Objective: Atrial fibrillation (AF) is the most common cardiac arrhythmia, associated with structural and functional atrial remodeling. Mice with cardiomyocyte (CM)-specific expression of the cAMP-dependent transcription factor CREM-IbΔC-X (TG), a model of AF along an extensive atrial remodeling, exhibit heterogeneous, depolarized resting membrane potentials (RMP) and prolonged action potentials (APs) in atrial CMs (ACMs). The pan class I HDAC inhibitor valproate decreased prolonged AP duration (APD), reversed atrial remodeling and delayed onset of AF in TG mice. Here, we investigated if genetic inactivation of the class I isoform HDAC2 (KO) is sufficient to reverse proarrhythmic AP alterations in TG mice using fluorescence microscopy.
Methods: Isolated ACMs from 6-8 weeks old male and female mice were loaded with the membrane potential dye FluoVoltTM to record action potentials via fluorescence microscopy. Fluorescence intensity at 470 nm was recorded with an IonOptix Myocyte Calcium and Contractility System. Atrial tissue homogenates were analysed by RNAseq and western blots.
Results: Baseline fluorescence correlated with changes in ACM size (ACM area in µm2±SD; CTR=1053±173, KO=1013±176, TG=1527±240*, TGxKO=1228±162; *p<0.05 vs CTR) and was increased by 100% in TG vs CTR ACMs (animal/ACM n=7-11/68-93) but reduced by 35% in TGxKO vs TG (n=11/93-103). The amplitude of an AP decreases, if the RMP rises. In TG vs CTR ACMs, the relative AP amplitude was significantly reduced by 37%, but restored in TGxKO vs CTR ACMs. APD50 and APD70 were prolonged by 50% and 35% in TG vs CTR. HDAC2 inactivation in TGxKO led to a significant reduction in APD50 and APD70 by 27% and 24% vs TG. HDAC2 inactivation alone had no effect on APDs (KO vs CTR). RNAseq (n=10/genotype) revealed numerous deregulated ion channel-encoding genes in TG vs CTR. However, HDAC2 inactivation in TGxKO mitigated the downregulation of depolarisation-related Na+ channel subunits Scn5a and Scn10a observed in TG. With respect to repolarisation, the downregulation of K+ channel subunits Kcnb1, Kcnc3, Kcnd3, Kcnj5 and Kcnj11 was attenuated in TGxKO vs TG. Moreover, HDAC2 inactivation in TGxKO even induced the expression of Scn4b and Kcne1 vs CTR, encoding Na+ and K+ channel subunits, respectively, which were not altered in TG vs CTR. In addition, HDAC2 inactivation in TGxKO limited the downregulation of Atp1a1, Atp1b1 and Kcnk3, encoding proteins that stabilize the RMP, which may also contribute to the reduced baseline fluorescence in TGxKO vs TG. When analyzed separately by sex in TGxKO vs TG, males exhibited increased expression of Kcnh2, Scn10a and Scn4b exclusively. In females, however, increased expression of Kcnb1 and Kcne1, as well as reduced expression of Kcne4, Kcnip3, Kcnt2 and Scn7a, was observed. Both sexes shared increased expression of the K+ channel subunits Kcnk3 and Kcnmb4 in TGxKO vs TG. Furthermore, NCX1 protein levels were increased in TG vs CTR, which has been shown to prolong APs. Again, this increase was counter regulated in TGxKO vs TG atria.
Conclusion: Genetic inactivation of HDAC2 normalizes proarrhythmic AP alterations in CREM-IbΔC-X transgenic mice by limiting the dysregulation of various Na+ and K+ channel subunits and NCX1, which control AP kinetics and RMP stability. Consequently, HDAC2 inhibition represents a promising strategy to restore electrophysiological properties in patients with atrial fibrillation.
Supported by the DFG and Deutsche Herzstiftung
