Frequent Premature Atrial Contractions Lead to Adverse Atrial Remodelling and Atrial Fibrillation in a Swine Model

Abstract

Background:

Frequent premature atrial complexes (PACs) are associated with future incident atrial fibrillation (AF), but whether PACs contribute to developing AF through adverse atrial remodelling has not been studied. This study aimed to explore the impact of frequent PACs from different sites on atrial remodelling in a swine model.

Methods:

Forty swine underwent baseline electrophysiologic studies and echocardiography followed by pacemaker implantations and paced PACs (50% burden) at a 250ms coupling intervals for 16 weeks in 4 groups: (1) lateral left atrium (LA) PACs via the coronary sinus (Lat-PAC; n=10); (2) interatrial septal PACs (Sep-PAC; n=10); (3) regular LA pacing at 130 beats/min (Reg-130, n=10); and (4) controls without PACs (CTRL, n=10). At the terminal study, repeat studies were performed followed by sacrifice, tissue histology and molecular analyses focusing on fibrotic pathways.

Results:

Lat-PACs were associated with a longer P-wave duration (93.0±9.0ms vs. 74.2±8.2ms vs. 58.8±7.6ms; p<0.001) and greater echocardiographic mechanical dyssynchrony (57.5±11.6ms vs. 35.7±13.0ms vs. 24.4±11.1ms; p<0.001) compared to Sep-PACs and CTRLs. After 16 weeks, Lat-PACs led to slower LA conduction velocity (1.1±0.2m/s vs. Sep-PAC 1.3±0.2m/s vs. vs. Reg-130 1.3±0.1m/s vs. CTRL 1.5±0.2m/s; p<0.001) without significant change in atrial ERP. The Lat-PAC group had a significantly increased in %LA fibrosis and up-regulated levels of extra cellular matrix proteins (lysyl oxidase, and collagen 1 and 8) as well as TGF-β1 signaling proteins (latent and monomer TGF-β1, and phosphorylation/total ratio of SMAD2/3) (p<0.05). The Lat-PAC group had the longest inducible AF duration (terminal–baseline: 131[IQR 30, 192] secs vs. Sep-PAC 16[6, 26] secs vs. Reg-130 22[11, 64] secs vs. CTRL −1[−16, 7] secs; p<0.001).

Conclusion:

In this swine model, frequent PACs resulted in adverse atrial structural remodelling with a heightened propensity to AF. PACs originating from the Lateral LA produced a greater atrial remodelling and longer induced AF duration than the septal origin PACs. These data provide evidence that frequent PACs can cause adverse atrial remodelling as well as AF and that the location of ectopic PACs may be clinically meaningful.

Introduction

Atrial fibrillation (AF) has been the most common arrhythmia encountered in clinical practice.^1–3 Premature atrial complexes (PACs) arising from the pulmonary veins are responsible for the initiation of AF;^1,2 however, maintenance of AF requires a ‘vulnerable’ atrial substrate. Numerous factors may contribute to the development of atrial remodelling, such as structural heart disease, sleep apnea, and obesity.³ Population-based cohort studies have demonstrated an association between frequent PACs and incident AF.^4,5 However, whether frequent PACs are only an epiphenomenon or contribute to the development of AF through adverse atrial remodelling or simply by an increased frequency of triggers has not been defined.

Frequent premature ventricular complexes (PVCs) have been shown to trigger a secondary ventricular cardiomyopathy.⁶ We previously demonstrated in a swine model of frequent PVCs that dyssynchronous PVCs lead to ventricular dilatation and a decreased left ventricular (LV) ejection fraction (EF).⁷ Given the association of PACs and AF, it is possible that an analogous process occurs in the atrium, whereby frequent dyssynchronous PACs contribute to atrial remodelling as well as AF maintenance. Therefore, we hypothesized that 1) frequent PACs would cause adverse atrial remodelling and greater AF maintenance, and 2) dyssynchronous PACs arising from the lateral left atrium (LA) would lead to greater atrial dyssynchrony, remodelling, and AF as compared to septal PACs or controls without PACs.

Methods

We studied 40 Yucatan mini-swine (female) with a 50% burden of paced PACs at a 250ms coupling interval in 4 groups: 1) paced PACs from the lateral LA via the coronary sinus (Lat-PAC group; n=10), 2) paced PACs from the interatrial septum (Sep-PAC group; n=10), 3) regular atrial pacing at a faster mean atrial rate (130 beats/min) than during PACs (Reg-130, n=10); and 4) a control group without pacing (CTRL, n=10). All animals underwent a comprehensive electrophysiologic and echocardiographic assessment at baseline and 16 weeks (terminal study), with the protocol summarized in Figure 1A and detailed below. The study was approved and overseen by the Laboratory Animal Resource Center at the University of California, San Francisco, CA. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Figure 1: Study protocol.

(A) Baseline and terminal study flowchart describing the protocol for the four groups of swine in the study (see Methods). Location of the pacing lead on anteroposterior fluoroscopy and gross pathology (yellow arrows) after sacrifice is shown for representative animals in the (B) lateral LA PAC and (C) septal PAC groups. PAC=premature atrial complex; LA = left atrial; LAA = left atrial appendage; LV = left ventricle; RAA = right atrial appendage; ERP = effective refractory period; EP=electrophysiologiy; PPM = pacemaker; TV = tricuspid valve.

Baseline electrophysiology study

After an overnight fast, anaesthesia was induced with an intramuscular injection of ketamine and acepromazine and maintained by inhalation of 1–5% isoflurane (1.5 – 2% for most animals), with each swine mechanically ventilated with 100% oxygen. Femoral venous access was obtained by a percutaneous puncture using the modified Seldinger technique with ultrasound guidance. A temporary, steerable mapping catheter was placed through a femoral vein sheath and atrial effective refractory periods (ERP) were measured at each of the following pacing sites: 1) right atrial free wall; 2) right interatrial septum; and 3) coronary sinus (Figure S1–A). Single paced atrial extrastimuli were delivered at a 400ms coupling interval from the prior atrial complex and decremented by 10ms until loss of capture, with a 5 second pause between extrastimuli; sensed extrastimuli that were delivered to mimic spontaneous PACs. The atrial ERP was defined as the longest coupling interval without atrial capture. After a 10-minute waiting period, AF induction was then attempted 3 times from the 3 atrial sites (total of 9 attempts) with burst atrial pacing (cycle length 50ms of 15 seconds) (Figure S1–B). All atrial pacing, including extrastimulus testing (ERP measurements) and burst pacing (AF induction), was performed using the same stimulus strength (strength 4mA; duration 2ms) for uniformity.

AF sustainability, which was defined as the average of the maximum AF duration at each of the 3 sites, and AF inducibility, which was defined as the percentage of an inducible AF duration of ≧5 seconds among a total of 9 AF induction attempts were assessed, respectively.

Pacemaker implantation and pacing protocol

The right neck was then cut down to expose the external jugular vein. The vein was tied distally, and after an anterior venotomy, two pacemaker leads were introduced directly. One bipolar active fixation lead (Medtronic 5076, Medtronic, Minneapolis, Minnesota) was placed in the right atrial appendage (RAA) for sensing. A second pacing lead was implanted in the distal coronary sinus aiming to maximize atrial dyssynchrony (Lat-PAC) (Figure 1B) or the interatrial septum (Sep-PAC) (Figure 1C). In the regular atrial pacing group (Reg-130), only a single lead was introduced into the distal coronary sinus. A biventricular pacemaker (Syncra or Viva CRT-P, Medtronic, Minneapolis, MN) was used to create paced PACs. The RAA lead was connected to the atrial port for sensing intrinsic atrial depolarizations, and the second lead was attached to the LV port for sequential atrial pacing and preventing oversensing. The right ventricular port was plugged.

After 1 week of recovery, the pacemakers were programmed from sensing-only (ODO) mode to DDD mode to create paced atrial bigeminy (50% PAC burden, Figure S2). For the Reg-130 group, the pacemakers were programmed to the SSI mode to allow constant atrial overdrive pacing at 130 beats/minute. In a preliminary study, the mean atrial rates during sinus rhythm and bigeminal PACs were 89±10 beat/min vs. 121±12 beat/min, respectively (Figure S3). Therefore, regular atrial pacing at 130 beats/min would allow higher atrial rates than those during bigeminal PACs.

For the sensing lead in the RAA, the atrial pacing threshold was set to the minimum level to avoid any unnecessary atrial pacing. The PAC coupling interval (sensed atrioventricular delay on the pacemaker) was programmed to a short coupling interval of 250ms. This aimed to ensure that the PACs were not conducted to the ventricle, thereby negating any potential confounding due to ventricular irregularity. The pacing output was set to ensure atrial capture of the bigeminal PACs, while avoiding ventricular and phrenic nerve capture. Pacemaker interrogations were performed at monthly intervals to confirm effective sensing, atrial capture, and absence of AF. Ten pigs underwent the same baseline and terminal electrophysiology study and echocardiography protocol but not a pacemaker implantation and were included as controls (CTRL, n=10).

Transthoracic echocardiography

Transthoracic echocardiography (TTE, M5Sc-D probe / Vivid E95 system, GE Healthcare, IL, USA) was performed at baseline and then monthly. All swine were sedated with midazolam (0.3–0.5 mg/kg), ketamine (12.5 mg/kg), and inhaled isoflurane (1–5%) by mask. Echo images were then acquired using a 3.5-MHz transducer placed in the left parasternal area in the right lateral decubitus position. The LA area was measured in a blinded fashion using images that were obtained from a modified apical four-chamber view at end-systole, taking care to exclude the LA appendage and pulmonary veins (Figure S4–A). The LA function was quantified by measuring the peak LA reservoir strain using 2D speckle tracking strain analysis software (EchoPAC version 201, GE Healthcare). A longitudinal strain curve was generated during sinus rhythm and gated for the atrial wall motion among 6 LA segments obtained from the modified apical 4-chamber view at a frame rate of ≥ 70 frames per second. The peak LA strain was assessed by measuring the average of the peak longitudinal strain across 6 LA segments using the QRS onset as a reference point (Figure S4–B). The LV function was also quantified by measuring the LV EF using M-mode in the parasternal short-axis view (Figure S5).

Electrical and mechanical atrial dyssynchrony

At baseline, electrical and mechanical atrial dyssynchrony were evaluated during sinus rhythm or regular pacing from the lateral LA pacing leads or interatrial septal pacing leads at 120 beats/min. The maximum P-wave duration was measured to assess the total atrial conduction time (electrical dyssynchrony) between sinus rhythm and that during regular atrial pacing from the septum and coronary sinus (LA). The maximum P-wave duration was measured from the earliest onset to the latest offset in all ECG leads at a sweep speed of 100mm/s (Figure 2A). Measurements were made using digital calipers on an electrophysiologic recording system. Furthermore, 2D speckle tracking strain analysis software (EchoPAC, version 202, GE Vingmed ultrasound) was used to assess mechanical intraatrial dyssynchrony. The difference in the time-to-peak of the earliest and latest activated segments among the 6 LA segments was measured and the mechanical regional incoordination was assessed during sinus rhythm and at the 2 different pacing sites (Figure 2B).

Figure 2: Measurements of LA dyssynchrony.

(A) Electrical dyssynchrony was assessed by measuring the max P-wave duration (total atrial conduction time) per pacing site. The max P-wave duration is measured from the earliest onset to the latest offset in all ECG leads using digital callipers on a polygraph system. (B) Mechanical dyssynchrony was assessed by measuring the regional coordination among 6 LA segments according to the pacing site. The difference in the time-to-peak of the earliest and latest activated segments among the 6 LA segments was measured during regular constant pacing (120ppm) from each pacing lead. Note that during septal pacing, the atrial segments on the septal side have the earliest time-to-peak activation, while during lateral pacing, those on the lateral side show the earliest time-to-peak activation.

Terminal study

After 16 weeks, the electrophysiology study and transthoracic echocardiography were repeated under general anaesthesia using the same protocol as the baseline study. Prior to the study, the pacemakers were turned off for 24 hours to prevent any acute effects of frequent PACs. If burst pacing induced AF did not convert to sinus rhythm spontaneously after 7 mins, electrical cardioversion was performed (this never occurred during baseline study). In addition, a transseptal puncture was undertaken guided by intracardiac echocardiography and fluoroscopy using a deflectable sheath (Agilis, Abbott Technologies, Minneapolis, MN) and BRK needle. After introducing the deflectable sheath into the LA, the mean LA pressure was obtained during sinus rhythm. The mean aortic pressure was also measured via the femoral artery sheath during sinus rhythm. A multipolar grid catheter (HD-Grid, Abbott) with 3–3-3-mm interelectrode spacing was then introduced into the right atrium (RA) as well as LA and high-density three-dimensional electroanatomical maps were created (NavX EnSite Velocity system version 3.0, Abbott) during sinus rhythm (Figure S6). Offline, the distribution of the unipolar electrogram voltage among the entire RA and LA points were also evaluated. Conduction velocity (CV) was assessed on the LA posterior wall using five electrogram pairs from each pacing site for each animal, while blinded to the group. The local activation time was measured perpendicular to the isochrones in areas of least isochronal crowding, with the CV measured as the surface distance between each point pair divided by the difference in the local activation time.

Hemodynamic impact of Non-conducted PAC vs. Conducted PACs

An acute study with five additional swine was conducted to examine the LA pressure during bigeminal non-conducted PACs versus conducted PACs, delivered from the coronary sinus, to better understand the hemodynamic impact of the PACs against a closed mitral valve. The LA pressure and aortic pressure were recorded during (A) sinus rhythm, (B) bigeminal non-conducted PACs at a coupling interval of 250ms, and (C) bigeminal conducted PACs at a coupling interval of 350ms which allowed conduction to the ventricle, using the same methods as the terminal study.

Histological analysis

The swine were euthanized, and full-thickness 2-cm² samples were obtained from the LA posterior wall, LA anterior wall, and RA appendages. Sections were preserved in buffered formalin and embedded with paraffin. Sections from each sample were then stained with Masson’s trichrome and quantification of fibrous tissue was performed on photomicrographs taken using brightfield microscopy. Fibrosis was then quantified by analysis of magnified (×20) images from each section using ImageJ v 1.52 software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, MD, USA, https://imagej.nih.gov/ij/, 1997–2019.), by counting the number of blue-stained pixels. Perivascular areas were avoided. Fibrous tissue content was expressed as a % of field area and averaged across 7 pictures in each LA site. Analyses were performed on coded specimens while blinded to pacing group assignment.

Molecular analysis (Immunoblots)

Five different biological replicates (tissue samples) were generated from the specific regions of the heart. Tissue chunks were harvested, snap frozen, and kept at −80°C until lysis. Tissue pieces (50mg) were aliquoted in 2ml reinforced tubes containing 2.8 mm ceramic beads and lysed with Fisherbrand^™ Bead Mill 24 Homogenizer using a lysis solution containing radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology 9806), protease inhibitors (Sigma-Aldrich P8340) and phosphatase inhibitors (Sigma-Aldrich 4906845001-PhosSTOP). All the lysates were clarified twice with ultracentrifugation, aliquoted and kept at −20°C. Rapid Gold Bicinchoninic acid assay kit (ThermoFisher Scientific) was used to quantify protein concentration. Proteins were denatured at 70°C for 10 minutes in SDS, and 10μg of samples were loaded on either 4–12% NuPAGE Bis-Tris gels or 3–8% NuPAGE Tris-Acetate gels from ThermoFisher Scientific. After gel electrophoresis, proteins were transferred to 0.45μM polyvinylidene difluoride (PVDF) membranes using a wet-transfer system (XCell II^™ Blot Module). Membranes were subsequently blocked with 5% milk for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies. Following primary antibodies were used: Extracellular proteins: Collagen 1 (ab138492, Abcam), Collagen 8 (102–11285, RayBiotech), Fibronectin (ab6328, Abcam), Periostin (ab92460, Abcam), Lox (ab174316, Abcam), Tgfβ related: Tgfβ (3711, Cell Signaling Technology), Tgfβ-r2(ab186838, Abcam), p-Smad2/3 & Smad 2/3(8828 & 8685, Cell Signaling Technology), Internal control: Gapdh (5174, Cell Signaling Technology). Equal protein loading and transfer onto PVDF membranes was verified with Ponceau S staining solution (Cell Signaling Solution 59803). Following the overnight primary antibody incubation, PVDF membranes were washed several times with TBST and incubated with horseradish peroxidase-linked secondary antibody (GE healthcare NA9340 or NA9310) for 1h at room temperature. SuperSignal^™ West Femto Maximum Sensitivity Substrate, an enhanced chemiluminescence reagent from ThermoFisher Scientific (34095), was used to visualize the bands. Semi-quantification (relative abundance) of proteins were conducted by comparing the relative intensity of bands against their respective Gapdh abundance, using ImageJ software. Samples were run, transferred to poly(vinylidene fluoride) (PVDF) membrane, exposed, and developed at the same time to minimize variability in loading and chemiluminescence substrate exposure. Equal protein loading was also validated in the aliquots using Coomassie and UV absorbance in gels. Normality test was performed using Shapiro–Wilk test. The statistical significance was calculated using non-parametric test, Kruskal-Wallis one-way ANOVA, followed by Benjamini, Krieger, & Yekutieli procedure. Statistical analysis for molecular data was performed in R Statistical Software (v 4.3.1).

Statistical analysis

Continuous variables were summarized using mean and standard deviation (if normally distributed), median and interquartile range (if skewed), while categorical variables were summarized as proportions. A comparison of means between continuous variables was performed using either an unpaired t-test or one-way analysis of variance (ANOVA) for normally-distributed data or the Kruskal-Wallis for skewed data. Normality was assessed by using the Shapiro-Wilk test. For ANOVA, a Tukey’s post hoc test was conducted for multiple comparisons. The data analysis was performed using Statistical Package for the Social Sciences for Windows (SPSS version 23, IBM). Two-tailed P values less than 0.05 were considered statistically significant.

Results

Forty swine underwent baseline and terminal EP studies; 5 underwent only acute hemodynamic study. The body weight was similar at baseline (CTRL 34.9±3.3kg vs. Reg-130 32.4±2.2kg vs. Sep-PAC 33.4±4.0kg vs. Lat-PAC 33.8±4.1kg; p=0.51) and at the terminal study (CTRL 48.0±5.7kg vs. Reg-130 46.2±2.6kg vs. Sep-PAC 49.0±2.6kg vs. Lat-PAC 48.0±4.7kg; p=0.50). Thirty swine underwent pacemaker implantation (Reg-130 n=10; Sep-PAC, n=10; Lat-PAC n=10) with atrial pacing/bigeminy from the assigned pacing site successfully established, while 10 swine served as CTRL without PACs.

Atrial dyssynchrony associated with the different PAC models

Baseline electrical atrial dyssynchrony was greater during lateral LA pacing than atrial septal pacing and sinus rhythm (P-wave duration: sinus rhythm 58.8±7.6ms vs. septal pacing 74.2±8.2ms vs. lateral pacing 93.0±9.0ms; p<0.001) (Figure 3A). Mechanical atrial dyssynchrony assessed by a 2D speckle tracking strain analysis was greater during lateral LA pacing than septal pacing and sinus rhythm (sinus rhythm 24.4±11.1ms vs. septal pacing 35.7±13.0ms vs. lateral pacing 57.5±11.6ms; p<0.001) confirming a greater electrical and mechanical dyssynchrony during lateral LA pacing (Figure 3B).

Figure 3: Electrical and mechanical atrial dyssynchrony.

(A) Electrical atrial dyssynchrony as assessed by the max P-wave duration according to the pacing site. (B) Mechanical dyssynchrony as assessed by 2D speckle tracking strain analysis.

Chronic Atrial structural remodelling

The change in the LA area after 16 weeks of paced atrial bigeminy is summarized in Figure 4A. The Lat-PAC group had the greatest increase in the LA area, followed by the Sep-PAC and Reg-130 groups, then the non-paced controls (terminal – baseline; CTRL 0.9±0.4cm² vs. Reg-130 3.0±0.8cm² vs. Sep-PAC 4.1±2.0cm² vs. Lat-PAC 5.9±1.2cm²; p<0.001) (Figure 4A). Changes in the LA area over time between the three groups are shown in Figure S7. A gradual LA enlargement was observed upon establishing PACs in either Sep-PAC or Lat-PAC group. The decrease in the peak reservoir strain was greatest in the Lat-PAC group, followed by the Sep-PAC, then the Reg-130 and CTRL groups (terminal – baseline; CTRL −0.7±4.2% vs. Reg-130 −8.6±3.0% vs. Sep-PAC −12.7±4.1% vs. Lat-PAC −17.3±3.2%; p<0.001) (Figure 4B). Echocardiographic parameters at the terminal study in each group are summarized in Table S1.

Figure 4: LA size and function after frequent PACs.

Change in the (A) LA area and (B) LA peak reservoir strain between the baseline and 16-week terminal studies in each group.

Chronic Atrial electrical remodelling

Overall, the change in average atrial ERP after 16-weeks of atrial bigeminy at the 3 sites (terminal – baseline) did not differ in either the Lat-PAC or Sep-PAC groups. The atrial ERP only decreased in the Reg-130 group (terminal - baseline: CTRL −3.2±23.7ms vs. Reg-130 −24.4±13.7ms vs. Sep-PAC 8.3±36.0ms vs. Lat-PAC −4.0±23.6ms; p=0.05) (Figure 5A). On the posterior LA wall, the Lat-PAC swine had significantly lower CVs at 16 weeks than Reg-130 and CTRL groups (CTRL 1.5±0.2m/s vs. Reg-130 1.3±0.1m/s vs. Sep-PAC 1.3±0.2m/s vs. Lat-PAC 1.1±0.2m/s: p<0.001) (Figure 5B). The distribution of unipolar voltage values in the LA and RA among the entire points is shown in Figure S8. The acquired points for the use of the analysis did not differ between the groups ([LA: CTRL 2016±542 vs. Reg-130 2322±520 vs. Sep-PAC 1786±629 vs. Lat-PAC 2109±648; p=0.47] and [RA: CTRL 2441±673 vs. Reg-130 2083±283 vs. Sep-PAC 2399±834 vs. Lat-PAC 2672±901; p=0.70]). The Lat-PAC group had a higher proportion of ‘low voltage zones’ in both the RA and LA, as defined by a % area below 1.0 mV, than the other groups with a leftward shift in the voltage distribution ([LA: CTRL 1.8±1.7%; vs. Reg-130 4.6±3.2% vs. Sep-PAC 7.4±11.2% vs. Lat-PAC 17.2±11.8%; p=0.02] and ([RA: CTRL 2.7±0.9% vs. Reg-130 7.5±1.9% vs. Sep-PAC 9.2±4.2% vs. Lat-PAC 15.1±8.6%; p=0.01]).

Figure 5: LA electrophysiology after frequent PACs.

(A) Change in the atrial effective refractory period (ERP) between the baseline and 16-week terminal studies in each group. The atrial ERPs are obtained by the average of the ERPs on the right atrial free wall, right atrial mid-septum and coronary sinus. (B) LA posterior wall conduction velocity during the terminal study in each group. LA= left atrial

Chronic change in hemodynamics

In the terminal study, both the Lat-PAC and Sep-PAC groups exhibited a significantly higher mean LA pressure than the CTRL group (Figure S9–A), despite no significant change in the mean aortic pressure (Figure S9–B).

LV function

Overall, the CTRL pigs exhibited a higher mean ventricular heart rate than the PAC pigs but no difference was found between the Sep-PAC and Lat-PAC groups (CTRL 88±11bpm vs. Sep-PAC 65±8bpm vs. Lat-PAC 66±9bpm; p<0.001). PACs were non-conducted to the ventricle in all pigs, and both the Lat-PAC or Sep-PAC groups had no change in the LVEF. In contrast, atrial constant pacing at 130/min for 16 weeks led to a significant small LVEF decline (terminal - baseline: CTRL 0.7±3.3% vs. Reg-130 −6.5±4.9% vs. Sep-PAC 0.5±4.0% vs. Lat-PAC −2.5±6.2%; p=0.006) (Figure S10).

AF induction

After 16 weeks of PACs, the increase in the average duration of induced AF between terminal and baseline study was greatest in the Lat-PAC group (CTRL −1 [IQR −16, 7] secs vs. vs. Reg-130 22 [11, 64] secs vs. Sep-PAC 16 [IQR 6, 26] secs vs. Lat-PAC 131 [IQR 30, 192] secs; p<0.001) (Figure 6A). Furthermore, the increase in the AF inducibility between terminal and baseline study was also greatest in the Lat-PAC group followed by the Sep-PAC and Reg-130 then CTRL (CTRL −2.2±16.2% vs. Reg-130 24.4±18.8% vs. Sep-PAC 32.6±23.2% vs. Lat-PAC 49.3±13.0%; p<0.001) (Figure 6B). All induced AF events were composed of irregular atrial activity and no regular atrial tachyarrhythmias were observed.

Figure 6: AF maintenance after frequent PACs.

(A) AF sustainability: change in duration of induced AF between baseline and 16-week terminal studies in each group. AF sustainability was defined as the average of maximum AF duration obtained on the right atrial free wall, right atrial mid-septum and coronary sinus. Cardioversion was performed after 7 minutes (420 secs) of sustained AF. (B) AF inducibility: change in the percentage of an inducible AF duration of ≧5 seconds among the 9 total AF induction attempts between the baseline and 16-week terminal studies in each group. AF = atrial fibrillation.

Hemodynamic impact of Non-conducted PAC vs. Conducted PACs

Figure S11 summarizes the LA pressure and aortic pressure during non-conducted PACs vs. conducted PACs. Interestingly, the magnitude of the additional “a” wave generated by the PAC atrial contraction was, paradoxically, much smaller during non-conducted (PAC-a in Figure S11–B) than conducted PACs (PAC-a in Figure S11–C), presumably due to less time for atrial filling. Figure S11–D summarizes the LA pressure values of sinus rhythm-a vs. non-conducted PAC-a vs. conducted PAC-a. While the LA pressure during conducted PACs (at a longer coupling interval) is higher than during non-conducted PACs, the difference did not reach statistical significance due to the small sample size (p=0.10).

Histopathology

The histological fibrosis in the LA and RA in each group is summarized in Figure 7. The lateral LA PAC group had the greatest degree of fibrosis, followed by septal PAC and Reg-130 then control pigs on either LA anterior wall (CTRL 3.9±1.7% vs. Reg-130 5.6±1.2% vs. Sep-PAC 5.8±1.6% vs. Lat-PAC 7.7±1.6%; p<0.001) (Figure 7E) or LA posterior wall (CTRL 4.0±1.5% vs. Reg-130 6.2±1.7% vs. Sep-PAC 6.6±1.4% vs. Lat-PAC 8.6±1.0%; p<0.001) (Figure 7F).

Figure 7: Histopathology analysis.

Representative histological slides for the (A) Control, (B) Regular 130, (C) Septal PAC and (D) Lateral left atrial (LA) PAC groups. Differences in the histological %fibrosis in the (E) LA anterior wall, (F) LA posterior wall, and (G) RA appendages by group.

Molecular analysis

The molecular analysis of fibrosis related proteins demonstrated that the chronic PAC model was characterized by upregulation of several extracellular matrix proteins as well as TGF-β1 signalling (Figure 8 and Figure S12). Collagen 1 levels were significantly higher in the lateral LA PAC group than septal PAC or CTRL groups. Collagen 8 was also abundant in the lateral LA PAC group than the control group with a p-value of 0.05, while no differences were observed on fibronectin and periostin proteins. Similarly, Lysyl oxidase (LOX), an essential protein involved in cross linking and stabilization of collagen,⁸ was more abundant in the lateral LA PAC group than the control group with a p-value of 0.05. Similarly, TGF-β signalling related proteins were also higher for lateral LA PAC, with increases in latent and active monomer TGF-β protein, as well as the intracellular second messenger of TGF-ß receptor activation, phosphorylated SMAD2/SMAD3. No statistical differences were observed for the TGF-β receptor type-2 levels. Those results suggested TGF-β1 mediated activation of SMAD signalling with a resultant increase in collagen and LOX proteins in the lateral LA PAC group.

Figure 8: Molecular analysis.

Left panel: Western Blot of extra cellular matrix proteins (pink) and TGFβ1 signaling proteins (yellow) in the control, Septal PAC, and Lateral LA PAC groups. Right panel: Comparison of the specific extra cellular proteins (above) and TGFβ1 signaling proteins (below) by the group.

Discussion

In a swine model of paced PACs we found; 1) frequent PACs lead to atrial dilatation, fibrosis, slow conduction and a longer duration of induced AF, 2) frequent PAC exposure for 16 weeks did not change the atrial ERP, while rapid regular atrial pacing led to a significant decrease, 3) mechanically dyssynchronous PACs from the lateral LA caused a greater degree of electrical and structural fibrotic remodelling than less dysynchronous septal PACs at the same PAC coupling interval, and 4) a resultant fibrotic atriopathy is driven by upregulation and activation of the TGF-β1 signalling pathway (Graphical Abstract). This suggests that frequent PACs can cause fibrotic atrial structural remodelling that leads to a milieu supporting AF.

PACs have been considered to be largely benign. However, recent population-based cohort studies demonstrated an association between frequent PACs and ischemic strokes or incident AF.^4,5,9–12 This relationship appears to be apparent at PAC burden significantly lower than the 50% used in our study, as low as >100 PACs per day¹³ or even the presence of PACs on a single ECG.⁵ Studying the natural history of humans with frequent PACs is difficult because AF may not develop for decades. Moreover, numerous factors such as structural heart disease, hypertension, and/or obesity may serve as the contributors to develop malignant atrial substrate which makes it difficult to evaluate the pure impact of PACs.^2,3 Therefore, our findings in a swine model provide novel insight into those studies that PACs themselves, without other comorbidities, can lead to progressive structural remodelling and AF. Furthermore, the described fibrotic atrial remodelling due to PACs may also play an important role in the progression from early paroxysmal AF to persistent AF.

The LA remodeling secondary to our chronic PAC model was best characterized by development of a fibrotic atrial substrate. LA fibrosis, which alters atrial tissue composition and function, is known as a key determinant of the AF substrate.^{14, 15} Previous case-control studies demonstrated an increased extracellular matrix deposition in patients with lone-AF or AF secondary to mitral valve disease. ^{14, 15} A recent human study demonstrated that extracellular matrix gene expression precedes the onset of AF.¹⁶ In our chronic PAC model, we demonstrated increased fibrosis on histology as well as significant upregulation of collagen and other extracellular matrix proteins, mediated by TGF-ß1 activation of the SMAD (phosphorylated SMAD 2/3) intracellular signalling pathway to stimulate collagen (Col1 and Col8) and extracellular matrix proteins (LOX) by cardiac fibroblasts.^17–20

Another interesting observation in our PAC model was that there was no change in atrial ERP, even after 16 weeks of PACs exposure. This suggests the mechanism of atrial remodelling due to PACs differs from prior atrial tachy-pacing models, in which the dominant effect on atrial remodelling was due to shortening of atrial ERP.^21–23 Notably, our regular rapid atrial pacing animals did have a significant shortening of atrial ERP, consistent with the findings of prior tachy-pacing models. On the other hand, models in which the AF substrate is induced by congestive heart failure,²⁴ obstructive sleep apnea,²⁵ or hypertension²⁶ are more similar to our chronic PAC model, all of which demonstrate structural remodelling with a similar electrophysiologic (slowed/heterogeneous conduction), histologic (increased fibrosis) and molecular (increased TGF-ß1 signalling) fingerprints.

Prior human experiments suggested less acute AF induction with coronary sinus compared to high right atrial PACs,²⁷ however our model is quite different in several respects. First, we examined the effect of chronic PACs on left atrial remodelling at AF induction. Second, we only utilized the coronary sinus in swine to access the vein of Marshall, which is quite large in swine and allows pacing from the base of the left atrial appendage (to avoid the complexity of transeptal puncture and chronic left atrial lead placement with its attendant stroke risk, as illustrated in Figure 1). This positioning is substantially different from pacing in the proximal or distal coronary sinus in humans.

Finally, the coupling interval in this PAC model was chosen to be shorter than the atrioventricular-nodal ERP and negate the impact of a rapid and/or irregular ventricular rate. One can speculate that the impact of atrial contractions during the PACs against a closed mitral valve may have also impacted the remodelling. However, when measuring the LA pressure during non-conducted PAC and conducted PACs, the magnitude of the PAC-a wave generated by the PAC atrial contraction was, paradoxically, much smaller during non-conducted PACs than conducted PACs. We believe the lower pressure during short-coupled, non-conducted PACs, compared to longer coupled conducted PACs, is due to the limited time for any atrial filling. When the LA is relatively empty, whether the mitral valve is open or closed does not seem to impact the acute pressure overload of a non-conducted PAC. Therefore, this short coupling interval PAC model seems to be differed from the mitral valve stenosis model, where the atrium contracts at a time with a high filling pressure.

Clinical implications

This study supports the notion that frequent PACs, particularly dyssynchronous ones arising from the lateral LA, are not simply an epiphenomenon, but may directly cause atrial myopathy and contribute to AF pathogenesis. Moreover, this supports the notion that frequent PACs may alone, in the absence of AF, contribute to atrial myopathy and predispose to stroke.^9–12 While the appropriate management strategy remains unproven, further studies will determine whether early PAC suppression and/or anticoagulation may prevent the development of adverse atrial remodelling and AF as well as reduce the risk of stroke. These data also suggest that PAC frequency is not sufficient as a predictor, but that the PAC location may also be critical to determining future AF risk.

Limitations

The use of a swine model has inherent limitations due to differences in physiology as compared to humans. However, this model enables a prospective assessment of the direct effects of high-burden PACs over a 16-week period under controlled conditions, which would be impossible in humans. While a 50% PAC burden is uncommonly seen in clinical practice, this model allowed us to test whether PACs would lead to atrial remodelling over a realistic 16-week time-period. The control animals were not instrumented to reduce costs, preserve pacemakers/leads, and minimize the number of invasive procedures. However, it is possible the presence of an atrial lead alone could affect atrial remodelling. We observed significant differences between these two PACs groups suggesting that other factors (other than the MV closure) impact the relative effects of PACs on remodelling. Further studies are required to characterize the PAC burden and duration required in humans for the development of atrial myopathy.

Conclusion

In a swine model, high-burden PACs for 16 weeks resulted in an atrial myopathy/atrial remodelling characterized by slow conduction and fibrosis development with a heightened propensity for AF. PACs led to a fibrotic atrial substrate without any change in the atrial ERP, suggesting a process distinct from tachy-pacing induced atrial remodelling. Lateral LA PACs appear to be associated with a greater degree of atrial myopathy/remodelling and longer duration induced AF than septal PACs. These data provide evidence that frequent PACs are not an epiphenomenon of AF and can cause an atrial substrate supporting AF. Whether early suppression of specific dyssynchronous PACs can prevent future AF and/or strokes requires further study.

Clinical perspectives:

What is new?

• Frequent premature atrial contractions (PACs) promote electrical (slow conduction) and structural (enlargement, fibrosis) changes in the atrium.

• These PAC induced structural/electrical changes (remodelling) promote a substrate that more easily maintains atrial fibrillation.

Clinical implications?

• Inhibition of fibrosis through pharmacologic or gene therapy could block the process that leads to the fibrotic atrial myopathy that promotes atrial fibrillation

• Elimination of frequent PACs, through medical therapy or catheter ablation, may represent a novel upstream therapy that reduces the burden of atrial fibrillation in the population.

Nonstandard Abbreviations and Acronyms

AF

atrial fibrillation

bpm

beats per minute

CV

conduction velocity

EF

ejection fraction

ERP

effective refractory period

LA

left atrium

LV

left ventricle

PAC

premature atrial complexes

PVC

premature ventricular complex

RA

right atrium

RAA

right atrial appendage

TGF

transforming growth factor

TTE

transthoracic echocardiography