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Inward Rectifier K+ Currents Contribute to the Proarrhythmic Electrical Phenotype of Atria Overexpressing Cyclic Adenosine Monophosphate Response Element Modulator Isoform CREM‐IbΔC‐X

BACKGROUND Transgenic mice (TG) with heart‐directed overexpresion of the isoform of the transcription factor cyclic adenosine monophosphate response element modulator (CREM), CREM‐IbΔC‐X, display spontaneous atrial fibrillation (AF) and action potential prolongation. The remodeling of the underlying ionic currents remains unknown. Here, we investigated the regulatory role of CREM‐IbΔC‐X on the expression of K+ channel subunits and the corresponding K+ currents in relation to AF onset in TG atrial myocytes. METHODS AND RESULTS ECG recordings documented the absence or presence of AF in 6‐week‐old (before AF onset) and 12‐week‐old TG (after AF onset) and wild‐type littermate mice before atria removal to perform patch clamp, contractility, and biochemical experiments. In TG atrial myocytes, we found reduced repolarization reserve K+ currents attributed to a decrease of transiently outward current and inward rectifier K+ current with phenotype progression, and of acetylcholine‐activated K+ current, age independent. The molecular determinants of these changes were lower mRNA levels of Kcnd2/3, Kcnip2, Kcnj2/4, and Kcnj3/5 and decreased protein levels of K+ channel interacting protein 2 (KChIP2 ), Kir2.1/3, and Kir3.1/4, respectively. After AF onset, inward rectifier K+ current contributed less to action potential repolarization, in line with the absence of outward current component, whereas the acetylcholine‐induced action potential shortening before AF onset (6‐week‐old TG mice) was smaller than in wild‐type and 12‐week‐old TG mice. Atrial force of contraction measured under combined vagal‐sympathetic stimulation revealed increased sensitivity to isoprenaline irrespective of AF onset in TG. Moreover, we identified Kcnd2, Kcnd3, Kcnj3, and Kcnh2 as novel CREM‐target genes. CONCLUSIONS Our study links the activation of cyclic adenosine monophosphate response element–mediated transcription to the proarrhythmogenic electrical remodeling of atrial inward rectifier K+ currents with a role in action potential duration, resting membrane stability, and vagal control of the electrical activity.


In vivo ECG measurements
Electrocardiogram (ECG) measurements were performed in isoflurane anesthetized mice to confirm the absence (in WT6w, TG6w and WT12w mice) and the presence of persistent atrial fibrillation (in TG12w). Mice were anesthetized with isoflurane (1.2% v/v) and nitrous oxide (66% v/v) while being placed on a warmed pad in supine position. Needle electrodes were attached to obtain limb leads (Einthoven). Short ECGs were recorded for 1 minute after achieving deep anesthesia (in 5 minutes) using a PowerLab hardware and LabChart Pro software (ADInstruments, Bella Vista, Australia). A section of 20 seconds at steady state of the ECG recordings were analyzed offline using Heart Rate Variability module (HRV) to measure the following parameters: mean heart rate (HR), standard deviation of NN intervals (SDNN), root mean square of the successive differences between neighboring RR intervals (RMSSD). After ECG measurements the hearts were used for cell isolation or biochemical assays.

Tissue isolation
The hearts were removed in ice cold calcium free Tyrode's solution. Both atria were quickly cut from the heart, frozen in liquid nitrogen and stored for further analysis using qRT-PCR, Western blot and Chromatin immunoprecipitation (ChIP) assays.

Isolation of atrial cardiomyocytes
Atrial cardiomyocytes were isolated from all 4 groups of mice, using the retrograde perfusion of the hearts to digest the connective tissue with collagenase Type II (230U/mg; Worthington, Lakewood, NJ, USA) as described before 16 . The TG mice exhibiting AF at the age of 5-7 weeks were randomly detected with low incidence. Due to the small number and the aim of our study they were not included or sufficient for further investigation. Prior to cell isolation, it was essential to perform ECG measurements on all mice to test for AF presence or absence for grouping the data after analysis, therefore, not all experimenters were blinded.
Moreover, due to the strong atrial phenotype, the quality of cell isolation was poor (few or no cells) in TG mice, meaning that more TG than WT mice had to be sacrificed for this study.
To measure the total outward K + currents, the cells were clamped at -80 mV followed by a 10 ms prepulse to -40 mV to inactivate the sodium channel and a series of 500 ms voltage steps from -80 to +70 mV in 10 mV increment. The depolarizing step was followed by a 300 ms pulse to -120 mV for assessing the inward tail currents flowing through the delayed rectifier currents, including HERG current (IKr). IKtot and IKend current amplitudes were measured as the peak current or the current at the end of the depolarizing pulse minus the mean current at -40 mV. The IKtail current amplitude was measured as the current at peak minus the current at the end of the 300 ms pulse to -120 mV. The transient outward channel (Ito) was inactivated using a 180 ms prepulse to -40 mV followed by a 5 s voltage steps from -40 to +70 mV in 10 mV increment. The Ito currents were obtained after offline subtraction of the depolarizing steps of the two voltage protocols and the amplitudes were measured as peak current minus the current at the end for the subtracted trace. The inward rectifier IK1 current was measured as the current at the end of the 500 ms pulses between -120 and -10 mV from -40 mV holding voltage. To assess the BaCl2 and ACh sensitive currents, a voltage ramp between -120 to +60 mV for 1 second, from the holding voltage of -40 mV was applied at 30 seconds interval. Action potentials (AP) were triggered at 1 Hz frequency with a suprathreshold current stimulus of 600 -1000 pA amplitude and 3 -6 ms duration. Three to five consecutive AP traces at steady state were averaged, and action potential amplitude, slope, and durations from the peak to 20, 50, 70 and 90 % repolarization using a macro for APD analysis designed by Dr. Jan S. Schulte (University of Münster, Germany) 21 for Origin 8.1 (OriginLab Corporation, Northampton, MA, USA).

Western blot
Atria from six to eight mice have been used from each group. Both left and right atria were

Quantitative real time PCR (qRT-PCR)
Eight animals have been used from each group. Total RNA was purified using TRIzol (15596018, ThermoFisher Scientific) according to the manufacturer instructions, and 1 g was reversely transcribed to cDNA that served as input for real-time PCR. The primers used

Measurement of atrial contraction
Left atria of all 4 experimental groups of mice were dissected in oxygenated ice cold physiological solution and attached to a force transducer for recording isometric contractions electrically driven by field stimulation at 1 Hz using suprathreshold rectangular pulses of 5 ms duration, as described previously 27 . Before the experimental protocol, atria were pre-stretched with a force of 5 mN and were allowed to equilibrate at 37°C for at least 30 minutes in a bath solution containing (in mM): 120 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 22.6 NaHCO3, 0.42 NaH2PO4, 0.28 ascorbic acid, 0.05 Na2EDTA and 5.5 glucose, adjusted to pH 7.4 with NaOH.