Chronic Volume Overload Caused by Abdominal Aorto-Venocaval Shunt Provides Arrhythmogenic Substrates in the Rat Atrium

Abstract

Atrial enlargement is thought to provide arrhythmogenic substrates, leading to the induction of atrial fibrillation (AF). In this study, we investigated the anatomical, molecular biological, and electrophysiological characteristics of remodeled atria in an animal model with chronic volume overload. We used rats that underwent abdominal aorto-venocaval shunt (AVS) surgery. In the in vivo studies, marked changes in electrocardiogram parameters, such as the P-wave duration, PR interval, and QRS width, as well as prolongation of the atrial effective refractory period were observed 12 weeks after the creation of AVS (AVS-12W), which were undetected at 8 weeks postoperative (AVS-8W) despite obvious atrial and ventricular enlargement. Moreover, the duration of AF induced by burst pacing in the AVS-12W rats was significantly longer than that in the Sham and AVS-8W rats. In the isolated atria, a longer action potential duration at 90% repolarization was detected in the AVS-12W rats compared with that in the Sham group. The mRNA levels of the K_v and K_ir channels in the right atrium were mostly upregulated in the AVS-8W rats but were downregulated in the AVS-12W rats. These results show that chronic volume overload caused by abdominal AVS provides arrhythmogenic substrates in the rat atrium. The difference in gene expression in the right atrium between the AVS-8W and AVS-12W rats may partly explain the acquisition of arrhythmogenicity.

INTRODUCTION

Atrial fibrillation (AF) is the most common sustained supraventricular arrhythmia, and various types of risk factors for AF, such as aging, diabetes, obesity, hypertension, and cardiac diseases, are suggested.¹⁾ It is widely recognized that hemodynamic overload to the heart due to such cardiovascular diseases progresses the atrial structural remodeling, leading to the development of AF.²⁾ In addition, the association of atrial structural remodeling with cellular mechanisms or development of AF has been widely analyzed in disease animal models such as congestive heart failure, mitral valve regurgitation, sterile pericarditis, atrioventricular block, chronic volume overload, and hypertension.³⁾ Among them, chronic volume overload strongly induces atrial dilatation in larger animal models of goat, sheep, and rabbit.^4–6) Notably, longer duration of AF (>1 week) was observed in the goat model of 4-weeks vascular shunt between the aorta and the left atrium.⁴⁾ Thus, arrhythmogenic substrates can be strongly generated in the pathophysiological process of chronic volume overload-induced atrial dilatation, leading to the induction of sustained AF.

Recently, we established a rat model that can deliver long-term cardiac overload by abdominal aorto-venocaval shunt (AVS) surgery to investigate atrial arrhythmogenicity.⁷⁾ Although marked atrial dilatation was observed in a rat model of 4-week AVS, the duration of AF induced by burst stimulation was unchanged compared with that in control animals in vitro. In another study using a rat model of 12-week AVS, the isolated pulmonary vein showed spontaneous electrical activity in 36% of preparations despite the fact that rare spontaneous electrical activity (0.04%) was observed in isolated preparations from control animals,⁸⁾ which may suggest that volume overload-induced remodeling progresses from 4–12 weeks to acquire arrhythmogenicity. To better understand the characteristics of atrial arrhythmogenic substrate remodeled by chronic volume overload in this animal model, we investigated the anatomical, molecular, biological, and electrophysiological characteristics of remodeled atria in rats with 8- and 12-week AVS. In addition, we adopted a transvenous electrode catheter for the atrium of closed-chest anesthetized rats to assess in vivo electrophysiology.

MATERIALS AND METHODS

All experiments were approved by the Toho University Animal Care and User Committee (Approval Nos. 14-53-161, 17-51-359) and performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society. A total of 73 male Wistar rats (Japan SLC, Inc., Hamamatsu, Japan) were used in this study. Animals were kept at 23 ± 1 °C under a 12-h light–dark cycle, with food and water available ad libitum.

Surgical Procedure of Abdominal Aorto-Venocaval Shunt

AVS was surgically created in 8-week-old Wistar rats (190–210 g) using the needle technique, as described previously.⁹⁾ Rats were anesthetized with 1.5% isoflurane vaporized with room air or pentobarbital sodium (50 mg/kg, intraperitoneally (i.p.)), and the vena cava and abdominal aorta were exposed by opening the abdominal cavity via a midline incision. An 18-gauge needle was inserted into the abdominal aorta and advanced through the medial wall into the vena cava to create an AVS. The aorta was temporarily clamped below the origin of the right renal artery, the needle was withdrawn, and the aortic puncture was immediately sealed with cyanoacrylate glue. To verify the patency of the shunt, the pulsatile flow of oxygenated blood into the inferior vena cava was visually observed. The abdominal cavity was closed using a standard technique with an absorbable suture. Experiments were performed at 8 weeks (AVS-8W, n = 13) or 12 weeks (AVS-12W, n = 30) after AVS surgery. Rats in the sham group (Sham, n = 30) underwent the same surgical procedure except for puncturing the vessels.

Measurement of Hemodynamics and Electrophysiological Parameters

Eight or 12 weeks after the surgery, the rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). The trachea was cannulated for artificial respiration (SN-480-7; Shinano, Tokyo, Japan). Tidal volume and respiratory rate were set at 10 mL/kg and 60 strokes/min, respectively. The right femoral artery was cannulated to measure the blood pressure. A surface lead II electrocardiogram (ECG) was obtained from the limb electrodes. A quad-polar electrodes catheter (3 French, SMC-304; Physio-Tech, Tokyo, Japan) was inserted through the right jugular vein and positioned at the atrial septum by watching the atrial ECG and the surface lead II ECG, where the peaks of the A-wave and P-wave overlapped. The electrograms were amplified using a bioelectric amplifier (AB-621G, Nihon Kohden, Tokyo, Japan) and fed into a computer-based data acquisition system (PowerLab, AD Instruments, New South Wales, Australia).

The atrial effective refractory period (ERP) was assessed by programmed electrical stimulation using a cardiac stimulator (SEN-7203, Nihon Kohden). The stimulation pulses were set rectangular in shape, with 1–2 V stimulation pulses (about twice the threshold voltage) and 3 ms duration. The pacing protocol consisted of 10 beats of basal stimuli with cycle lengths of 120 and 100 ms, followed by an additional stimulus at various coupling intervals. Starting in the late atrial diastole, the coupling interval was shortened by consecutive reductions until the additional stimulus could no longer elicit a response. Atrial ERP was defined as the shortest coupling interval that still produced an electrical response.

AF was induced by pacing at the atrial septum with burst pacing (5V output; 3-ms pulse width; 15-ms cycle length for 20 s) 10 times via the catheter using a stimulator (SEN-7203, Nihon Kohden). AF was defined as a period of rapid irregular atrial rhythm, resulting in an irregular ECG baseline. The duration and cycle length of the AF were measured using an atrial electrogram.

Anatomical Assessment

After the electrophysiological examinations, the whole heart was rapidly excised and washed thoroughly with cold saline to remove the contaminating blood, and the heart was then separated into three parts: atrium, left ventricle, and right ventricle. Each part was weighed, and the thickness of the ventricular septal and left and right ventricle walls were measured using a caliper.

Quantitative Real-Time PCR

To determine the level of expression of the genes of Na_v1.5, Ca_v1.2, Ca_v1.3, K_v1.5, K_v4.2, K_v4.3, K_Ca3.1, K_Ca1.1 beta subunit 1, K_Ca2.2, K_Ca2.3, K_ir3.1, K_ir2.1, K_ir2.2, connexin 43 (Cx43), transient receptor potential canonical type 6 (TRPC6), transforming growth factor (TGF)-β1, and TGF-β2, quantitative RT-PCR was performed with SsoAdvanced SYBR Green Supermix (Bio-Rad, CA, U.S.A.) in a CFX96 System (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ATP5PB were used for the normalization of the mRNA expression data. Briefly, total RNA was isolated from the right and left atria and ventricles of the Sham-8W (n = 6), AVS-8W (n = 7), Sham-12W (n = 6), and AVS-12W (n = 9) rats using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Venlo, Netherlands). The cDNA was synthesized from RNA samples using the iScript cDNA Synthesis Kit (Bio-Rad) for Gene Amp PCR System 9700 (Applied Biosystems, MA, U.S.A.). The primers used for this reaction were purchased from TaKaRa Biomedical, Inc. (Shiga, Japan). The primer sequences used in this study are listed in Supplementary Table 1.

Measurement of Action Potential

The action potential was recorded to better understand the electrophysiological changes in the atrium of rats with 12-week AVS that showed longer duration of AF. The action potentials were recorded using standard microelectrode techniques.⁸⁾ Twelve weeks after the AVS surgery, the left atria were rapidly isolated from the Sham (n = 8) and AVS-12W (n = 10) rats, which were pinned down on the bottom of the recording chamber with a volume of 20 mL. The extracellular solution had the following composition (mM concentration): NaCl 118.4, KCl 4.7, CaCl₂ 2.5, MgCl₂ 1.2, KH₂PO₄ 1.2, NaHCO₃ 24.9, and glucose 10; it was gassed with 95% O₂–5% CO₂ and maintained at 36.0 ± 0.5 °C (pH 7.4). Action potentials were recorded in pulmonary vein tissue preparations by standard microelectrode penetration from the luminal side. The glass microelectrodes filled with 3 M KCl had resistances of 20–30 M. The output of a microelectrode amplifier with high input impedance and capacity neutralization (MEZ-8201, Nihon Kohden) was digitized using an A/D converting interface (Power Lab/4SP, AD Instruments) and analyzed using the Chart 7 software (AD Instruments).

Histological Assessment

Microscopic assessment was performed to better understand the histological changes in the atrium of rats with 12-week AVS that showed longer duration of AF. Twelve weeks after the AVS surgery, the heart was rapidly excised from one Sham rat and one AVS-12W rat under anesthesia with pentobarbital sodium (50 mg/kg, i.p.). The heart was washed thoroughly with cold saline to remove contaminating blood and was separated into three parts: the atrium, left ventricle, and right ventricle. The parts were fixed with 10% formalin neutral buffer solution and processed into paraffin blocks. The paraffin-embedded tissue blocks were cut into 4-µm-thick sections, which were then stained with Elastica–Masson stain.

Statistical Analysis

All data are expressed as mean ± standard error of the mean (S.E.M.). The statistical significance of the parameters among the three models was evaluated using the Tukey–Kramer test. The significance of differences between the two models was determined by unpaired t-test or Welch’s t-test correction. Statistical significance was set at p < 0.05.

RESULTS

Hemodynamic and Electrophysiological Parameters in Anesthetized Rats

Table 1 summarizes the hemodynamic parameters in the Sham, AVS-8W, and AVS-12W groups. The diastolic blood pressure in the AVS-8W and AVS-12W groups was significantly lower than that in the Sham group.

Table 1. Hemodynamic and Electrophysiological Parameters in Rats with Abdominal Aorto-Venocaval Shunt

Parameter	Sham	AVS-8W	AVS-12W
Hemodynamic parameters
Heart rate (BPM)	367 ± 15	391 ± 15	375 ± 13
Systolic BP (mmHg)	141 ± 5	135 ± 3	138 ± 6
Diastolic BP (mmHg)	115 ± 4	90 ± 4**	89 ± 4**
Electrophysiological parameters
P-wave duration (ms)	14 ± 1	14 ± 1	17 ± 1*
PR interval (ms)	44 ± 1	43 ± 1	52 ± 1**‡‡
QRS width (ms)	15 ± 0	17 ± 1	19 ± 1**
QT interval (ms)	80 ± 2	80 ± 3	85 ± 2
R-wave amplitude (mV)	0.6 ± 0.0	0.9 ± 0.1**	0.8 ± 0.1*

Data are means ± S.E.M. of the Sham (n = 9), AVS-8W (n = 6) and AVS-12W (n = 10) groups. BP; blood pressure. * p < 0.05, ** p < 0.01 vs. Sham, ^‡‡ p < 0.01 vs. AVS-8W.

Morphometric Parameters of the Rat Heart

The tissue weight of the heart, heart/body weight ratio, and wall thickness are summarized in Table 2. The weight of each cardiac tissue in the AVS-8W and AVS-12W groups was significantly greater than that in the Sham group. The thickness of the ventricular septal, left, and right ventricular wall in the AVS-8W and AVS-12W groups were greater than those in the Sham group, although not significantly different.

Table 2. Morphological Parameters of the Heart Obtained from Rats with Abdominal Aorto-Venocaval Shunt

Parameter	Sham	AVS-8W	AVS-12W
Body weight (g)	374 ± 19	330 ± 7	417 ± 6*‡‡‡
Tissue weights
Heart weight (mg)	929 ± 33	1277 ± 58*	1784 ± 107***‡‡
Heart (mg)/body (g) weight ratio	2.5 ± 0.1	3.9 ± 0.2***	4.3 ± 0.2***
Atrial weight (mg)	86 ± 6	161 ± 18*	213 ± 20***
RV weight (mg)	192 ± 9	275 ± 18*	406 ± 26***‡‡‡
LV weight (mg)	651 ± 25	830 ± 26*	1165 ± 64***‡‡‡
Tissue thickness
RV wall thickness (mm)	1.3 ± 0.0	1.4 ± 0.1	1.5 ± 0.1
Septal thickness (mm)	2.9 ± 0.1	3.1 ± 0.2	3.3 ± 0.1
LV wall thickness (mm)	4.8 ± 0.2	4.6 ± 0.3	5.3 ± 0.2

Data are means ± S.E.M. of the Sham (n = 9), AVS-8W (n = 6) and AVS-12W (n = 10) groups. RV; right ventricle, LV; left ventricle. * p < 0.05, *** p < 0.001 vs. Sham, ^‡‡ p < 0.01, ^‡‡‡ p < 0.001 vs. AVS-8W.

mRNA Expression Levels of Cardiac Ion Channels and Others

The gene expression levels determined by real-time PCR in the atrial tissues obtained from AVS-8W and AVS-12W rats are summarized in Fig. 1, and those in the ventricular tissues are shown in Supplementary Fig. 1.

Fig. 1. Fold Changes in Gene Expression Measured by Real-Time PCR in the Atrial Tissues Obtained from AVS-8W and AVS-12W Rats

Results are expressed as fold changes over Sham-8W or Sham-12W rats as each of control groups. GAPDH and ATP5PB served as the internal control in the left atrium and right atrium, respectively. Data are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. each of control groups.

In the right atrium, higher mRNA levels of Ca_v1.3, K_Ca1.1 beta subunit 1, K_Ca2.3, TRPC6, and TGF-β2 were detected both in the AVS-8W and AVS-12W groups than in the Sham group. Higher mRNA levels of Ca_v1.2, K_Ca3.1, K_ir3.1 (p = 0.061), K_ir2.1 (p = 0.058), and Cx43 were observed in the AVS-8W group; in contrast, these genes were significantly downregulated in the AVS-12W group. Higher mRNA levels of Na_v1.5, K_v4.3, and TGF-β1 were detected only in the AVS-8W group. The lower mRNA levels of K_v1.5, K_v4.2, K_Ca2.2, and K_ir2.2 were observed only in the AVS-12W group.

In the left atrium, higher mRNA levels of K_Ca1.1 beta subunit 1, TRPC6, and TGF-β1 and lower mRNA levels of K_v1.5 and K_Ca3.1 were detected in both the AVS-8W and AVS-12W groups than in the Sham group. The lower mRNA levels of K_v4.2, K_Ca2.2 and K_ir2.2 were observed only in the AVS-8W group. Higher mRNA levels of Na_v1.5, Ca_v1.3, K_Ca2.3, and K_ir2.1 and lower mRNA level of Cx43 were detected only in the AVS-12W group.

Electrophysiological Parameters in Anesthetized Rats

Figure 2 shows the typical tracings of surface ECG and right atrial electrogram in the Sham, AVS-8W, and AVS-12W rats, whereas Table 1 summarizes the electrophysiological parameters in those groups. The P-wave duration, PR interval, QRS width, and R-wave amplitude of the ECG were significantly greater in the AVS-12W group than those in the Sham group.

Fig. 2. Typical Tracings of Surface Lead II-ECG and Right Atrial Electrogram

Typical tracings of surface lead II electrocardiogram and right atrial electrogram in the Sham (left), AVS-8W (middle), and AVS-12W groups (right), respectively. ECG, electrocardiogram; RA, right atrial electrocardiogram.

Inducibility of Atrial Fibrillation

The representative electrocardiograms of AF induced by burst pacing in the AVS-12W rat are shown in Fig. 3A, whereas the duration and cycle length of AF in the Sham, AVS-8W, and AVS-12W groups are summarized in Figs. 3B and C. The AF duration in the AVS-12W group was significantly longer than that in the Sham and AVS-8W groups. The cycle length of AF in the AVS-8W and AVS-12W groups was significantly longer than that in the Sham group. As shown in Fig. 3D, the atrial ERP in the AVS-12W group was significantly longer than that in the Sham group.

Fig. 3. Atrial Arrhythmogenicity in the AVS-8W and AVS-12W Rats

(A) Representative electrocardiograms of AF induced by burst pacing in AVS-12W rat. (B) The duration of AF, (C) the cycle length of AF, and (D) the atrial ERP in basal pacing cycle lengths of 120 ms (left) and 100 ms (right) in the Sham, AVS-8W, and AVS-12W groups, respectively. Data are means ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Sham, ^‡‡‡ p < 0.001 vs. AVS-8W. AF, atrial fibrillation; RA, right atrial electrocardiogram; ERP, effective refractory period; CL, cycle length.

Action Potential Parameters in the Isolated Rat Atrium

Typical tracings of the action potentials recorded from the left atria are depicted in Fig. 4A, and the action potential parameters are summarized in Table 3. The overshoot and amplitude in the AVS-12W group were significantly lower than those in the Sham group, and the resting potential in the AVS-12W group was slightly positive compared with that in the sham group, although the difference was not statistically significant. Shorter action potential duration at 20% repolarization (APD₂₀) and longer action potential duration at 90% repolarization (APD₉₀) in the AVS-12W group were detected compared with those in the Sham group.

Fig. 4. Typical Tracings of Atrial Action Potentials and Histology of the Atrial Tissues Processed by Elastica–Masson Staining

(A) Typical tracings of action potentials recorded from the left atrium in the Sham rat (left) and AVS-12W model rat (right). All preparations were driven at 1 Hz. (B) Histology of the right atrial tissues (a, b) and left atrial tissues (c, d) processed by Elastica–Masson staining. a and c, Sham rat; b and d, AVS-12W rat. APD₉₀, action potential duration at 90% repolarization; RP, resting potential.

Table 3. Action Potential Parameters in the Isolated Left Atrium from Rats with Abdominal Aorto-Venocaval Shunt

Parameter	Sham	AVS-12W
RP (mV)	−77.8 ± 0.9	−75.6 ± 1.2
OS (mV)	27.0 ± 1.4	20.6 ± 2.0*
AMP (mV)	104.8 ± 2.1	96.2 ± 3.0*
V̇max (V/s)	238.2 ± 16.6	216.5 ± 15.0
APD20 (ms)	6 ± 1	4 ± 0**
APD50 (ms)	16 ± 2	14 ± 2
APD90 (ms)	71 ± 5	90 ± 7*

Data are means ± S.E.M. of the Sham (n = 8) and AVS-12W (n = 10) groups. RP; resting potential, OS; overshoot, AMP; amplitude, V̇_max; maximum rate of phase 0 depolarization, APD₂₀, APD₅₀, APD₉₀; action potential duration at 20, 50, 90% repolarization, respectively. * p < 0.05, ** p < 0.01 vs. Sham.

Histological Characteristics of the Rat Heart

Typical photomicrographs of longitudinal sections of the right and left atrial tissues are shown in Fig. 4B, which were obtained from rats in the Sham and AVS-12W groups. Hypertrophy of atrial myocytes and interstitial and perivascular fibrosis were observed in AVS rats. The histology in the ventricular tissues is shown in Supplementary Fig. 2. Hypertrophy of ventricular myocytes and epicardial, perivascular, and interstitial fibrosis were observed in the AVS rats.

DISCUSSION

This study analyzed the AF inducibility in the AVS rat, which has been used as an experimental model of congestive heart failure associated with chronic volume overload. Although structural changes were observed in the heart of the AVS-8W rats, including atrial enlargement, no significant changes were detected in cardiac electrophysiological parameters such as ECG, atrial ERP, or AF duration. In the AVS-12W rats, marked electrophysiological changes, including the prolonged P-wave duration and atrial ERP in addition to structural changes, leading to a significantly longer duration of AF induced by burst pacing. Meanwhile, significant changes in the mRNA levels of various ion channels were detected in the AVS-8W and AVS-12W rats. To comprehensively understand the pathophysiological changes in the rat atrium for induction of AF after AVS surgery, the possible relationship between gene expression and structural and electrophysiological changes is discussed.

Cardiohemodynamic Property of the AVS Rats

A decrease in diastolic blood pressure was observed in the AVS-8W and AVS-12W groups (Table 1), indicating the maintenance of the fistula between the abdominal aorta and vena cava during the experiment. Ryan et al.¹⁰⁾ have demonstrated that the left ventricular end-diastolic pressure increased at 2 d and remained elevated (10–15 mmHg) throughout the postoperative 4–15 weeks, whereas an increase in end-systolic dimension was detected ≥4 weeks after the surgery, suggesting the involvement of left ventricular systolic and diastolic dysfunction in the AVS rats. Moreover, elevation of central venous pressure to 4–8 mmHg has been reported in another study.¹¹⁾ Such hemodynamic changes caused by abdominal fistula might induce sustained atrial volume overload, leading to atrial enlargement in the AVS-8W and AVS-12W rats.

Atrial Ion Channel Gene Expression after the Surgery of AVS

In the AVS-12W rat, downregulation of various types of K_v and K_ir channels at the mRNA level was most evident in the right atrium, and similar changes were observed in the left atrium and both ventricles (Fig. 1, Supplementary Fig. 1). The results essentially resemble previous observations in AF patients and animal models of AF.^12,13) In contrast, it is noteworthy that mRNA levels were upregulated in most of the K_v and K_ir channels of the right atrium at 8 weeks after AVS surgery. Among the four-chambered hearts, the right atrium had a greater impact of elevated central venous pressure at 8 weeks after AVS surgery, leading to the activation of gene signals in relation to mechanical stretch, which might not create arrhythmogenic substrates in the atrium. Thus, the difference in gene expression in the right atrium between the AVS-8W and AVS-12W rats may partly explain the acquisition of arrhythmogenicity. However, we could not determine the responsible genes in this study. In addition, the ion-channel subunit mRNA does not necessarily reflect protein expression or ionic-current properties. Further extensive studies with a combination of molecular biology and cell electrophysiology are needed to identify the genes responsible for arrhythmogenesis during the establishment of atrial remodeling.

Structural Remodeling in the Atrium Induced by AVS

As shown in Table 2, Fig. 4 and Supplementary Fig. 2, increment of the atrial and ventricular weights and hypertrophy were observed after the creation of fistula using the needle technique due to drastic cardiac preload, which are in good accordance with previous studies.^14–18) Furthermore, a significant atrial fibrosis was confirmed in the AVS-12W rat (Fig. 4B), being reflected by the increment of mRNA levels of TGF-β1 and TGF-β2 (Fig. 1), which is presumably stimulated by a mechanical stretch in the cardiac fibroblasts via the TGF-β signaling pathway.¹⁹⁾ Notably, the higher expression levels of TRPC6 channels, a TRPC subfamily member that functions as a Ca²⁺-permeable cation channel, were observed in all sections of the heart of the AVS-8W and AVS-12W rats (Fig. 1). Recently, TRPC6 channel has been suggested to be a positive regulator of Ca²⁺-calcineurin-nuclear factor of activated T cells signaling following stimulation of Gq-coupled receptors such as endothelin, angiotensin II, or α₁-adrenergic receptors, which is a key component of pathological cardiac hypertrophy and fibrosis.²⁰⁾ Since an upregulation of endothelin-1 mRNA level²¹⁾ as well as an activation of renin-angiotensin-aldosterone system²²⁾ and sympathetic nervous system²³⁾ has been reported in the AVS rat heart, TRPC6 may contribute to atrial remodelings relating Gq protein-coupled receptor-mediated Ca²⁺ signaling pathways in this animal model. In addition, TRPC6 channel can be critical for fibroblasts differentiation into myofibroblasts associated with mechanical stretch.^24–27) Also, roles of the upregulation of TRPC6 channels for fibrosis have been suggested in some pathophysiological processes, including glomerulosclerosis, pulmonary fibrosis, and Crohn’s disease.^28–30) Thus, similar mechanisms may be involved in the rat dilated atrium caused by long-term volume overload to the heart.

Electrical Remodeling Induced by AVS

In previous studies dealing with electrical remodeling, it has been widely recognized that rapid atrial pacing abbreviated the electrical refractory period and action potential duration in the atrium,^31–34) which is explained by the reduction of L-type Ca²⁺ channel currents.³⁴⁾ In this study, a lower mRNA level of K_v1.5, representing I_Kur, was detected in the left atrium of the AVS-8W and AVS-12W rats (Fig. 1), which may partly explain the prolonged action potential duration in the left atrium. Meanwhile, mRNA levels of Ca_v1.2 decreased in the right atrium of the AVS-12W rat as well as that of the rapid atrial pacing-induced electrical remodeling.^34–36) A significant reduction in mRNA levels was more often observed in the right atrium of the AVS-12W rat than in the left atrium, such as K_v1.5, K_v4.2, K_ir3.1, K_ir2.1, and K_ir2.2, representing I_Kur, I_to, I_{K, ACh}, and I_K1, respectively, possibly affecting the membrane potential and/or action potential in the right atrium. Because gene expression of cardiac ion channels, such as K_v1.5, K_v2.1, K_v4.2, and K_v4.3, is known to be regulated by intracellular calcium ([Ca²⁺]i) or intracellular signaling,³⁷⁾ TRPC6 channels may contribute to ion channel remodeling caused by AVS. The mRNA level of Na_v1.5, significantly increased in the left atrium. However, no significant difference was detected in the maximum rate of phase 0 depolarization of the atrial action potential between the Sham and AVS-12W groups (Table 3), suggesting that the function of voltage-dependent Na⁺ channels remains normal after the creation of AVS.

Electrophysiology of the Atrium in the AVS Rat

The ECG parameters were affected by AVS surgery (Table 1, Fig. 2). Based on our results regarding the reduction of mRNA levels of Cx43 (Fig. 1), it can be speculated that the increased P-wave duration, PR interval, and QRS width in the AVS-12W rat are associated with gap junction remodeling. In addition, the P-wave duration might also be altered by atrial fibrosis in the AVS-12W rat, since the function of voltage-dependent Na⁺ channels in the left atrium was shown to be maintained based on the mRNA level of Na_v1.5 and phase 0 depolarization of the atrial action potential in this study. Hence, it is suggested that the obvious atrial fibrosis and remodeling of Cx43 are responsible for the development of apparent intra-atrial conduction disturbance. It is noteworthy that marked ECG changes were detected only in the AVS-12W rat despite obvious increments in atrial and ventricular tissue weights and altered gene expression in the AVS-8W rat (Tables 1, 2, Figs. 1, 2). This may suggest that the electrical remodeling observed in the atrium is preceded by structural remodeling after AVS surgery.

Proarrhythmic Substrate in the AVS Rat Atria

In the AVS-12W rat, AF could be induced by burst pacing (Fig. 3A), in contrast to previous studies with combined electrophysiological protocol with cholinergic or hypoxic stimulation.^38,39) Irregularity in fractionated atrial electrogram in addition to organized atrial activity with regularity may suggest an association of microreentry and macroreentry in the atria of the AVS-12W rat. The duration of AF in the AVS-12W rats was significantly longer than that in the AVS-8W group (Fig. 3B), suggesting that arrhythmogenicity was totally acquired in the atria between 8 and 12 weeks after the AVS surgery, where atrial fibrosis and Cx43 remodeling might occur according to the prolonged P-wave duration, PR interval, and QRS width (Table 1). Prolongation of atrial refractoriness is generally considered a major factor in preventing reentrant arrhythmia. Although the ERP at the atrial septum was prolonged in this study, atrial ERP heterogeneity may also play an important role in increasing atrial variability to AF.^40,41)

As shown in Fig. 4B, fibrosis was more often observed in the atria of the AVS rat. In the isolated left atrium from the AVS-12W rat, a slight positive shift in the resting potential (+2.2 mV) was detected (Fig. 4A, Table 3). The influence of AVS on the action potentials of the atrium is essentially in accordance with our previous study using the pulmonary-vein myocardium in the same animal model, where a positive shift of the resting potential (+4.4 mV) was observed.⁸⁾ The electrophysiological properties of the atrium as well as anatomical changes may be important to better understand the mechanism of AF in AVS rats. In addition, the results of PCR analysis in AVS-12W rats showed nonuniform remodeling in the right and left atria, such as Na_v1.5 (encoded by Scn5a), Ca_v1.2 (Cacna1c), K_v4.2 (Kcnd2), K_Ca2.2 (Kcnn2), K_ir3.1 (Kcnj3), K_ir2.1, (Kcnj2), and K_ir2.2 (Kcnj12). Such differences within atria might contribute to heterogeneity in atrial electrophysiology, leading to conduction delay and heterogeneous remodeling, which should be clarified further in future studies.

In summary, chronic volume overload caused by an abdominal AVS provides arrhythmogenic substrates in the rat atrium, accompanied by prolonged P-wave duration. The difference in gene expression in the right atrium between the AVS-8W and AVS-12W rats may partly explain the acquisition of arrhythmogenicity.

Acknowledgments

This work was supported, in part, by JSPS KAKENHI Grant Nos. JP15K08598 (to A.T.) and 20K22924 (to M.A.).

Conflict of Interest

The authors declare no conflict of interest. Tetsuo Kuze, Takeshi Hasegawa, Kohei Kumada, and Toshiki Chiba are employees of TOA EIYO LTD.

Supplementary Materials

This article contains supplementary materials.

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