Holiday heart syndrome (HHS) is an alcohol-induced myocardial pathology that occurs during the hangover period following episodic consumption of a large amount of alcohol over a short time interval (3–7 days). Clinically, it manifests as paroxysms of supraventricular cardiac arrhythmias, predominantly atrial fibrillation (AF). Unlike alcoholic cardiomyopathy, which develops against the background of chronic alcohol abuse and is also accompanied by cardiac arrhythmias, arrhythmias in HHS occur even in practically healthy individuals due to electrophysiological changes in cardiomyocytes caused by alcohol intoxication during the hangover period [1]. Alcohol has been shown to sharply shorten the effective refractory period and reduce the action potential of atrial cardiomyocytes, thereby creating electrophysiological conditions for re-entry and subsequent abnormal circulation of excitation, i.e., the formation of the arrhythmogenic re-entry mechanism [2].

Previously, using our developed rat model of HHS, synchronous multichannel cardioelectrochronotopography (64 atrial leads) demonstrated for the first time that in rats with HHS, a focus of abnormal depolarization forms in the left atrial appendage, while excitation waves from the ectopic focus do not spread to either the right atrium or the ventricles. The mismatch in the propagation of the excitation wave across the atrial epicardium, associated with the presence of two initial excitation foci — the physiological focus in the superior vena cava region of the right atrium (sinus node) and the abnormal focus in the left atrial appendage — represents an arrhythmogenic factor that may contribute to the development of AF via the re-entry mechanism [3]. In tissue biopsies taken from the abnormal depolarization focus located in the left ventricle of rats with HHS, real-time PCR for the first time revealed hyperexpression of Epac regulatory proteins.

Historically, the only allosteric effector of cAMP was thought to be the enzyme discovered in 1968 — cAMP-dependent protein kinase or protein kinase A (PKA). However, in 1998, a cAMP-dependent protein was identified that, without PKA involvement, activated small GEFases (cAMP-GEFs) of the Rap family belonging to the Ras superfamily of proteins [4]; it was named cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF) or exchange protein directly activated by cAMP (Epac) — a regulatory/signaling protein [5]. Currently, two isoforms of Epac proteins have been identified — Epac1 or cAMP-GEF-I and Epac2 or cAMP-GEF-II — which are encoded by different genes [6].

Both Epac1 and Epac2 proteins are expressed in various organs and tissues of the body, including cardiomyocytes. In cardiomyocytes, the localization of Epac proteins differs, which largely accounts for the distinctions in their functional activity. Epac1 proteins are located near the inner surface of the cell membrane, on the mitochondrial membrane, on the perinuclear membrane, and possibly in the cell nucleus, whereas Epac2 proteins are localized in the Z-line region adjacent to T-tubules, near which clusters of sarcoplasmic reticulum (SR) cisterns are concentrated [7].

Under normal physiological conditions, Epac1 signaling proteins primarily regulate the inotropic activity of cardiomyocytes, their electromechanical coupling, and apoptosis. However, under pathological conditions, hyperexpression of Epac1 signaling proteins plays a key role in initiating myocardial hypertrophy, remodeling, and fibrosis [8]. Recently, it has been reported that Epac1 proteins exhibit proarrhythmic activity [9].

Epac2 proteins, due to their localization in cardiomyocytes [7], regulate their rhythmic activity, and their excessive activity underlies the development of cardiac arrhythmias [10, 11]. These findings allow us to confidently state that Epac proteins can be considered an original biotarget for the development of drugs for the prevention/relief of supraventricular tachyarrhythmias pathognomonic for HHS.

To advance this direction, we used molecular docking to design a group of potential Epac protein inhibitors that bind to this protein in its inactive form.

The synthesis of potential Epac protein inhibitors was carried out according to the scheme shown in Fig. 1, where X = CO or SO₂.

Fig. 1. Synthesis scheme of Epac protein potential inhibitors

As a result of these studies, a compound, N,2,4,6-tetramethyl-N-(pyridin-4-yl)benzenesulfonamide hydrochloride — code ZMEI-3 — was synthesized from a series of trimethylbenzenesulfonic acid derivatives (Fig. 2).

Fig. 2. N,2,4,6-tetramethyl-N-(pyridin-4-yl)benzenesulfonamide hydrochloride

In vitro and in vivo experiments showed that ZMEI-3 suppresses cardiomyocyte automaticity and exhibits pronounced antiarrhythmic activity. Furthermore, in model experiments reproducing alcoholic cardiomyopathy (ACM) in rats, it was shown that under conditions of established ACM, systematic therapy with ZMEI-3 contributes to a statistically significant (p = 0.0002) increase in left ventricular contractility compared to controls, and consequently reduces the severity of chronic heart failure pathognomonic for ACM [12].

Somewhat later, in rats with HHS, we identified for the first time the morphological substrate responsible for the desynchronization of the excitation wave in the atria. It was shown that animals with HHS develop perivascular and interstitial fibrosis, as well as fatty infiltration of the myocardium in the atria and interventricular septum, which can be considered the morphological substrate underlying the formation of supraventricular tachyarrhythmias. It is known from the literature that under conditions of ischemic cardiac injury, activation of the Epac1‑coupled Rap/PKCδ/P38MAPK signaling cascade promotes the development of myocardial fibrosis [13], and pharmacological inhibition of Epac1 significantly suppresses fibroblast proliferation by reducing the expression levels of profibrotic genes and components of the neddylation pathway (the pathway conjugating the NEDD8 protein with target proteins) in fibroblasts [14].

The aim of this study was to investigate the possibility of using Epac regulatory protein inhibitors to prevent alcohol‑induced myocardial fibrosis.

Animals. Experiments were performed on 20 white outbred male rats weighing 180–200 g, obtained from the “Stolbovaya” branch of the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency, which possessed a veterinary certificate of quality and health status and had undergone a 15‑day quarantine in the vivarium of the Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies. The animals were kept in standard individual plastic cages with pelleted feed provided ad libitum under a regulated 12/12 light cycle (lights off at 08:00 am). Animal housing conditions complied with GOST 33215‑2014 “Guidelines for the maintenance and care of laboratory animals. Rules for the arrangement of premises and organization of procedures” (reissue) and GOST 33216‑2014 “Guidelines for the maintenance and care of laboratory animals. Rules for the maintenance and care of laboratory rodents and rabbits” (reissue). All work with laboratory animals was performed in accordance with generally accepted standards for animal handling adopted at the Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies, international rules (European Communities Council Directive of November 24, 1986 (86/609/EEC)), and the “Rules for Working with Animals” approved by the Bioethics Committee of the Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies. The animals received standard pelleted feed PK‑120‑1 (Laboratorsnab LLC, Russian Federation). Each animal was intact and used in the experiment only once.

HHS model. For 10 days, the animals received a 10% aqueous ethanol solution as their sole source of fluid, followed by drinking water for 10 days, and then again a 10% aqueous ethanol solution for the next 10 days. In terms of pure ethanol, the average alcohol consumption by rats during the experiment ranged from 5.0 to 6.5 g/kg per day.

Study design. The animals were randomized into 5 groups:Group 1 (n = 6) — intact control (animals with free access to tap water for 30 days);Group 2 (n = 6) — HHS model (control for Group 3);Group 3 (n = 8) — HHS + ZMEI‑3 (animals receiving ZMEI‑3 at a dose of 2 mg/kg, intraperitoneally (i.p.) in 0.3 mL of apyrogenic water for injection, starting from the beginning of the second “binge” for 10 days);Group 4 (n = 7) — HHS model (control for Group 5);Group 5 (n = 8) — HHS + ZMEI‑3 (animals with HHS receiving ZMEI‑3 at a dose of 2 mg/kg, i.p. in 0.3 mL of apyrogenic water for injection, after the end of the second “binge” for 10 days).Animals in Groups 2 and 4 received i.p. injections of 0.3 mL of apyrogenic water for injection according to the same schedule.

Morphological studies. During postmortem examination and assessment of the internal organs, the heart was removed and placed in a fixing solution — 10% buffered formalin at a volume ratio of 1:20. After fixation, frontal segments including the atria and ventricles were cut from the heart. The excised fragments were placed in embedding cassettes, and then histological processing was carried out according to a standard protocol using an automatic carousel-type tissue processor (Leica TP 1020, Leica Microsystems, Germany). After processing, the myocardial sections were embedded in homogenized paraffin‑like medium Paraplast (Leica Biosystems Richmond, USA). A modular tissue embedding system with a graphic display (Tissue‑Tek® TEK, Sakura, Japan) was used for paraffin embedding. Histological sections of 5–6 μm thickness were obtained using a specially equipped microtomy workstation (Bio‑Optica Milano SPA, Italy) and a rotary microtome (Accu‑Cut SRM 200, Sakura, Japan). Glass slides with the sections were dried. Subsequently, paraffin‑embedded sections stained with gallocyanin‑eosin and Van Gieson’s picrofuchsin were coverslipped using synthetic mounting medium Bio Mount (Bio‑Optica Milano SPA, Italy). The finished microslides of the atrial and ventricular fragments were examined under transmitted light using a Nikon Eclipse 55 I microscope (Japan) at magnifications of ×40, ×100, ×200, and ×400. Images were documented with a Nikon DS‑Fi1c camera using NIS Elements BR imaging software for Nikon.

To quantify the intensity of the identified pathological changes, a scoring system was used. For this purpose, in 10 randomly selected microscopic fields of the interatrial septum microslides, the intensity of the detected changes was scored as follows:

Statistical analysis. Since measurements were made on ordinal scales and more than two samples were compared, a non‑parametric analogue of one‑way analysis of variance followed by Dunn’s multiple comparisons test was used for statistical analysis. Results are presented as medians with lower and upper quartiles. In all cases, the critical significance level α = 0.05.

The ventricular myocardium of intact rats has a characteristic cytoarchitecture. Microscopy of sections stained with gallocyanin‑eosin at low magnification clearly visualizes the epicardium, myocardium, and endocardium. The endocardium lines the inner surfaces of the cardiac chambers and valves. The myocardium is represented by interconnected striated muscle cells — cardiomyocytes — arranged in layers. Loose connective tissue, blood vessels, and nerves are found between the myocardial muscle structures. The epicardium is a connective tissue plate firmly fused with the myocardium. In ventricular heart preparations stained with Van Gieson’s picrofuchsin, the myocardium appears yellow. Single layers of crimson‑colored connective tissue are observed between the myocardial muscle structures (Fig. 3 a). Connective tissue of the walls of arteries and arterioles is also visible on the preparations.

Fig. 3. Microscopic picture of rat hearts: intact (a, d, i, n); control rats with HHS syndrome (b, e, k, o); rats with HHS syndrome that received ZMEI-3 starting from the beginning of the 2nd “binge” (c, f, l, p); rats with HHS syndrome that received ZMEI-3 after the end of the 2nd “binge” (d, g, m, r).

Microscopic analysis of the structure of the atria and interatrial septum of intact rats stained for connective tissue with Van Gieson’s picrofuchsin revealed their typical cytoarchitecture (Fig. 3 d, i, n). The myocardium appears yellow on the preparations. Layers of connective tissue, connective tissue of the walls of arteries and arterioles, as well as endocardial connective tissue, all stained crimson, are observed between the myocardial muscle structures.

The microscopic picture of the atria and ventricles of control Groups 2 and 4 does not fundamentally differ.

The ventricular myocardium of rats with HHS also exhibits characteristic cytoarchitecture. Microscopy of sections stained with gallocyanin‑eosin at low magnification clearly visualizes the epicardium, myocardium, and endocardium. The endocardium lines the inner surfaces of the cardiac chambers and valves. The myocardium is represented by interconnected striated muscle cells — cardiomyocytes — arranged in layers. Loose connective tissue, blood vessels, and nerves are found between the myocardial muscle structures. A noticeable increase in the space between cardiomyocytes and capillaries is observed. The epicardium is a connective tissue plate firmly fused with the myocardium.

In ventricular heart preparations stained with Van Gieson’s picrofuchsin, the myocardium appears yellow. Layers of crimson‑colored connective tissue are observed between the myocardial muscle structures (Fig. 3 b). Connective tissue of the walls of arteries and arterioles is also visible.

Thus, as can be seen from the data obtained, the structure of the ventricular myocardium of intact rats and rats with HHS shows no significant visual differences. They also do not differ in connective tissue density.

The microscopic picture of the atria of rats with HHS differs significantly from that of intact animals. In atrial microslides of control rats, thick layers of connective tissue stained crimson with picrofuchsin are visible in the left atrium and the interatrial septum (Fig. 3 e, k). In addition, the interatrial septum and left atrium of control rats are infiltrated with fatty inclusions and vacuoles (Fig. 3 e, k). Connective tissue of the walls of arteries and arterioles is also visualized. The intensity of fibrosis and the number of fatty inclusions in the right atrial myocardium are visually lower (Fig. 3 o).

According to current concepts, atrial fibrosis leading to structural remodeling is a complex multifactorial process that ultimately results in the onset and maintenance of AF [15, 16]. It can be assumed that our previously demonstrated formation of ectopic excitation foci in the left atrium, particularly in its appendage [3], which plays a significant role in the initiation of arrhythmogenesis [17], is associated with alcohol‑induced fibrosis of the left atrial tissue. In turn, pronounced fibrosis of the interatrial septum may underlie the desynchronization of excitation waves in the atria and the formation of a re‑entry wave in the left atrium, i.e., the re‑entry phenomenon, which is the main electrophysiological mechanism responsible for the development of AF [18] — the primary clinical manifestation of HHS [19].

Furthermore, the presence of fatty inclusions in the interatrial septum (Fig. 3 k) may also contribute to the desynchronization of excitation waves in the atria, since it has recently been shown that an increase in the number of epicardial adipocytes is directly associated with the risk of developing AF [20].

Microscopic analysis of the ventricular myocardium of alcohol‑exposed rats treated with ZMEI‑3 revealed its characteristic cytoarchitecture. When stained with gallocyanin‑chrome alum, the epicardium, myocardium, and endocardium are visualized in the heart wall. The endocardium lines the inner surfaces of the chambers and valves. The myocardium consists of cardiomyocytes arranged in layers. Loose connective tissue, blood vessels, and nerves are found between cardiomyocytes. The epicardium is represented by a connective tissue plate.

When the myocardium of the ventricles of alcohol‑exposed rats treated with ZMEI‑3 is stained with Van Gieson’s method, a typical cytoarchitecture is visualized. The microscopic picture of the ventricular myocardium of rats in this group differs little from that of control alcohol‑exposed animals. Yellow myocardium with crimson layers of connective tissue is also visualized (Fig. 3 c, d).

The extent and severity of connective tissue elements in the ventricular myocardium of intact rats, rats with HHS, and rats treated with ZMEI‑3, regardless of the treatment schedule, do not show significant visual differences.

In atrial microslides of alcohol‑exposed rats treated with ZMEI‑3, it is evident that the right and left atria, as well as the interatrial septum, like the hearts of control animals, contain layers of connective tissue stained crimson with picrofuchsin (Fig. 3 p, r, f, z, l, m). However, these layers are significantly thinner than in control animals, and small fatty inclusions are encountered only in isolated cases. Connective tissue of the walls of arteries and arterioles is visible as thin crimson layers. No significant visual differences were found between the atrial microslides of rats treated with ZMEI‑3 according to the different schedules. Nevertheless, the density of connective tissue in the interventricular septum of rats treated with ZMEI‑3 from the beginning of the second “binge” visually appears lower (Fig. 3 l), whereas in animals treated with ZMEI‑3 after the end of the second “binge”, fewer fatty inclusions are visually observed (Fig. 3 m).

Table 1. Effect of ZMEI‑3 on the degree of manifestation of connective tissue elements and fatty inclusions in the atria and interatrial septum of rats with Holiday Heart Syndrome (HHS)

Parameter, pointsIntact animals n = 6HHS control, n = 6HHS + ZMEI‑3 (2 mg/kg i.p.) n = 8HHS control deferred, n = 7HHS + ZMEI‑3 (2 mg/kg i.p.) deferred, n = 8Connective tissue in the left atrium1.0* [0.0+1.0]2.0 [1.0+3.0]2.0 [1.0+3.0] p1 = 0.34492.0 [1.0+3.0]1.0 [0.5+1.0] p2 = 0.0721Fatty inclusions in the left atrium1.5* [1.0+2.0]3.0 [3.0+4.0]3.0 [1.0+3.0] p1 = 0.41353.0 [2.0+4.0]0.0* [0.0+3.0] p2 = 0.0034Connective tissue in the interatrial septum1.0* [0.0+1.0]4.0 [3.0+4.0]1.5* [0.0+2.0] p1 = 0.00134.0 [2.0+4.0]1.5* [0.5+2.0] p2 = 0.0451Fatty inclusions in the interatrial septum1.0* [0.0+1.0]2.5 [2.0+4.0]1.0 [0.0+3.0] p1 = 0.14183.0 [2.0+4.0]2.0 [0.0+3.5] p2 = 0.6126Connective tissue in the right atrium1.0* [0.0+1.0]3.0 [2.0+3.0]1.0* [1.0+1.5] p1 = 0.00792.0 [1.0+3.0]1.0 [0.5+1.5] p2 = 0.2810Fatty inclusions in the right atrium1.0* [0.0+1.0]3.5 [0.0+4.0]2.50 [0.0+3.0] p1 = 0.41354.0 [2.0+4.0]0.0* [0.0+3.0] p2 = 0.035

Notes: * – p1 < 0.05; p1 is indicated relative to the HHS control group; p2 is indicated relative to the deferred HHS control group.

The obtained results indicate that in animals with HHS, experimental therapy with ZMEI‑3, regardless of the administration schedule, substantially reduces the intensity of incorporation of both connective tissue elements and adipocytes into the atrial myocardium and interatrial septum, which may underlie the antiarrhythmic effect of the compound.