Electrophysiological study in rabbit isolated heart
New Zealand white female rabbits (weight 2.5-3.0 kg) were anesthetized using xylazine (16 mg/kg by IM, Huamu; China) and ketamine (40 mg/kg by IM, CAHG; China). Rabbit hearts were excised and placed in a modified Krebs-Henseleit (K-H) solution (pH=7.4, gassed with 95% O2 and 5% CO2) composed of the following (in mM; Sigma, USA): 118 NaCl, 2.8 KCl, 1.2 KH2PO4, 2.5 CaCl2, 0.5 MgSO4, 2.0 pyruvate, 5.5 glucose, 0.57 Na2EDTA, and 25 NaHCO3. The aorta was cannulated, and the heart was perfused at a rate of 20 mL/min with K-H solution warmed to 37 °C according to the Langendorff system. Coronary perfusion pressure was monitored from a side port of the aortic cannula. A bipolar Teflon-coated electrode was placed on the right atrial appendage to pace the heart with an electrical stimulator (EP-4, St. Jude Medical; USA), 0.6 ms in width and 3–5-fold threshold amplitude. The concentration-response relationships of IVA on heart rate were tested in spontaneously beating hearts, while monophasic APs (MAPs) and arrhythmic events in the atria were obtained from hearts paced at a fixed pacing cycle length (CL) of 350 ms or 570 ms for hearts after SANs were thermo-ablated. After 20-30 minutes of equilibrium, the hearts were randomly assigned into groups treated with either increasing concentrations (0.01-10 µM) of IVA at the spontaneous heart rate (HR) or paced to measure EP parameters at a fixed pacing cycle lengths of 350 ms and 570 ms, or in the absence and presence of low-concentration anemone toxin-II (ATX-II) or acetylcholine (ACh) to increase late sodium current (INa) or vagal tone and mimic the pathological conditions, respectively. ATX-II (2 nM) and ACh (0.3 µM) were confirmed not to cause atrial arrhythmias in any of the hearts studied, similar to our previous report 10.