2.3 Electrophysiological studies
An in vivo electrophysiological study was performed as previously described (Feng et al., 2020; Xu et al., 2012; Xu et al., 2010). After the mice were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (7.5 mg/kg), a 1.2-Fr quadripolar catheter with a 2-mm electrode distance (Unique Medical, Tokyo, Japan) was inserted into the right atrium via the cervical vein. The electrode catheter was placed at a site where the amplitude of the intra-atrial electrogram was higher than that of the intraventricular electrogram. To detect the atrial effective refractory period (AERP), a programmable stimulator (SEN-7203; Nihon Kohden, Tokyo, Japan) was used to deliver approximately twice the threshold current at a 2-ms duration. AERPs were measured at basic cycle lengths involving an eight basic stimulus drive train (S1 × 8) followed by an extra stimulus (S2) at 5-ms decrements. The AERPs were defined as the shortest S1–S2 intervals that could be captured. For AF induction, an atrial burst pacing protocol was performed via two poles on the electrode catheter using a programmable stimulator with 6V amplitude, 20 ms cycle length, 6 ms pulse duration, and 30 s stimulation time. Atrial burst pacing was consecutively performed five times in each mouse to calculate the AF induction rate. The AF induction rate was calculated by dividing the number of times AF was induced by the number of times burst pacing was performed. The AF duration was defined as the interval between the initiation and spontaneous termination of AF. Additionally, an in vitro electrophysiological study was performed using HL-1 cells. HL-1 cells were seeded in precoated multi-electrode array (MEA) dishes (6wellMEA200/30iR-Ti-tcr; Multi Channel Systems, Germany) using the same methods as previously mentioned for HL-1 cell culture. The HL-1 seeded MEA dish was set in a chamber controlled at 37°C and stimulated with ±7500 mV bipolar stimulation at 3 Hz 30 times using MEA2100 (MC-Rack, Multi Channel Systems). Data were captured using LabChart software. Waveforms were generated and filtered at 100 Hz to eliminate electrical noise. The action potential duration (APD) was measured from the waveform of the application. The frequency of abnormal delayed after depolarization (DAD) was a calculation of how many channels out of all the channels showed the occurrence of DADs.