Termination of Vernakalant-Resistant Atrial Fibrillation by Inhibition of Small-Conductance Ca2+-Activated K+ Channels in Pigs

Supplemental Digital Content is available in the text.

recorded at baseline and following drug application, normalized to full block by dofetilide and used to establish the IC 50 .
Data were sampled at 10 kHz, 8th order Bessel filter, cut-off frequency 3 kHz, and 80% Rs compensation. Only experiments with a whole cell seal of > 500 MΩ were used.
Data acquisition was performed with the Pulse software (HEKA Elektronik, Lambrecht/Pfalz, Germany).

Tissue samples
After premedication with zoletil pig mixture (250 mg dry tiletamin+zolazepam, 6.5 ml xylazine 20 mg/ml, 1.25 ketamine 100 mg/ml, 2.5 ml butorphanol 10 mg/ml, and 2 ml methadone 10 mg/ml) 1 ml/10 kg given IM, the pigs were euthanized by intravenous injection of pentobarbital 200 mg/ml. The hearts were immediately excised and placed in ice-cold cardioplegic solution (NaCl 110.0 mM, KCl 16.0 mM, MgCl 2 16.0 mM, CaCl 2 1.2 mM, NaHCO3 10.0 mM). Cardiac tissue from each of the four chambers was obtained from long-term AT pigs (n=6) or controls (n=6) and rapidly snap-frozen in liquid nitrogen. Tissue was kept at -80°C for later RNA extraction.
Approximately 40mg of atrial and ventricular tissue specimens were lysed in QIAzol reagent (QIAGEN, Maryland, USA) using a Precellys 24 (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA including small RNAs was purified according to the manufacturer's instruction using the miRNeasy Mini kit (QIAGEN, Hilden, Germany). RNA samples were treated with DNAse. The RNA concentration and purity was determined by spectrophotometry (NanoDrop2000, Thermo Scientific, Wilmington, USA) using the absorbance ratio of A260/A280.

Expression profiling
Reverse transcription (RT) reactions were performed following the manufacturer's instructions using the Precision Thermal Cycler (Struers KEBO Lab, Albertslund, DK) as follows: 25°C for 5 minutes, 42°C for 20 minutes and 75°C for 10 minutes. To assess the levels of mRNAs from the genes KCNN1, KCNN2 and KCNN3 in the samples, quantitative realtime polymerase chain reaction (qPCR) was carried out in duplicates using Custom real-time PCR assays with double dye probe (Taqman style) and PrecisionPLUS MasterMix with ROX (PrimerDesign Ltd., Southampton, UK) according to manufacturer's instruction. GAPDH and GPI were used as reference genes for qPCR normalization and no template controls (NTCs) were run simultaneously to assess contamination. The qPCR steps were conducted with Precision BrightWhite real-time PCR 96-well plates (PrimerDesign Ltd., Southampton, UK) on a CFX Connect Real-Time System (BIO-RAD, Hertfordshire, UK) as follows: 95°C for 2min followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute with FAM dye fluorescence read at the end of each cycle. Threshold cycle (Ct) values were obtained using Bio-Rad CFX96 Managed 3.0 software and assuming a single threshold mode. The data was transferred to a spreadsheet for calculation of ΔCts and the relative expression of the genes in both groups was calculated using the 2 -ΔCt method.