BackgroundAntiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 transcription factor (PITX2) might play an important role in regulating gene expression and electrical function of the adult left atrium (LA).ObjectivesAfter determining LA PITX2 expression in AF patients requiring rhythm control therapy, the authors assessed the effects of Pitx2c on LA electrophysiology and the effect of antiarrhythmic drugs.MethodsLA PITX2 messenger ribonucleic acid (mRNA) levels were measured in 95 patients undergoing thoracoscopic AF ablation. The effects of flecainide, a sodium (Na+)-channel blocker, and d,l-sotalol, a potassium channel blocker, were studied in littermate mice with normal and reduced Pitx2c mRNA by electrophysiological study, optical mapping, and patch clamp studies. PITX2-dependent mechanisms of antiarrhythmic drug action were studied in human embryonic kidney (HEK) cells expressing human Na channels and by modeling human action potentials.ResultsFlecainide 1 μmol/l was more effective in suppressing atrial arrhythmias in atria with reduced Pitx2c mRNA levels (Pitx2c+/–). Resting membrane potential was more depolarized in Pitx2c+/– atria, and TWIK-related acid-sensitive K+ channel 2 (TASK-2) gene and protein expression were decreased. This resulted in enhanced post-repolarization refractoriness and more effective Na-channel inhibition. Defined holding potentials eliminated differences in flecainide’s effects between wild-type and Pitx2c+/– atrial cardiomyocytes. More positive holding potentials replicated the increased effectiveness of flecainide in blocking human Nav1.5 channels in HEK293 cells. Computer modeling reproduced an enhanced effectiveness of Na-channel block when resting membrane potential was slightly depolarized.ConclusionsPITX2 mRNA modulates atrial resting membrane potential and thereby alters the effectiveness of Na-channel blockers. PITX2 and ion channels regulating the resting membrane potential may provide novel targets for antiarrhythmic drug development and companion therapeutics in AF.

Understanding the mechanism of re-entrant arrhythmias in the past 30 years has allowed the development of almost curative therapies for many rhythm disturbances. The complex, polymorphic arrhythmias of atrial fibrillation (AF) and sudden death are, unfortunately, not yet well understood, and hence still in need of adequate therapy. AF contributes markedly to morbidity and mortality in aging Western populations. In the past decade, many genetically altered murine models have been described and characterized. Here, we review genetically altered murine models of AF; powerful tools that will enable a better understanding of the mechanisms of AF and the assessment of novel therapeutic interventions.

Atrial fibrillation (AF) is a common arrhythmia. Better prevention and treatment of AF are needed to reduce AF‐associated morbidity and mortality. There are several major mechanisms that cause AF in patients, including a genetic predisposition to develop AF. Genome‐wide association studies have identified genetic variants associated with AF populations, with the strongest hits clustering on chromosome 4q25, close to the gene for the homeobox transcription factor PITX2. The effect of these common gene variants on cardiac PITX2 mRNA is currently under study. PITX2 protein regulates right–left differentiation of the embryonic heart, thorax and aorta. PITX2 is expressed in the adult left atrium, but much less so in other heart chambers. Pitx2 deficiency results in electrical and structural remodelling, and impaired repair of the heart in murine models, all of which may influence AF through divergent mechanisms. PITX2 levels and single nucleotide polymorphisms on chromosome 4q25 may also be a predictor of the effectiveness of anti‐arrhythmic drug therapy.

BackgroundThe left atrial posterior wall (LAPW) is potentially an important area for the development and maintenance of atrial fibrillation. We assessed whether there are regional electrical differences throughout the murine left atrial myocardium that could underlie regional differences in arrhythmia susceptibility.MethodsWe used high-resolution optical mapping and sharp microelectrode recordings to quantify regional differences in electrical activation and repolarisation within the intact, superfused murine left atrium and quantified regional ion channel mRNA expression by Taqman Low Density Array. We also performed selected cellular electrophysiology experiments to validate regional differences in ion channel function.ResultsSpontaneous ectopic activity was observed during sustained 1Hz pacing in 10/19 intact LA and this was abolished following resection of LAPW (0/19 resected LA, P<0.001). The source of the ectopic activity was the LAPW myocardium, distinct from the pulmonary vein sleeve and LAA, determined by optical mapping. Overall, LAPW action potentials (APs) were ca. 40% longer than the LAA and this region displayed more APD heterogeneity. mRNA expression of Kcna4, Kcnj3 and Kcnj5 was lower in the LAPW myocardium than in the LAA. Cardiomyocytes isolated from the LAPW had decreased Ito and a reduced IKACh current density at both positive and negative test potentials.ConclusionsThe murine LAPW myocardium has a different electrical phenotype and ion channel mRNA expression profile compared with other regions of the LA, and this is associated with increased ectopic activity. If similar regional electrical differences are present in the human LA, then the LAPW may be a potential future target for treatment of atrial fibrillation.

Background: Kinase oxidation is a critical signalling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, type-1 protein kinase A (PKARIα) can be reversibly oxidized, forming interprotein disulfide bonds within the holoenzyme complex. However, the effect of PKARIα disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored. Methods: Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARIα disulfide formation by immunoblot. To determine the impact of disulfide formation on PKARIα catalytic activity and sub-cellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in prkar1 knock-out mouse embryonic fibroblasts, neonatal myocytes or adult LV myocytes isolated from 'redox dead' (Cys17Ser) PKARIα knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes whereas I/R-injury was assessed ex vivo. Results: In both humans and mice, myocardial PKARIα disulfide formation was found to be significantly increased (2-fold in humans, p=0.023; 2.4-fold in mice, p<0.001) in response to I/R in vivo. In mouse LV cardiomyocytes, disulfide-containing PKARIα was not found to impact catalytic activity, but instead led to enhanced A-kinase-anchoring protein (AKAP) binding with preferential localization of the holoenzyme to the lysosome. Redox-dependent regulation of lysosomal two pore channels (TPC) by PKARIα was sufficient to prevent global calcium release from the sarcoplasmic reticulum in LV myocytes, without affecting intrinsic ryanodine receptor leak or phosphorylation. Absence of I/R-induced PKARIα disulfide formation in "redox dead" knock-in mouse hearts resulted in larger infarcts (2-fold, p<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, p<0.001), which was prevented by administering the lysosomal TPC inhibitor Ned-19 at the time of reperfusion. Conclusions: Disulfide-modification targets PKARIα to the lysosome where it acts as a gatekeeper for TPC-mediated triggering of global calcium release. In the post-ischemic heart, this regulatory mechanism is critical for protecting from extensive injury and offers a novel target for the design of cardioprotective therapeutics.