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.