Regular Article
Aldosterone Robustly Promotes Atrial Fibrillation in the Presence of Chronic Volume Overload in Rats
2024 Volume 47 Issue 9 Pages 1525-1531
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2024 Volume 47 Issue 9 Pages 1525-1531
We investigated the modulatory effects of aldosterone on atrial remodeling induced by an abdominal aorto-venocaval shunt (AVS) in rats, as patients with primary hyperaldosteronism are suggested to have a higher risk of developing atrial fibrillation (AF). The rats were divided into four groups based on the basis of whether they underwent AVS surgery, received aldosterone using an intraperitoneally implanted osmotic minipump, or both. Aldosterone was started at 0.5 µg/h during the AVS surgery, and morphological and electrophysiological assessments were performed four weeks after AVS creation. The atrial structural changes induced by AVS, including atrial cell hypertrophy and fibrosis, were not modulated by aldosterone, whereas P-wave duration was longer in aldosterone-treated AVS rats than in non-treated rats. Although the average AF duration induced by burst pacing was 10–25 s in the untreated, aldosterone-treated, and AVS rats, the AF duration was approximately 100 s in the aldosterone-treated AVS rats. Meanwhile, there was no significant difference in the atrial effective refractory period among the four experimental groups. Notably, premature atrial contractions (PAC) were frequently observed in aldosterone-treated sham rats, while paroxysmal AF, in addition to PAC, was detected in aldosterone-treated AVS rats, which was not induced in non-treated AVS rats. These findings suggest that aldosterone robustly promotes AF, particularly in the presence of chronic volume overload.
Atrial fibrillation (AF) is the most common cardiac arrhythmia, and its incidence and prevalence are increasing with an aging population.1) The pathogenesis of AF is diverse, and electrical and structural remodeling of the atria can be induced by congestive heart failure, leading to AF development and perpetuation.2,3) Heart failure is a progressive disease associated with abnormal levels of neurohumoral factors, including catecholamines, angiotensin II, and aldosterone, which promote ventricular remodeling and dysfunction.4) Although an association between AF and aldosterone has been reported in patients with primary hyperaldosteronism, showing a 12-fold greater AF risk than hypertensive controls,5) the modulatory effects of aldosterone on heart failure-related AF have not been sufficiently investigated.
We recently established a rat model that can deliver long-term cardiac volume overload by abdominal aorto-venocaval shunt (AVS) surgery to investigate atrial arrhythmogenicity. Our in vitro and in vivo studies have shown that AVS induces atrial enlargement, hypertrophy, and fibrosis,6) leading to prolonged P-wave and AF durations, depending on the time elapsed after the surgery.6,7) In this study, we investigated the modulatory effects of aldosterone on the atrial remodeling induced by 4-weeks of cardiac volume overload in rats with AVS.
All experiments were approved by the Toho University Animal Care and User Committee (Approval Number: 14-53-161). They were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society. A total of 52 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 and Implantation of Osmotic MinipumpEight-week-old rats were anesthetized using pentobarbital sodium (50 mg/kg, intraperitoneally). The rats were then carefully monitored for the depth of anesthesia and respiratory status. AVS was performed using the needle technique with an 18-gauge needle after exposure of the vena cava and abdominal aorta by opening the abdominal cavity through a midline incision.6,7) The sham operation involved the same surgical procedure, except for vessel puncture. Intraperitoneal administration of aldosterone was started at a release rate of 0.5 µg/h for 4 weeks using an osmotic minipump (ALZET pump 2ML4; Durect Corp., Cupertino, CA, U.S.A.). Rats were divided into four groups based on whether they received aldosterone: AVS surgery, sham operation (Sham, n = 12), sham operation + aldosterone (Sham + Aldo, n = 15), AVS operation (AVS, n = 13), and AVS operation + aldosterone (AVS + Aldo, n = 12).
Measurement of Hemodynamics and Electrophysiological ParametersFour weeks after surgery, the rats were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally) 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). Pentobarbital sodium was added when required to maintain the depth of the anesthesia. Blood pressure was measured in the right femoral artery, and a surface lead II electrocardiogram (ECG) was obtained from the limb electrodes. After setting the sheath introducer at the right jugular vein, a quadpolar electrode catheter (3 French, SMC-304; Physio-Tech, Tokyo, Japan) was inserted and positioned at the atrial septum by observing the atrial electrocardiogram and surface lead II ECG, where the peaks of the A-wave and P-wave overlapped. 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). Spontaneously occurring premature atrial contractions (PAC) and paroxysmal AF were monitored before electrophysiological testing.
The atrial effective refractory period (AERP) was measured using a stimulator (SEN-7203, Nihon Kohden) after the equilibration period of 20–60 min. The stimulation pulses were programmed to have a rectangular shape with 1–2 V stimulation pulses (approximately twice the threshold voltage) and a 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 late atrial diastole, the coupling interval was successively shortened until an electrical response was no longer elicited. The AERP is defined as the shortest coupling interval producing an electrical response.
AF was induced by burst pacing (5V 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 with an irregular ECG baseline, the duration of which was measured using atrial electrography. AF was induced ten times, and its duration and cycle length were determined using an atrial electrogram.
Anatomical and Histological AssessmentsFor anatomical assessment, the rats were euthanized after electrophysiological testing and their hearts were immediately removed and thoroughly washed with cold saline to eliminate blood contamination. The hearts were dissected into the atria, left ventricle, and right ventricle, and weighed. The thicknesses of the ventricular septum and the left and right ventricular walls were measured using calipers. For histological assessment, the hearts were rapidly excised from the rats without electrophysiological testing under pentobarbital anesthesia. The heart was washed thoroughly with cold saline to remove contaminating blood and then separated into four parts: the right and left atria and the right and left ventricles. Sections were fixed in 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 hematoxylin-eosin and Elastica–Masson stains. Histopathological findings in cardiac tissues were confirmed by microscopic examination. Cellular changes in necrosis include loss of nuclei and hypereosinophilic cytoplasm on samples with hematoxylin and eosin stain, which can be seen as a darker stain of the cytoplasm.
DrugsAldosterone (Sigma-Aldrich, St. Louis, MO, U.S.A.) was dissolved in polyethylene glycol 400. Pentobarbital sodium (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was dissolved in saline solution.
Statistical AnalysisAll data are expressed as mean ± standard error of the mean (S.E.M.). The statistical significance of the parameters among the four groups was evaluated using the Tukey–Kramer test. Statistical significance was set at p < 0.05.
Figure 1 shows typical traces of surface lead II ECGs in Sham, Sham + Aldo, AVS, and AVS + Aldo rats, and Table 1 summarizes the electrophysiological and hemodynamic parameters in these groups. The PR and QT intervals in Sham + Aldo and AVS + Aldo rats were significantly longer than those in Sham and AVS rats. The P-wave duration in AVS + Aldo rats tended to be longer than that in AVS rats (p = 0.083) and was significantly longer than that in Sham + Aldo rats. There were no significant differences in the QRS width between the groups with and without aldosterone. Diastolic blood pressure in rats that underwent AVS was significantly lower than that in rats that did not. There were no significant differences in the systolic or diastolic blood pressure between the groups with and without aldosterone.
| Sham | Sham +Aldo | AVS | AVS +Aldo | |
|---|---|---|---|---|
| n | 6 | 8 | 7 | 6 |
| Electrophysiological parameters | ||||
| P-wave duration (ms) | 15 ± 1 | 17 ± 1 | 17 ± 0 | 20 ± 1† |
| PR interval (ms) | 41 ± 2 | 49 ± 1** | 43 ± 1 | 52 ± 3** |
| QRS width (ms) | 17 ± 1 | 18 ± 1 | 18 ± 1 | 20 ± 1 |
| QT interval (ms) | 66 ± 3 | 88 ± 6** | 69 ± 2 | 90 ± 5* |
| R-wave amplitude (mV) | 0.6 ± 0.1 | 0.5 ± 0.1 | 0.9 ± 0.0† | 0.9 ± 0.1†† |
| Hemodynamic parameters | ||||
| Heart rate (BPM) | 434 ± 15 | 399 ± 11 | 461 ± 3 | 416 ± 21 |
| Systolic BP (mmHg) | 156 ± 5 | 152 ± 6 | 159 ± 3 | 144 ± 6 |
| Diastolic BP (mmHg) | 116 ± 4 | 110 ± 4 | 96 ± 2†† | 87 ± 6†† |
Data are means ± S.E.M. BP, blood pressure. * p < 0.05, ** p < 0.01 vs. each animal model without aldosterone (Sham vs. Sham + Aldo rats and AVS vs. AVS + Aldo rats). †p < 0.05, ††p < 0.01 vs. each animal model without AVS operation (Sham vs. AVS rats and Sham + Aldo vs. AVS + Aldo rats).
The heart tissue weights and wall thicknesses of the rats are summarized in Table 2. Heart weight, heart/body weight ratio, atrial weight, and right and left ventricular weights in rats that underwent AVS were significantly greater than those in rats that did not undergo surgery. The atrial and ventricular weights in rats receiving aldosterone tended to be greater than those without receiving it both in the Sham and AVS groups, although their differences were statistically insignificant. There were no significant differences in wall thickness among the four experimental groups, except for the ventricular septal wall thickness in the Sham + Aldo group compared with that in the Sham group.
| Sham | Sham +Aldo | AVS | AVS +Aldo | |
|---|---|---|---|---|
| n | 6 | 8 | 7 | 6 |
| Body weight (g) | 279 ± 6 | 279 ± 7 | 274 ± 6 | 290 ± 7 |
| Tissue weights | ||||
| Heart weight (mg) | 772 ± 20 | 816 ± 16 | 1135 ± 29††† | 1187 ± 54††† |
| Heart (mg)/body (g) weight ratio | 2.8 ± 0.1 | 2.9 ± 0.0 | 4.1 ± 0.1††† | 4.1 ± 0.1††† |
| Atrial weight (mg) | 63 ± 3 | 72 ± 2 | 119 ± 5††† | 132 ± 10††† |
| RV weight (mg) | 134 ± 6 | 147 ± 6 | 194 ± 4††† | 213 ± 11††† |
| LV weight (mg) | 575 ± 15 | 597 ± 10 | 823 ± 23††† | 842 ± 37††† |
| Tissue thickness | ||||
| RV wall thickness (mm) | 1.4 ± 0.1 | 1.6 ± 0.1 | 1.7 ± 0.1 | 1.6 ± 0.1 |
| Septal thickness (mm) | 2.6 ± 0.1 | 3.0 ± 0.1* | 2.9 ± 0.1 | 3.1 ± 0.2 |
| LV wall thickness (mm) | 3.4 ± 0.1 | 3.6 ± 0.1 | 3.6 ± 0.1 | 3.7 ± 0.1 |
Data are means ± S.E.M. RV, right ventricle, LV, left ventricle. * p < 0.05 vs. animal model without aldosterone (Sham vs. Sham + Aldo rats). †††p < 0.001 vs. each animal model without AVS operation (Sham vs. AVS rats and Sham + Aldo vs. AVS + Aldo rats).
Typical photomicrographs of the right and left atrial tissues are shown in Figs. 2A and 2B, respectively, and the number of individuals with histological findings is summarized in Table 3. Myocardial hypertrophy and interstitial fibrosis were detected in the right and left atria of the rats that underwent AVS. These characteristics did not differ between groups with and without aldosterone. In contrast to the atria, ventricular fibrosis was more often observed in rats receiving aldosterone than that without receiving it both in the Sham and AVS groups, which is summarized in Supplementary Fig. 1 and Supplementary Table 1.
| Histopathological findings | Sham | Sham +Aldo | AVS | AVS +Aldo |
|---|---|---|---|---|
| n | 6 | 7 | 6 | 6 |
| Right atrium | ||||
| Hypertrophy | - | - | 3/6 | 4/6 |
| Fibrosis, interstitium/perivessel | - | - | 3/6 | 2/6 |
| Fibrosis, focal | - | - | - | - |
| Necrosis with inflammatory cell infiltration | - | - | - | - |
| Left atrium | ||||
| Hypertrophy | - | - | 4/6 | 5/6 |
| Fibrosis, interstitium/perivessel | - | - | 2/6 | 2/6 |
| Fibrosis, focal | - | - | - | - |
| Necrosis with inflammatory cell infiltration | - | 1/7 | - | - |
Data represent the number of individuals in which each histological finding was observed. (-), no individuals observed.
Figure 3A shows representative electrograms of AF induced by burst pacing in an AVS + Aldo rat, and the AF duration and cycle length are summarized in Fig. 3B. There was no significant difference in AF duration between the Sham and AVS rats. The AF duration in AVS + Aldo rats was approximately eight times longer than that in AVS rats. AF lasting longer than 10 min was detected in three out of six AVS + Aldo rats, whereas it was not observed in any of the other three experimental groups. As shown in Fig. 3C, there was no significant difference in the AERP among the four experimental groups.
(A) Representative electrocardiograms of AF induced by burst pacing in AVS + Aldo Rat, (B) Duration (left) and cycle length (right) of burst pacing-induced AF, and (C) the AERP in basal pacing cycle lengths of 120 ms (left) and 100 ms (right) in the Sham, Sham + Aldo, AVS, and AVS + Aldo groups, respectively. Data are means ± S.E.M. *** p < 0.001 vs. AVS, †††p < 0.001 vs. Sham, ‡‡‡p < 0.001 vs. Sham + Aldo rats. ECG, electrocardiogram; RA, right atrial electrocardiogram; AERP, atrial effective refractory period; CL, cycle length.
Figures 4A and 4B show typical traces of supraventricular arrhythmias. The incidences of PAC and paroxysmal AF are summarized in Table 4. PACs were detected in one out of six sham rats and two out of seven AVS rats, whereas paroxysmal AF was not observed in these experimental groups. PACs were detected in all Sham + Aldo and AVS + Aldo rats, whereas paroxysmal AF was detected in one of eight Sham + Aldo rats and five of six AVS + Aldo rats.
(A) Typical electrograms of premature atrial contraction. The black arrows represent the occurrence of premature atrial contraction. The premature atrial contraction occurred at intervals of 173 ms after the sinus beat of the 200 ms cycle. The premature atrial contractions were observed 8 times per 10 min. (B) Typical electrograms of spontaneously induced paroxysmal AF. ECG, electrocardiogram; RA, right atrial electrocardiogram.
| Sham | Sham +Aldo | AVS | AVS +Aldo | |
|---|---|---|---|---|
| Premature atrial contraction | 1/6 (17%) | 8/8 (100%) | 2/7 (29%) | 6/6 (100%) |
| Paroxysmal AF | 0/6 (0%) | 1/8 (13%) | 0/7 (0%) | 5/6 (83%) |
Data represent the incidence of supraventricular arrhythmias observed during the experimental condition of the absence of artificial stimuli for the atria, including electrical pacing. AF, atrial fibrillation.
In this study, chronic administration of aldosterone potently prolonged the AF duration in rats during the atrial remodeling induced by 4 weeks of cardiac volume overload. Meanwhile, aldosterone hardly affected AVS-induced atrial structural changes, including atrial cell hypertrophy and fibrosis and atrial enlargement, whereas the hormone markedly increased the incidence of atrial arrhythmias, namely PAC and paroxysmal AF, which were not induced by chronic volume overload alone due to AVS.
Rationale of the Dose of Aldosterone and Its Electrophysiological EffectsIn this study, chronic administration of aldosterone at 0.5 µg/h to the Sham rats for 4 weeks significantly prolonged the PR and QT intervals without elevating blood pressure (Table 1). ECG changes may be consistent with those of primary aldosteronism.8) Meanwhile, in sham rats receiving aldosterone, atrial morphology and histology were not modified by aldosterone, in contrast to ventricular fibrosis (Fig. 2, Table 3, Supplementary Fig. 1, Supplementary Table 1). A previous report has shown that aldosterone at 1.5 µg/h for 8 weeks in healthy rats induced atrial electrophysical and histological changes, such as prolonged P-wave duration, increased atrial fibroblasts and atrial interstitial collagen, hypertrophy of atrial myocytes, and locally disturbed atrial conduction.9) Thus, the dose of aldosterone in this study had little effect on the atria compared to the ventricles in the normal heart.
Although the chronic effects of aldosterone 0.5 µg/h in the AVS rats were similar to its effects in the sham rats (Table 1), the P-wave duration in the AVS + Aldo rats was longer than that in the AVS rats (p = 0.083), as shown in Fig. 1, which is meaningful for understanding atrial arrhythmogenic properties. There was no significant difference in atrial weight between the AVS and AVS + Aldo rats (Table 2). Since AVS-induced atrial cell hypertrophy and interstitial fibrosis, as observed in our previous studies,6,7) were not modified by chronic administration of aldosterone (Fig. 2, Table 3), intra-atrial conduction disturbance might be a cause of the prolonged P-wave duration.
The current dose of aldosterone (0.5 µg/h) is speculated to provide similar levels of blood aldosterone concentrations in patients with primary aldosteronism (approximately 150–1500 pg/mL),10) based on the previous report using 1.5 µg/h of aldosterone in rats (2339 pg/mL).9)
Promotion of Induction of Atrial Arrhythmias in the AVS Rats by AldosteroneChronic administration of aldosterone to AVS rats markedly prolonged the AF duration induced by burst pacing (average AF duration, >100 s), as shown in Fig. 3B (AVS + Aldo group); however, such effects were not observed in Sham + Aldo rats, indicating that the AF-promoting effect of aldosterone is more likely to be exerted in the presence of pathological conditions of volume overload. Previous reports using other rat disease models, such as myocardial infarction or pulmonary hypertension, have shown that the average AF duration was within 1 min, despite having reentrant substrates such as shortened ERPs and conduction delays.11–13) In our recent study, the average duration of AF was 15 s in the same AVS model with chronic volume overload for 3 months.7) Thus, the present results suggest that aldosterone acts as a robust promoter of AF under the pathological conditions of chronic volume overload in rats.
AF cycle length or AERP is an important indicator for understanding the sustainability of AF in this animal model. Since the indices were unaffected by aldosterone (Figs. 3B, 3C), the mechanism of AF prolongation observed in AVS + Aldo rats cannot be explained by the effects of the reentrant substrate alone, although the P-wave duration in AVS + Aldo rats was longer than that in AVS rats. A more important observation from this study was that PAC and paroxysmal AF, as shown in Fig. 4, occurred spontaneously in most AVS + Aldo rats, the incidence of which is summarized in Table 4. These results suggest that chronic aldosterone administration strongly and repeatedly generates arrhythmogenic triggers in the volume-overloaded atria. We speculate that one of the mechanisms exerting a longer duration of burst pacing-induced AF in AVS + Aldo rats is associated with a “Trigger-based mechanism” newly proposed by Inoue et al.,14) in which triggers play a major role in the AF persistence. This mechanism is based on electrophysiological analysis of persistent AF patients with immediate recurrence of AF; the activity of the AF triggers is so elevated that AF is initiated frequently, leading to “new” AF before the “old” AF terminates, which results in persistent AF.
Possible Mechanism of Aldosterone-Induced AF Prolongation in the AVS Rats and Clinical ImplicationIn this study, 4 weeks of volume overload by AVS itself, as well as aldosterone administration, had little effect on atrial refractoriness such as AERP. In our previous study using a 12-week AVS model, the mRNA levels of Kv and Kir channels in the right atrium were mostly downregulated, and the associated prolongation of action potential duration and AERP were observed.7) Thus, such a change in ion channel expression may not induce atrial electrical changes in the early phase of AVS-induced atrial remodeling.
Aldosterone has been demonstrated to increase ICa,L, ICa,T, and INa and decrease Ito via its mineralocorticoid receptor-mediated action over 24 h in ventricular myocytes in vitro15–18) and in vivo19,20) studies, which may be related to the prolongation of PR and QT intervals on ECG (Table 1). Since a dose of aldosterone three times higher (1.5 µg/h) than that used in the present study has been reported to have no effect on AERP,9) the aldosterone at a dose of 0.5 µg/h used in this study is unlikely to interact with ion channels associated with atrial refractoriness. In contrast, previous studies have reported that aldosterone increases the occurrence of delayed afterdepolarization (DADs) in ventricular myocytes isolated from mice exposed to aldosterone for 48 h,21) which may partly explain the cellular mechanisms of aldosterone-induced PAC in this study. In addition, an association between the downregulation of FK506-binding proteins (FKBP12 and 12.6) and regulatory proteins of the cardiac ryanodine receptor macromolecular complex by aldosterone has been suggested. This may lead to aberrant Ca2+ release during diastole.21)
Aldosterone-induced PAC can be a trigger for AF, and the structural changes within 4 weeks of the AVS surgery, such as interstitial fibrosis, can provide the reentrant substrate necessary for AF maintenance. Thus, the coexistence of these two factors in AVS + Aldo rats may have led to the development of paroxysmal AF and sustained AF induced by burst-pacing, which can explain that aldosterone promotes AF in the early stages of atrial remodeling induced by volume overload. In addition, the present results suggest that patients with primary aldosteronism may have a higher probability of developing AF when atrial remodeling begins due to cardiac diseases, such as heart failure. This finding may also provide important information for elucidating the mechanisms underlying aldosterone- or heart failure-related AF.
Aldosterone robustly promotes AF, particularly in the presence of chronic volume overload.
The authors thank Dr. Tetsuo Kuze and Mr. Takeshi Hasegawa at TOA EIYO LTD. for technical advice. This work was supported in part by JSPS KAKENHI Grant Numbers JP15K08598 (to A.T.) and JP23K07564 (to M.A.).
The authors declare no conflict of interest.
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