Anti-atrial Fibrillatory Effects of the TRPC3 Channel Inhibitor Pyrazole-3 in Rats with Atrial Enlargement Induced by Chronic Volume Overload

Abstract

The effect of transient receptor potential canonical-3 (TRPC3) channel inhibitor pyrazole-3 (Pyr3) on the stability of atrial fibrillation (AF) was analyzed in a rat model of chronic volume overload. Male Wistar rats underwent aorto-venocaval shunt (AVS) surgery and received Pyr3 treatment intraperitoneally for 12 weeks. Morphological and electrophysiological assessments were performed at the end of the treatment period. Longer P-wave duration, atrial conduction time, and AF duration, in addition to greater atrial tissue weight, were detected in AVS rats compared with sham rats. Chronic Pyr3 administration prevented AVS-induced prolongation of P-wave duration and atrial conduction time, partially prevented atrial weight increase, and abbreviated AVS-induced prolongation of AF duration to similar levels as those in sham rats, without affecting the atrial effective refractory period. These results suggest that the TRPC3 inhibitor Pyr3 can ameliorate sustained AF associated with chronic volume overload by improving atrial conduction defects in AVS rats. Pharmacological inhibition of TRPC3 channels by Pyr3 thus represents a promising therapeutic strategy for preventing AF development related to structural modeling.

INTRODUCTION

Transient receptor potential (TRP) channels are a large family of transmembrane ion channels that can sense a wide range of external and internal stimuli.¹⁾ As TRP channels are involved in diverse physiological processes, they have been recognized as promising drug targets for a variety of diseases, including cardiovascular, respiratory, neurological, and inflammatory bowel diseases.²⁾ In a mouse model, pyrazole-3 (Pyr3; Fig. 1), a selective transient receptor potential canonical-3 (TRPC3) inhibitor, has been reported to prevent the progression of pressure overload-induced cardiac hypertrophy³⁾ and dilated cardiomyopathy,⁴⁾ indicating its potential benefit in structural heart disease. Recently, chronic administration of Pyr3 was shown to reduce the duration of burst pacing-induced atrial fibrillation (AF) in dogs with atria exposed to atrial tachypacing.⁵⁾ These findings also suggest that Pyr3 may have therapeutic potential in attenuating the electrical remodeling processes underlying AF onset.⁵⁾ The pathophysiology of AF is known to involve structural remodeling events, including atrial enlargement and fibrosis, and electrical remodeling due to AF itself, both of which play crucial roles in generating a substrate favorable for the development and persistence of AF.^6,7) Given that AF substrate advances with progressive atrial structural remodeling,⁸⁾ it is crucial to analyze the effects of Pyr3 on AF associated with atrial enlargement induced by increased preload to the heart.

Fig. 1. The Chemical Structure of Pyr3

Pyr3 is a representative selective inhibitor of the TRPC3 channels. Pyr3: pyrazole-3.

We recently established a rat AF model by surgery of an abdominal aorto-venocaval shunt (AVS), resulting in long-term cardiac volume overload.^9,10) In AVS rats, structural remodeling precedes electrical remodeling in the generation of an arrhythmogenic substrate in the atria, with atrial enlargement and hypertrophy developing within 2 weeks after post-AVS, and AF occurring by 12 weeks. We investigated the effect of the TRPC3 inhibitor Pyr3 on the stability of AF in a rat model of atrial enlargement induced by chronic volume overload. To better understand the effect of Pyr3 on the progression of atrial remodeling required for sustained AF, we used two animal models with different arrhythmogenic stages caused by exposure to volume overload for 4 or 12 weeks.

MATERIALS AND METHODS

All experiments were approved by the Toho University Animal Care and Use Committee (Approval Nos.: 15-54-160 for the 4-week AVS model using pentobarbital as an anesthetic and 17-51-359 for the 12-week AVS model using isoflurane as an anesthetic) and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society. Forty male Wistar rats (Japan SLC, 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 for AVS Introduction and Osmotic Minipump Implantation

Eight-week-old rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection) or 1.5% isoflurane vaporized in ambient air. AVS surgery was performed using the 18-gauge needle technique after exposure of the vena cava and abdominal aorta by opening the abdominal cavity through a midline incision, as described previously.^9–11) The sham operation involved the same surgical procedure but excluded the vessel puncture. Pyr3 was synthesized in our facility, dissolved in polyethylene glycol 400, and administered continuously at a release rate of 1.25 µg/h (equivalent to 0.1 mg/kg/d) for 4 or 12 weeks using an osmotic minipump (ALZET pump 2ML4; Durect, Cupertino, CA, U.S.A.) implanted intraperitoneally during creation of the AVS. The rats were divided into 4- and 12-week groups based on drug administration duration. Each group was further subdivided into sham operation (Sham), AVS operation (AVS), and AVS operation + Pyr3 (AVS + Pyr3) groups. Experiments were performed for 4 weeks (Sham, n = 3; AVS, n = 5; and AVS + Pyr3, n = 5) or 12 weeks (Sham, n = 14; AVS, n = 12; and AVS + Pyr3, n = 8) after the start of drug administration. Twelve weeks of drug administration was maintained by replacing minipumps every 4 weeks.

Measurement of Hemodynamics and Electrophysiological Parameters

At 4- or 12-weeks post-surgery, rats were re-anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection) or 1.5% isoflurane vaporized in ambient air, and artificially ventilated via a tracheal cannula with a tidal volume of 10 mL/kg and a respiratory rate of 60 strokes/min (SN-480-7; Shinano, Tokyo, Japan). Blood pressure was measured in the right femoral artery, and a surface lead II electrocardiogram (ECG) was obtained from limb electrodes. A quad-polar electrode catheter (3 French, SMC-304; Physio-Tech, Tokyo, Japan) was inserted into the right jugular vein, for atrial electrogram recording and electrical stimulation, and positioned at the atrial septum by observing the atrial electrocardiogram and surface lead II ECG, where the A-wave and P-wave peaks overlapped. The electrogram was amplified using a bioelectric amplifier (AB-621G; Nihon Kohden, Tokyo, Japan) and fed into a computer-based data acquisition system (PowerLab; AD Instruments, NSW, Australia).

Atrial effective refractory period (ERP) and conduction time were measured using a stimulator (SEN-7203; Nihon Kohden) after equilibration for 20–60 min. Stimulation pulses were delivered in the form of rectangular pulses (1–2 V; approximately twice the threshold voltage) with a pulse duration of 3 ms. 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 late atrial diastole, the coupling interval was successively shortened until an electrical response was no longer elicited. The atrial ERP was defined as the shortest coupling interval that produced an electrical response. During basal stimuli with a cycle length of 120 or 100 ms, the local atrial conduction time was measured from the pacing artifact to the onset of the first initial deflection recorded by the electrode catheter. Three consecutive electrical responses from the 8th to 10th beats of the 10-beat basal stimulus were used to obtain an average value.

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

Anatomical Assessments

For anatomical assessment, rats were euthanized after electrophysiological testing, and their hearts were immediately excised and thoroughly washed with cold saline to eliminate blood contamination. Hearts were dissected into the atria, and the left and right ventricles, and weighed. The thicknesses of the ventricular septa and the left and right ventricular walls were measured using calipers.

Statistical Analysis

All data are expressed as means ± standard error of the mean (S.E.M.). The statistical significance of parameter differences among the three models within each drug administration period was evaluated using the Tukey–Kramer test. Statistical significance was set at p < 0.05.

RESULTS

Stability of Atrial Fibrillation

Representative electrocardiograms of AF induced by burst pacing in the 12-week model are shown in Fig. 2. The effects of Pyr3 on AF duration are summarized in Fig. 3A. In the 4-week model, no significant differences were observed in AF duration among the three experimental groups. In the 12-week model, the duration of AF in the AVS group was 208 s, approximately 15-fold longer than that in the Sham group. In the AVS + Pyr3 group, the duration of AF was significantly shorter than that in the AVS group, and similar to that in the Sham group.

Fig. 2. Representative Electrocardiogram of Burst Pacing-Induced AF in Sham, AVS, and AVS + Pyr3 Groups in the 12-Week Model

AF: atrial fibrillation; AVS: aorto-venocaval shunt; ECG: electrocardiogram; RA: right atrial electrocardiogram; BP: blood pressure; Pyr3: pyrazole-3.

Fig. 3. Effect of Pyr3 on Atrial Arrhythmogenicity in AVS Rats

(A) AF duration and (B) AF cycle length of burst pacing-induced AF in Sham, AVS, and AVS + Pyr3 groups in 4- and 12-week models. Data are expressed as the means ± S.E.M. of the Sham (n = 3), AVS (n = 5), and AVS + Pyr3 (n = 5) groups in the 4-week model, and of the Sham (n = 10), AVS (n = 9), and AVS + Pyr3 (n = 8) groups in the 12-week model. AF: atrial fibrillation; AVS: aorto-venocaval shunt; Pyr3: pyrazole-3.

The effect of Pyr3 on AF cycle length is summarized in Fig. 3B. In the 4-week model, no significant differences were observed in the AF cycle length among the three experimental groups. A typical example of AF cycle length in a 12-week model is shown in Fig. 2. AF cycle length was significantly longer in the AVS group than in the Sham group. In the AVS + Pyr3 group, the AF cycle length tended to be shorter than in the AVS group (p = 0.068).

Electrophysiological and Hemodynamic Parameters

Figure 4 shows typical traces of surface lead II ECGs in the 4- and 12-week models. Table 1 summarizes the electrophysiological and hemodynamic parameters of each study group. In the 4-week model, no significant differences were observed in the ECG or hemodynamic parameters between the Sham and AVS groups, except for R-wave amplitude and heart rate. In the 12-week model, the AVS group exhibited significantly prolonged P-wave duration, PR interval, QRS width, QT interval, and R-wave amplitude, compared with the Sham rats.

Fig. 4. Effect of Pyr3 on Surface Lead II Electrocardiograms in AVS Rats

Representative surface lead II electrocardiograms in Sham, AVS, and AVS + Pyr3 groups in 4- and 12-week models. AVS: aorto-venocaval shunt; Pyr3: pyrazole-3.

Table 1. The Effect of Pyr3 on Electrophysiological and Hemodynamic Parameters in the AVS Rats

	4-week			12-week
	Sham	AVS	AVS + Pyr3	Sham	AVS	AVS + Pyr3
Electrophysiological parameters
P-wave duration (ms)	18 ± 1	20 ± 1	18 ± 0	18 ± 1	25 ± 1***	22 ± 1*‡
PR interval (ms)	44 ± 1	43 ± 2	40 ± 1	44 ± 1	55 ± 2***	53 ± 1***
QRS width (ms)	19 ± 2	17 ± 1	18 ± 1	18 ± 1	22 ± 1*	21 ± 1
QT interval (ms)	72 ± 3	69 ± 2	73 ± 3	83 ± 3	96 ± 4*	95 ± 2*
R-wave amplitude (mV)	0.8 ± 0.0	1.0 ± 0.1*	1.0 ± 0.1	0.7 ± 0.0	1.0 ± 0.0**	1.0 ± 0.1**
Hemodynamic parameters
Heart rate (BPM)	355 ± 26	416 ± 9*	411 ± 12	342 ± 7	307 ± 15	336 ± 9
Systolic BP (mmHg)	130 ± 9	148 ± 6	145 ± 11	146 ± 3	120 ± 7**	119 ± 6**
Diastolic BP (mmHg)	92 ±10	88 ± 4	90 ± 11	102 ± 3	63 ± 6**	68 ± 6**

Data are expressed as the means ± S.E.M. of the Sham (n = 3), AVS (n = 5), and AVS + Pyr3 (n = 5) groups in the 4-week model, and of the Sham (n = 10), AVS (n = 9), and AVS + Pyr3 (n = 8) groups in the 12-week model. BPM, beats per minute, BP, blood pressure. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Sham, ^‡p < 0.05 vs. AVS.

The effects of Pyr3 on P-wave duration are summarized in Table 1. In the 4-week model, no statistically significant differences were detected in ECG parameters, including P-wave duration or hemodynamic parameters, between the AVS and AVS + Pyr3 groups. In the 12-week model, P-wave duration was significantly shorter in the AVS + Pyr3 group than in the AVS group. No significant differences were observed in other ECG parameters between the AVS and AVS + Pyr3 groups.

Atrial Conduction Time and Atrial ERP

Table 2 shows the influence of AVS on atrial conduction time in the 4- and 12-week models. In the 4-week model, atrial conduction times tended to be longer in the AVS group than in the Sham group at both pacing cycle lengths of 120 and 100 ms. In the 12-week model, atrial conduction times in the AVS group were significantly longer than those in the Sham group at both pacing cycle lengths, with a greater increase observed at 100 ms. The effects of Pyr3 on atrial conduction time are presented in Table 2. In the 4- and 12-week models, atrial conduction time in the AVS + Pyr3 group was shorter than that in the AVS group. The reduction was statistically significant at 100 ms in the 12-week model.

Table 2. The Effect of Pyr3 on Atrial Conduction Time and Effective Refractory Period in the AVS Rats

		4-week			12-week
		Sham	AVS	AVS + Pyr3	Sham	AVS	AVS + Pyr3
Atrial conduction time (ms)	CL = 120 ms	3.1 ± 0.7	4.5 ± 0.2	3.7 ± 0.8	2.7 ± 0.9	6.2 ± 0.6**	4.2 ± 0.5
	CL = 100 ms	2.9 ± 0.6	4.8 ± 0.9	3.7 ± 0.9	2.9 ± 0.8	7.4 ± 0.9***	4.1 ± 0.5‡
Atrial ERP (ms)	CL = 120 ms	41 ± 2	44 ± 2	56 ± 6	35 ± 2	44 ± 3	45 ± 4
	CL = 100 ms	42 ± 1	45 ± 2	55 ± 6	36 ± 2	45 ± 3	44 ± 3

Data are expressed as the means ± S.E.M. of the Sham (n = 3), AVS (n = 5), and AVS + Pyr3 (n = 5) groups in the 4-week model, and of the Sham (n = 10), AVS (n = 9), and AVS + Pyr3 (n = 8) groups in the 12-week model. CL, cycle length; ERP, effective refractory period. ^**p < 0.01, ^***p < 0.001 vs. Sham, ^‡p < 0.05 vs. AVS.

Table 2 shows the influence of AVS on the atrial ERP and the effects of Pyr3 in the 4- and 12-week models. No significant difference was observed in the atrial ERP among the three experimental groups in both the 4-week and 12-week models.

Morphological Parameters

Table 3 summarizes the influence of AVS on heart tissue weights and wall thicknesses, and the effects of Pyr3 in the 4- and 12-week models. In both the 4- and 12-week models, heart weight, heart/body weight ratio, atrial weight, and left and right ventricular weights in the AVS group were significantly greater than those in the Sham group. The AVS group also demonstrated significantly increased right ventricular wall thickness in the 4-week model, and left ventricular wall thickness in both the 4- and 12-week models, relative to the respective Sham group. The effects of Pyr3 on heart tissue weight are presented in Table 3. In the 4-week model, atrial and ventricular weights in the AVS + Pyr3 group were not significantly different from those in the AVS group, either in absolute values or when normalized to body weight. In the 12-week model, the atrial weight and left and right ventricular weights in the AVS + Pyr3 group tended to be lower than those in the AVS group, and this tendency was also observed when values were normalized to body weight, although the differences were not statistically significant.

Table 3. The Effect of Pyr3 on Morphological Parameters in the AVS Rats

	4-week			12-week
	Sham	AVS	AVS+Pyr3	Sham	AVS	AVS+Pyr3
Body weight (g)	286 ± 12	296 ± 12	266 ± 2	395 ± 11	393 ± 15	361 ± 9
Tissue weights
Heart weight (mg)	749 ± 21	1143 ± 58**	1010 ± 72**	938 ± 17	1742 ± 147***	1506 ± 79***
Heart/body weight ratio (mg/g)	2.6 ± 0.1	3.9 ± 0.2**	3.8 ± 0.2	2.4 ± 0.1	4.4 ± 0.3***	4.2 ± 0.2***
Atrial weight (mg)	58 ± 5	125 ± 13*	111 ± 18*	84 ± 3	224 ± 33***	187 ± 16**
Atrial/body weight ratio (mg/g)	0.12 ± 0.0	0.42 ± 0.04**	0.42 ± 0.06*	0.20 ± 0.01	0.56 ± 0.07***	0.51 ± 0.04***
RV weight (mg)	121 ± 1	219 ± 16**	207 ± 17*	150 ± 4	296 ± 31***	248 ± 14**
RV/body weight ratio (mg/g)	0.43 ± 0.02	0.74 ± 0.05**	0.78 ± 0.06**	0.37 ± 0.01	0.83 ± 0.08***	0.75 ± 0.04***
LV weight (mg)	597 ± 21	799 ± 33**	692 ± 39	704 ± 13	1106 ± 75***	979 ± 45**
LV/body weight ratio (mg/g)	2.0 ± 0.1	2.7 ± 0.1**	2.6 ± 0.1*	1.7 ± 0.0	3.0 ± 0.2***	2.9 ± 0.1***
Tissue thickness
RV wall thickness (mm)	1.2 ± 0.1	1.7 ± 0.0***	1.4 ± 0.0**‡‡	1.2 ± 0.0	1.4 ± 0.1	1.3 ± 0.1
Septal thickness (mm)	2.4 ± 0.2	2.7 ± 0.1	2.6 ± 0.1	2.8 ± 0.1	3.0 ± 0.1	2.9 ± 0.1
LV wall thickness (mm)	3.1 ± 0.1	3.7 ± 0.1*	3.5 ± 0.1*	3.6 ± 0.1	4.0 ± 0.1**	3.6 ± 0.1‡‡

Data are expressed as the means ± S.E.M. of the Sham (n = 3), AVS (n = 5), and AVS + Pyr3 (n = 5) groups in the 4-week model, and of the Sham (n = 10), AVS (n = 9), and AVS + Pyr3 (n = 8) groups in the 12-week model. RV, right ventricle; LV, left ventricle. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. Sham, ^‡‡p < 0.01 vs. AVS.

Right ventricular wall thickness in the 4-week model, and left ventricular wall thickness in the 12-week model in the AVS + Pyr3 group were significantly lower than those in the AVS group.

DISCUSSION

To better understand the effect of Pyr3 on the progression of atrial remodeling required for sustained AF, we used two animal models representing distinct remodeling stages: a moderately remodeled atrium at 4 weeks without sustained AF, and a severely remodeled atrium at 12 weeks with sustained AF. The representative TRPC3 inhibitor Pyr3 potently ameliorated AF duration induced by burst pacing in 12-week AVS rats, whose atria were severely remodeled by chronic volume overload, compared with 4-week rats. Pyr3 also prevented the prolongation of atrial conduction time and P-wave duration in these rats, suggesting that Pyr3 may ameliorate sustained AF by improving atrial conduction abnormalities associated with chronic volume overload. Furthermore, chronic administration of Pyr3 for 12 weeks did not affect hemodynamic parameters or electrocardiographic indices related to intraventricular conduction and ventricular repolarization time in AVS rats. These results indicate that Pyr3 is well tolerated and may have a favorable safety profile under pathological conditions induced by chronic volume overload.

Electrophysiological Effects of Pyr3 in the AVS Rat Atria

The increased atrial conduction time and P-wave duration in 12-week AVS rats relative to those in the Sham group were effectively ameliorated by chronic administration of Pyr3, as shown in Tables 1 and 2. These results indicate that atrial conduction defects occurred due to 12 weeks of volume overload, and that Pyr3 prevented this progression. Slowed atrial conduction contributes to the maintenance of reentry by prolonging the conduction time around a reentrant circuit, thereby allowing sufficient time for all regions within the circuit to recover excitability.⁸⁾ Therefore, conduction defects are one of the fundamental determinants of reentry, and the amelioration of conduction defects by Pyr3 is likely to be associated with its anti-AF effects. Factors that contribute to slowed atrial conduction include disruption of structural integrity due to fibrosis, impaired cell-to-cell coupling resulting from altered connexin expression or function in intercalated disks, and reduced phase-0 Na⁺-current (I_Na), which provides the electrical energy for conduction.⁸⁾ Our preliminary study demonstrated that a single intravenous administration of Pyr3 at a dose approximately 10 times higher than that used in the present study had no effect on the P-wave duration, which reflects intra-atrial conduction time, and showed no anti-AF effect (Supplementary Fig. 1). Thus, the amelioration of conduction defects by Pyr3 is primarily associated with its chronic effects. Furthermore, our previous study confirmed the relationships between connexin 43 gene downregulation and TGF-β gene upregulation in the atria and the intercellular conduction defects observed in 12-week AVS rats, despite the intact function of voltage-gated Na⁺ channels, as demonstrated by the normal maximum rate of phase 0 depolarization of the atrial action potential.¹⁰⁾ These findings suggest that the amelioration of conduction defects in the 12-week AVS model by Pyr3 is presumably not simply associated with Na⁺ channel activity. Other mechanisms, such as improvement of interstitial fibrosis and/or disruption of intercellular conduction, should be explored based on a general explanation of the establishment of atrial conduction defects.⁸⁾

Anti-AF Effects of Pyr3 in the AVS Rat Atria

Myocardial conduction defects are known to contribute to reentry, which becomes the primary cause of AF.¹²⁾ As shown in Table 2 and Fig. 3A, atrial conduction time in the 4-week AVS model was approximately 1.5 times that in the Sham rats, slightly prolonging AF duration. By contrast, the 12-week AVS model showed a markedly prolonged AF duration, indicating that conduction defects more than twice the atrial conduction time are required to induce sustained AF. Treatment with Pyr3 in the 12-week AVS model reduced atrial conduction time prolongation to approximately 1.6-fold that of the Sham group, indicating that its beneficial effect on atrial conduction defects can be presumed to contribute to its anti-AF properties. Indeed, the amelioration of the longer AF cycle in the 12-week AVS model by Pyr3 may partly reflect the restoration of myocardial conduction defects. The mitigating effect of Pyr3 on the myocardial conduction defects is a noteworthy mechanism that differs from that of conventional agents, as most anti-arrhythmic drugs generally exert an anti-AF action by prolonging atrial ERP.¹³⁾ In addition, the partial inhibition of atrial enlargement by Pyr3 shown in Table 3 leads to a reduction in the number of reentry circuits that can be accommodated within atrial tissue.⁸⁾ Notably, this effect was evident in the 12-week AVS model with severely remodeled atria, but not in the 4-week AVS model with moderately remodeled atria, suggesting that the inhibitory effect of Pyr3 on structural remodeling may become apparent at a more progressed stage of atrial remodeling required for sustained AF. Thus, the anti-AF effect of Pyr3 is presumably supported by its ameliorative effects on both conduction defects and atrial enlargement caused by chronic volume overload.

TRPC3 channels, the molecular targets of Pyr3, have been shown to mediate calcium-dependent hypertrophic signaling¹⁴⁾ and mechanical stress-induced cardiac fibrosis, as well as to promote reactive oxygen species (ROS) production, partly through Rac1 signaling in cardiomyocytes.^4,15,16) In cardiac fibroblasts, TRPC3 also contributes to fibrotic remodeling by activating the GEF-H1–RhoA signaling pathway, thereby promoting myofibroblast differentiation and collagen synthesis.¹⁶⁾ Consistent with these mechanisms, Pyr3 has been reported to suppress mechanical stretch-induced ROS production in cardiomyocytes⁴⁾ and TGF-β-induced fibrotic responses in cardiac fibroblasts.¹⁶⁾ These findings suggest that, by inhibiting TRPC3 in both cell types, Pyr3 may suppress structural remodeling processes that underlie AF in the pathophysiological condition of AVS.

Pharmacological targets of upstream therapy for sustained AF have been clinically analyzed using non-antiarrhythmic drugs, including inhibitors of the renin-angiotensin-aldosterone system, glucocorticoids, statins, omega-3 polyunsaturated fatty acids, antioxidants, and sodium-glucose cotransporter 2 inhibitors. However, no recommendations have been made for the use of these upstream therapies to prevent AF, because large randomized controlled trials have failed to reduce recurrent AF based on recent guidelines for the diagnosis and management of AF.^17–19) Interestingly, Pyr3 has been reported to prevent the progression of AF associated with electrical remodeling caused by AF itself,⁵⁾ in addition to our present study; ameliorative effect on AF associated with structural remodeling due to chronic volume overload. Therefore, Pyr3 may become a promising therapeutic tool with multifaceted inhibitory effects on both AF development associated with structural remodeling due to underlying cardiac disease and AF progression with electrical remodeling due to AF itself.

To further explore the translational potential of Pyr3, its safety and pharmacokinetic profiles must also be considered. Pyr3 contains a trichloroacrylamide moiety with alkylating properties and has shown cytotoxic effects in cancer cells at micromolar concentrations.²⁰⁾ However, no significant toxicity was observed in renal fibroblasts²¹⁾ or in vivo at 0.1 mg/kg/d—the same dose used in our study.^3–5) Although human dose extrapolation has not been performed, the dosing was based on previously effective regimens. In the present study, Pyr3 was well tolerated, with no adverse changes in ECG or hemodynamic profiles. Although body weight gain in Pyr3-treated AVS animals was slightly lower than that in untreated AVS animals, the difference was not statistically significant. Previous studies have also shown that the same dose of Pyr3 does not affect body weight gain,^3,22) suggesting that the observed difference in our study likely falls within the range of biological variability rather than representing a true pharmacological effect. Metabolic instability remains a limitation,²³⁾ though more metabolically stable TRPC3 inhibitors have recently been reported.²⁴⁾ These findings collectively provide important preclinical insights that may support future drug development targeting TRPC3.

CONCLUSION

The present study demonstrates that the TRPC3 inhibitor Pyr3 can ameliorate sustained AF associated with chronic volume overload by improving atrial conduction defects in AVS rats. These results suggest that TRPC3 channel-targeting agents such as Pyr3 represent a multifaceted therapeutic strategy to prevent the development of AF subsequent to structural remodeling, in addition to previously reported amelioration of the progression of AF based on the AF-induced electrical remodeling.⁵⁾

Acknowledgments

This work was supported in part by JSPS KAKENHI (Grant No.: JP23K07564 to M. A.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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