Rationale: Post-operative atrial fibrillation (POAF) is a common and troublesome complication of cardiac surgery. POAF is generally believed to occur when post-operative triggers act on a pre-existing vulnerable substrate, but the underlying cellular and molecular mechanisms are largely unknown. Objective: To identify cellular POAF-mechanisms in right-atrial samples from patients without a history of atrial fibrillation undergoing open-heart surgery. Methods and Results: Multicellular action potentials, membrane ion-currents (perforated patch-clamp) or simultaneous membrane-current (ruptured patch-clamp) and [Ca 2+ ]i-recordings in atrial cardiomyocytes, along with protein-expression levels in tissue homogenates or cardiomyocytes, were assessed in 265 atrial samples from patients without or with POAF. No indices of electrical, profibrotic, or connexin remodeling were noted in POAF, but Ca 2+ -transient amplitude was smaller while spontaneous sarcoplasmic-reticulum (SR) Ca 2+ -release events and L-type Ca 2+ -current alternans occurred more frequently. Ca 2+ /calmodulin-dependent protein kinase-II (CaMKII) protein-expression, CaMKII-dependent phosphorylation of the cardiac ryanodine-receptor channel type-2 (RyR2) and RyR2 single-channel open-probability were significantly increased in POAF. SR Ca 2+ -content was unchanged in POAF despite greater SR Ca 2+ -leak, with a trend towards increased SR Ca 2+ -ATPase activity. POAF patients also showed stronger expression of activated components of the NLRP3-inflammasome system in atrial whole-tissue homogenates and cardiomyocytes. Acute application of interleukin-1beta caused NLRP3-signaling activation and CaMKII-dependent RyR2/phospholamban hyperphosphorylation in HL-1-cardiomyocytes and enhanced spontaneous SR Ca 2+ -release events in both POAF-cardiomyocytes and HL-1-cardiomyocytes. Computational modeling showed that RyR2-dysfunction and increased SR Ca 2+ -uptake are sufficient to reproduce the Ca 2+ -handling phenotype and indicated an increased risk of proarrhythmic delayed afterdepolarizations in POAF-subjects in response to interleukin-1beta. Conclusions: Pre-existing Ca 2+ -handling abnormalities and activation of NLRP3-inflammasome/CaMKII signaling are evident in atrial cardiomyocytes from patients who subsequently develop POAF. These molecular substrates sensitize cardiomyocytes to spontaneous Ca 2+ -releases and arrhythmogenic afterdepolarizations, particularly upon exposure to inflammatory mediators. Our data reveal a potential cellular and molecular substrate for this important clinical problem.

Chronic β-adrenergic stimulation is regarded as a pivotal step in the progression of heart failure which is associated with a high risk for arrhythmia. The cAMP-dependent transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) mediate transcriptional regulation in response to β-adrenergic stimulation and CREM repressor isoforms are induced after stimulation of the β-adrenoceptor. Here, we investigate whether CREM repressors contribute to the arrhythmogenic remodeling in the heart by analyzing arrhythmogenic alterations in ventricular cardiomyocytes (VCMs) from mice with transgenic expression of the CREM repressor isoform CREM-IbΔC-X (TG). Patch clamp analyses, calcium imaging, immunoblotting and real-time quantitative RT-PCR were conducted to study proarrhythmic alterations in TG VCMs vs. wild-type controls. The percentage of VCMs displaying spontaneous supra-threshold transient-like Ca2+ releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca2+ waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca2+ transport and NCX1 protein level in TG. An increase in INCX and decrease in Ito and its accessory channel subunit KChIP2 was associated with action potential prolongation and an increased proportion of TG VCMs showing early afterdepolarizations. Finally, ventricular extrasystoles were augmented in TG mice underlining the in vivo relevance of our findings. Transgenic expression of CREM-IbΔC-X in mouse VCMs leads to distinct arrhythmogenic alterations. Since CREM repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of CRE-dependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-016-0532-y) contains supplementary material, which is available to authorized users.

Background: Small-conductance Ca 2+ -activated K + (SK)–channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel antiarrhythmic option. However, the regulation of SK-channels in human atrial cardiomyocytes and its modification in patients with AF are poorly understood and were the object of this study. Methods and Results: Apamin-sensitive SK-channel current (I SK ) and action potentials were recorded in human right-atrial cardiomyocytes from sinus rhythm control (Ctl) patients or patients with (long-term persistent) chronic AF (cAF). I SK was significantly higher, and apamin caused larger action potential prolongation in cAF- versus Ctl- cardiomyocytes. Sensitivity analyses in an in silico human atrial cardiomyocyte model identified I K1 and I SK as major regulators of repolarization. Increased I SK in cAF was not associated with increases in mRNA/protein levels of SK-channel subunits in either right- or left-atrial tissue homogenates or right-atrial cardiomyocytes, but the abundance of SK2 at the sarcolemma was larger in cAF versus Ctl in both tissue-slices and cardiomyocytes. Latrunculin-A and primaquine (anterograde and retrograde protein-trafficking inhibitors) eliminated the differences in SK2 membrane levels and I SK between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced I SK amplitude and abolished the difference between Ctl- and cAF-cardiomyocytes, indicating that reduced calmodulin-Thr80 phosphorylation due to increased protein phosphatase-2A levels in the SK-channel complex likely contribute to the greater I SK in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased I SK and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced I SK -upregulation. Conclusions: I SK is upregulated in patients with cAF due to enhanced channel function, mediated by phosphatase-2A-dependent calmodulin-Thr80 dephosphorylation and tachycardia-dependent enhanced trafficking and targeting of SK-channel subunits to the sarcolemma. The observed AF-associated increases in I SK , which promote reentry-stabilizing action potential duration shortening, suggest an important role for SK-channels in AF auto-promotion and provide a rationale for pursuing the antiarrhythmic effects of SK-channel inhibition in humans.

Transgenic mice with heart directed expression of transcription factor CREM‐IbΔC‐X (TG) develop atrial alterations including dilatation, impaired electrical conduction and impaired regulation of intracellular Ca2+ preceding spontaneous‐onset atrial fibrillation. Here, we studied whether CREM‐IbΔC‐X is linked to proarrhythmic alterations in ventricular cardiomyocytes (CMs). Action potentials were prolonged in TG CMs along with reduced mRNA of the Ito underlying channel subunit Kv4.2 as compared to wild‐type (WT) CMs. In TG vs. WT CMs Ca2+ transient amplitude was unaltered under basal conditions but increased under stimulation with isoproterenol. Under both conditions Ca2+ release was retarded but the Ca2+ decay was accelerated in TG vs. WT CMs. The frequency of Ca2+ sparks was enhanced while spark amplitude was reduced in TG vs. WT CMs. Stress stimulation protocols revealed an enhanced rate of spontaneous Ca2+ releases in TG vs. WT CMs. Hence, human cardiac isoform CREM‐IbΔC‐X is linked to an increased susceptibility to arrhythmia possibly by regulating the transcription of genes modulating AP shape and duration and intracellular Ca2+ cycling. Supported by IZKF Münster

Protein abundance in cardiomyocytes is controlled at both transcription and translation levels. While changes in transcription in cardiac proteins such as the sarcoplasmic reticulum Ca 2þ ATPAse (SERCA2a) in response to both physiological and pathological stimuli have been extensively studied, dynamic modulation of translation and the underlying mechanisms have received little attention. We have developed MR-PLISH (mRNA-rRNA Proximity Ligated in Situ Hybridization), a new imaging method for visualization of translation of endogenous specific mRNA transcripts in cardiomyocytes. This method is based on combined use of RNA in situ hybridization and the proximity ligation assay (PLA) technology and allows us to detect single mRNAs interacting with 18S ribosomal RNA (Rn18s). Using this approach, as well as RNAscopeÒ for single molecule detection of mRNA, we examined the effects of depleting intra-SR Ca 2þ with thapsigargin (TG) on transcription and translation of the mRNA for SERCA2a (Atp2a2) in cardiomyocytes. Under baseline conditions both total and 18s rRNA-hybridized Atp2a2 displayed a cell-wide distribution. Treatment with thapsigargin caused no significant changes in abundance or distribution of total Atp2a2 mRNA; however, it resulted in a significant increase in 18s rRNA-hybridized Atp2a2, consistent with an increased rate of active protein synthesis. These results suggest that intra-SR Ca 2þ modulates localized translation of SERCA2a mRNA in cardiomyocytes. Thus, SERCA2A translation maybe subject to regulation via a local feedback loop to compensate for reduction in intra-SR Ca 2þ levels.