Animal Model Generation
The cardiomyocyte-specific ETV1 null mice, hereby referred to as ETV1-CKO mice (ETV1f/fMyHCCre/+), were generated using CRISPR/Cas9 technique by Saiye Biotechnology Co., LTD (Suzhou, China). Mice were housed and cared at PLA General Hospital Laboratory (Beijing, China). All our experimental procedures were approved by the Ethics Committee of Fujian Provincial Hospital and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (Publication No. 23, revised 1996).
Electrophysiology Study
A subcutaneous Ⅱ-lead ECG of was used to obtain ECG recordings in the in vivo studies. Intraesophageal burst pacing was used to assess the susceptibility to AF. AF was defined as a rapid, fragmented f-waves with irregular AV node conduction and ventricular rhythm lasting for at least 10 s immediately after a burst pacing cycle. The stimulation voltage was 2 times the diastolic threshold, the pulse width was 2 ms. Firstly paced for 10 s with a circle length of 100ms, followed by burst stimulation for 10-12 s with a circle length of 30 ms, until 10 consecutive burst stimulation or AF was induced. The occurrence and duration of AF in each group were observed and recorded.
HE staining and Masson staining
Atrial structure and fibrosis changes were observed by HE staining and Masson staining.
Atrial muscle cells were immersed in 4% paraformaldehyde for 4 h and then transferred to 70% ethanol. Individual lobes of atrial myocyte biopsy material were placed into a treatment cassette, dehydrated through a series of alcohol gradients, and embedded in paraffin blocks. Prior to immunostaining, 4μm tissue sections were dewaxed in xylene and washed in PBS by reduced ethanol rehydration followed by hematoxylin and eosin staining (HE staining). After staining, sections were dehydrated by increasing concentrations of ethanol and xylene.
In the masson staining, the sections were dewaxed to water and fixed in Bouin solution overnight, and rinsed with running water until the sections were colorless. It was counterstained with Weigert's hematoxylin for 10 min. Fully washed and turned blue. Place the spring red liquid for 5 min and rinse slightly in distilled water.1% phosphomolybdate acid was colored for 5min and the microscopic control cytosol and myofibers appeared in bright red. Bright green dye solution was then stained for 3,5,7, and 10 min. 1% Ⅱal acetate 1 min, 95% alcohol and 100% alcohol were dehydrated.
Single Atrial Myocyte Preparation
The Langendorff system was used to prepare single atrial myocytes. The hearts of mice were perfused in Ca2+-free Tyrode’s solution immediately after removal. The formula of Ca2+-free Tyrode solution was composed of NaCl 140 mM, KCl 4 mM, MgCl 21 mM, HEPES 10 mM, with the PH adjusted to 7.40 with NaOH. First, the isolated hearts were perfused with Tyrode buffer solution for 3 min. Then, the isolated heart was perfused with Tyrode’s solution containing 1 mg/ml collagen Ⅱ (Worthington, USA) and 0.25 mg/ml trypsin (Gibco, USA) for 11-13 min. The left and right atrial appendages were removed and digested in the enzyme solution to form single atrial cells. Then, isolated atrial myocytes were stored in a low Ca2+ (0.5 mM) Tyrode solution. Finally, atrial myocytes were moved to a Tyrode’s solution containing 1.8 mM Ca2+ for measuring intracellular Ca2+ and membrane currents/potentials.
Confocal Ca2+ imaging and intracellular Ca2+ Quantification
Confocal calcium imaging was used to detect intracellular Ca2+ changes by recording Ca2+ sparks and Ca2+ waves as well as the timing of Ca2+ peak and Ca2+ decay. Fluo-4 AM (Thermo Fisher Scientific, USA), a non-ratiometric Ca2+ indicator that is commonly used with 488 nm excitation, was added to acute isolated atrial myocytes and incubated in black light avoidance boxes. After de-esterification, a laser-scanning confocal microscope (SP5, Leica Microsystems, German) was used to record spontaneous Ca2+ release events (SCaEs) and the constant rate of Ca2+ cycling under different conditions. Small dishes were placed in the confocal microscope fixation slot and given S1S1 stimulation with 1Hz, and Ca2+ sparks and Ca2+ waves were observed. Then Using Myocyte Calcium & Contractility Recording System (IonOptix, USA), S1S1 stimulation with 1Hz was given to observe Ca2+ release and Ca2+ relevant indicators: Ca2+ release amplitude (△F/F0), half time for Ca2+ release (t to peak 50%), half time for Ca2+ elimination (t to bl 50%), elimination time constant (sin exp tau), etc. Among them, △F/F0 and t to peak 50% reflect Ca2+ release function of sarcoplasmic reticulum, t to bl 50% and sin exp tau reflect the sarcoplasmic reticulum Ca2+ recovery function.
Patch Clamp Experiments
Patch-clamp experiments were used to record action potentials and L-type Ca2+ currents (ICa,L) in atrial myocytes. The Axon Multiclamp 700B Amplifier (Molecular Devices, USA) was connected to the computer. Signal acquisition was completed using a digidata 1440A acquisition interface (Molecular Devices, USA) controlled by pCLAMP programs (version 10.2).
To record the action potential, cells were kept at 37℃ and bathed in a solution that contained NaCl 140 mM, CaCl2 1 mM, MgCl2 1 mM, HEPES 10 mM, KCL 4 mM and glucose 5 mM; it was at pH 7.36 and was adjusted with CsOH. The glass pipettes were filled with a solution of KCl 20 mM, kaspartate 120 mM, MgCl2 1 mM, HEPES 10 mM, Na2ATP 4 mM and glucose 10 mM at pH7.3, which was adjusted with CsOH.
ICa,L were recorded at 37℃. Tetrodotoxin (5µM) was added to block sodium current when recording calcium currents. the pipette solution contained CsCl2 120 mM, CaCl2 1 mM, MgCl2 5 mM, EGTA 11 mM, HEPES 10 mM, and Na2ATP 5 mM, adjusted to pH 7.2 with CsOH. The external solution contained NaCl 140 mM, CaCl2 2 mM, MgCl2 1 mM, KCl 4 mM, HEPES 10, and glucose 10 mM, adjusted to pH 7.4 with CsOH.
Action potentials were recorded after giving a 2.5 ms/1 nA depolarization stimulation under current-clamp mode. Calculate the resting membrane potential (RMP), AP duration at 50 and 90% of repolarization (APD50 and APD90 respectively) and triggered activity. Triggered activity was defined as an unstimulated action potential arising from a DAD or an EAD. The ICa,L was obtained under the voltage clamp mode. ICa,L was recorded from a holding potential of -40 mV to +50 mV with a 10 mV voltage step, and the depolarization pulse was set to 200ms. Current amplitude data of each cell were normalized to its cell capacitance (current density, pA/pF) and current voltage relationship (I-V curve) was plotted. Voltage-dependent activation and steady-state inactivation profiles were fitted to a Boltzmann equation a =1/{1 +exp[-(Vm-V1/2 )/ k]}, where a is the normalized conductance, Vm is the test potential, V½ is the potential at which current is half activated/inactivated, and k is the slope factor. Electrophysiological data were analyzed by Clampfit 10.4 (Axon Instruments) and Origin (Microcal software).
Western Blot Analysis
Western blot was used to investigate the expression of the ion channel proteins Cav1.2, NCX1.1, and the calcium transport-related proteins RyR2, p-RyR2 (site S2808), SERCA2a, and CaMKⅡ. Total protein was obtained from 12-week-old ETV1-CKO mice and WT mice. Protein samples were equally loaded on 8% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with the specified primary antibody overnight at 4℃. Protein blots were performed under primary antibody incubation including Anti-RYR2 (abcam), Anti-p-RyR2 (abcam), anti-CaMKⅡ (CST), antiphosphorylated CaMKⅡ (abcam), Anti-ETV1 (Invitrogen), Anti-NCX1.1 (abcam), and Anti-SERCA2a (abcam). After 1-h incubation with the required secondary antibody, the particular signals were revealed by the chemiluminescence detection reagent Western lightning plus-ECL. Western Blotting data densitometric analysis was conducted using ImageJ software.
Statistical Analysis
Data were processed using Origin 8.5 and Image J software. The data were presented as the mean ± SD, with n denoting the number of cells analysed. One-way ANOVA with Bonferroni post hoc analysis or student’s t-test was used for comparison between groups. Fisher’s exact test was used to compare the incidences of atrial tachyarrhythmia and oscillation in membrane potentials (DADs/EADs) between the groups. SPSS 17.0 was used for analyses, with p<0.05 considered statistically significant.