAT.³⁰

One molecule that might function to connect NFAT and Ca_v3.2 is Csx/Nkx2.5. Chen and Cao recently demonstrated that Csx/Nkx2.5 is a direct target of NFAT that co-ordinates with other transcription factors such as GATA4 to regulate Csx/Nkx2.5 during cardiogenesis.³² Our previous research demonstrated that overexpression of Csx/Nkx2.5 by adenovirus-mediated gene transfer markedly increased the spontaneous beating rate, I_Ca.T and mRNA expression of Ca_v3.2.¹⁷ Our results therefore suggest that the effects of binge ethanol drinking to cause TCC channel upregulation are mediated through cPKC/GSK3β/NFAT and Csx/Nkx2.5 signaling pathways (Figure 8). This finding raises the question of whether other signals that phosphorylate GSK3β, such as protein kinase B (Akt), regulate AF susceptibility. Because PI3-kinase and Akt are known to play a role in the inactivation of GS3β by phosphorylation, which occurs after activation of the insulin receptor, transient hyperglycemia and/or during an insulin surge may account for AF epidemiology. Actually, diabetes mellitus is an independent risk factor for AF. The potential role of TCC changes in linking insulin dysregulation to AF substrates may be an interesting topic for future exploration.

Figure 8.

Schematic illustration of ethanol-protein kinase C (PKC) pathways that participate in T-type calcium channel (TCC) upregulation via GSB3β-calcineurin-NFAT-Csx signals for atrial fibrillation (AF) promotion; a possible molecular model of “holiday heart syndrome”.

Physiological and Pathophysiological Transcription by Nkx/Csx2.5

Csx/Nkx2.5 is an evolutionarily conserved transcription factor of the homeobox gene family, which plays a crucial role in cardiogenesis, and whose expression continues throughout adulthood.³³ A recent study identified a distinct role of Csx/Nkx2.5 in the formation and identity of PV cardiomyocytes.³⁴ The fact that perinatal loss of Csx/Nkx2.5 results in a large reduction in cardiac TCC levels (both Ca_v3.1 and Ca_v3.2) in the heart³⁵ suggests that PV automaticity can be controlled by Csx/Nkx2.5 even in adult animals.

In fetal alcohol syndrome (FAS), the genomic action of ethanol is postulated to play a prominent role. FAS is characterized by organ defects, fetal growth restriction, neurodevelopmental delays, and craniofacial malformations, with a high incidence of cardiac conduction defects, ventricular septal defects and valvular diseases.³⁶ The cardiac anomalies in FAS are almost identical to those observed in mice with Csx/Nkx2.5 mutations.³⁶ Our data raise the interesting possibility that ethanol-induced Csx/Nkx2.5 abnormalities might be involved in inducing FAS.

Study Limitations

Although the present study results provide compelling evidence for a contribution of TCC upregulation to ethanol-induced PV automaticity and AF inducibility, we cannot exclude the possibility that other changes may also contribute. It is possible that the effects of ethanol-dependent TCC upregulation on AF-susceptibility in rats are different from those in humans, especially in diseased hearts. Accordingly, whether increased ethanol-PKC signaling and upregulation of TCC contribute to the development of clinical AF remains to be determined.

Conclusions

Here, we developed a novel animal model of binge alcohol-induced AF susceptibility. This model was used to assess underlying cellular and molecular mechanisms. Ethanol activated intracellular Ca²⁺-dependent PKC isoforms, which inhibited GSK3β through enhanced phosphorylation, thereby increasing nuclear NFAT translocation and Csx/Nks2.5 expression, leading to upregulation of TCCs. Blockade of TCCs or suppression of TCC-upregulating pathways prevented AF promotion by binge alcohol. The present study thus provides novel insights into the molecular mechanisms underlying the previously enigmatic “holiday heart syndrome”.

Acknowledgments

The authors would like to thank Dr. F. Hamada and Miss Y. Akiyoshi for their technical support.

Sources of Funding

This work was supported, in part, by KAKEN grants #21590934 to K.O., #21.09356 to Y.W., and the Canadian Institutes of Health Research and Canadian Heart Foundation grants to S.N.

Disclosures

N.T., S.N. are members of Circulation Journal’ Editorial Team.

IRB Information

Oita University granted an exemption for this study to require ethics approval.

Supplementary Files

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-20-0288

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