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.