Methods
Clinical study participants
This is a prospective, randomized, double-blind clinical study. The research subjects were patients with NVAF (n = 74) or paroxysmal supraventricular tachycardia (PSVT, n = 73). All patients who met the indications in the guidelines for radiofrequency ablation of arrhythmias were selected, and all underwent radiofrequency ablation for the first time. We conducted a sub-group analysis to evaluate the associations between AF and BRD4. All participants provided written informed consent, and all protocols were performed in accordance with the Medical Science Ethics Committee of the Affiliated Hospital of Guizhou Medical University.
Ascertainment of AF and PSVT
AF was diagnosed by the ECG and/or dynamic ECG results before radiofrequency ablation. AF was defined according to the guidelines [12]. PSVT was diagnosed by the ECG and/or intracardiac electrophysiology results, and include atrioventricular node reentrant tachycardia (AVNRT) and atrioventricular reentrant tachycardia (AVRT).
Research groups and clinical indicators
Patients with AF were defined as the AF group; patients with paroxysmal supraventricular tachycardia were defined as the PSVT group; patients with AF with normal left atrial voltage were defined as the NLAV group; patients with AF with left atrial low-voltage areas (LAVs) were defined as the LAV group; patients with AF who maintained sinus rhythm after ablation were defined as the NreAF group; and patients with AF who had recurrence AF/atrial tachycardia/atrial flutter after ablation were defined as the reAF group.
For all enrolled patients, to detect BRD4 levels by qRT-PCR before radiofrequency ablation, 2 ml of elbow venous blood was extracted in the morning after a state of abstinence from drinking and fasting for at least 8 h. Transthoracic echocardiography was completed in parallel for all candidates prior to radiofrequency ablation, to measure the left atrium anterior and posterior diameter (LAAPD) and left ventricular ejection fraction (LVEF). For patients with NVAF, transesophageal echocardiography was completed in the 24 h prior to radiofrequency ablation to exclude left atrial and/or left atrial appendage thrombosis while measuring the left atrial appendage peak emptying velocity (LAA-PEV). For patients with NVAF, left atrial high-density matrix mapping was performed to observe the bipolar voltage at each point in the left atrium during radiofrequency ablation. After ablation, all patients with AF underwent follow-up at 1, 3, 6, 12, and 18 months by standard 12-lead ECG and/or 72-h dynamic ECG.
BRD4 levels of human peripheral venous blood
We used qRT–PCR to detect the levels of BRD4 [21]. The following primers were used:BRD4:5′-CCCTGAAGCCGTCCACACT-3′ (forward) and 5′-TTCTCAGCTTGAGGTTTCCTTTTC-3′ (reverse); GAPDH:5′-GATCCCTCCAAAATCAAGTGG-3′ (forward) and 5′-GGAGGCATTGCTGATGATCT-3′ (reverse). The 2-∆∆Ctmethod was used to analyze the relative expression of target genes.
Cardiac parameters measured by echocardiography
A Philips iE33 color Doppler ultrasonic diagnostic instrument and quantitative analysis software QLab 8.1 were used to measure LAAPD,LVEF, and LAA-PEV. For transthoracic echocardiography, the two-dimensional probe (S5-1) was selected and the frequency was adjusted to 2.0–3.5 MHz. The LAAPD was measured using the method of two-dimensional radial line through long axial section of the left ventricle parasternal, and the LVEF was calculated using the biplane Simpson method. For transesophageal echocardiography, the real-time three-dimensional transesophageal matrix probe (X7-2t) was selected and the frequency was adjusted to 2–7 MHz, while the LAA-PEV was measured by the pulsed wave doppler(PW) method. In patients with sinus rhythm, the flow spectrum of the left atrial appendage (LAA) was a regular bidirectional waveform:positive waveform was an empty wave,and negative waveform was a filling wave. The peak value of the positive waveform was recorded, and was named LAA-PEV (Fig. 1). In patients with AF, the flow spectrum of the LAA was an irregular serrated waveform, and the peak value of its positive waveform was also selected as LAA-PEV (Fig. 2).
High density matrix mapping of the left atrium
Under sinus rhythm, the Abbott EnSite VelocityTM 5.0 intelligent heart 3D mapping system was used to construct the left atrium 3D model with the assistance of EnSite VelocityTM body surface electrodes. The InquiryTM AFocus IITM 10-pole adjustable bending pulmonary vein catheter was used to detect the bipolar voltage at each point in the left atrium. The WorkMateTM ClarisTMelectrophysiological recording system was used to statistically analyze the local bipolar voltage at each point in the left atrium, and the overall average value of the bipolar voltage at each point in the left atrium was calculated. A local bipolar voltage >0.5 mV in the left atrium was defined as normal voltage, while a bipolar voltage <0.5 mV was defined as a low voltage area (Fig. 3 and Fig. 4). To facilitate recording and description, the left atrium was divided into anterior, apical, and posterior anatomical regions.
Follow-up after ablation
After ablation, all patients with AF completed follow-up at 1, 3, 6, 12, and 18 months by standard 12-lead ECG and/or 72-h dynamic ECG. Three months after ablation was defined as the “blank period.” Recurrence of AF was defined as atrial flutter/AF/tachycardia with a duration >30 s, which was occurring more than 3 months after ablation. In this study, the time point for recurrence assessment was set at 12 months after ablation. If the follow-up time was less than 12 months, the actual follow-up time was recorded. All patients had a follow-up time of at least 6 months.
Animal experiments
All protocols were performed in accordance with the Animal Experiment Ethics Committee of Guizhou Medical University. Thirty-six male SD rats aged 6–8 weeks were equally assigned to three groups according to the principle of randomness and double blindness; the groups included the blank control group (CTL), AF model group (AF + CTL), and AF model + JQ-1 group (AF + JQ-1). SD rats in the AF + JQ-1 group were intravenously injected with (+)-JQ-1 for 3 weeks through the caudal vein; the dosage was 8 mg/kg and the frequency was once a week [19]. All of the SD rats underwent transesophageal electrophysiological examination under anesthesia. SD rats in the AF + CTL and AF + JQ-1 groups also underwent rapid atrial pacing to induce AF for 3 weeks. The ECG trace was continuously recorded by a biologic signal recording and analysis system which filtered out the signals below 10 Hz and above 100 Hz.The AF inducibility
was performed by the transesophageal burst rapid pacing with a stimulating amplitude of 2-fold atrial capture threshold. Four consecutive bursts of rapid electrical stimulation for 30s (20, 30, 40, and 50 Hz) were applied to induce AF with 3-minute pause[22]. AF was defined as an abnormal ECG showing rapid and fragmented P wave with absolute irregular RR intervals (the time elapsed between two successive R-waves of the QRS signal on the electrocardiogram) for at least 2 seconds, and AF could be induced at least two times of five sets of pacing[22-24] (Fig. 5).The mean incidence and duration of AF in each group were recorded. The mean incidence of AF of each rat was the number of times that AF was successfully induced divided by 5, and the unit was percentage (%). The mean duration of AF was the average of the sum of the duration of AF of each rat, and the unit of value was seconds (s).
After the above steps, the SD rats were anesthetized and killed, and the left atrium tissue samples were collected and marked for subsequent experiments. Six left atrium tissue samples were collected from each group, and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into sections (5um thick). Sections of left atrium were stained by Masson’s staining to detect collagen as previously reported[23-24]. The level of cardiac fibrosis was determined by the percentage of the fibrosis area to the total area (% cardiac fibrosis, collagen volume fraction) using the ImageJ software[23-24].Six left atrium tissue samples were collected from each group, and western blot was used to detect the expression levels of BRD4, TGF-β1, P-SMad2/3, SMAD2/3, Smad7, Cx43, and type III collagen in each group.
Statistical analysis
The participant characteristics are summarized as the mean standard deviation (SDs) or percentage and count. Non-normally distributed measurement data were represented by the median (25 to 75 percentiles) and the rank sum test was used for inter-group comparison.χ2 tests were used to examine the association between categorical variables and AF. Spearman correlation test was used for correlation analysis, and t -tests or ANOVA were used for continuous variables. The test-level α = 0.05, GraphPad Prism 5.0 was used to compile the graphs, and ImageJ software was used for gray value analysis. SPSS version 24.0 software was used for all statistical analyses. All probability values for statistical tests used P< 0.05 as a statistically significant value and are two-tailed.