Objectives To determine whether Gal-3 mediates sustained atrial fibrillation (AF)-induced atrial structural and electrical remodeling and contributes to AF perpetuation. Background Galectin-3 (Gal-3) mediates extracellular matrix remodeling in heart failure, but its role in AF progression remains unexplored. Methods We examined intracardiac blood samples from patients with AF (N=55) to identify potential biomarkers of AF recurrence. In a sheep model of tachypacing-induced AF (N=20), we tested the effects of Gal-3 inhibition during AF progression. Results In patients, intracardiac serum Gal-3 levels were greater in persistent than paroxysmal AF and independently predicted atrial tachyarrhythmia recurrences after a single ablation procedure. In the sheep model, both Gal-3 and TGF-β1 were elevated in the atria of persistent AF animals. The Gal-3 inhibitor GM-CT-01 (GMCT) reduced both Gal-3 and TGF-β1-induced sheep atrial fibroblast migration and proliferation in vitro. GMCT (12 mg/kg twice/week) prevented the increase in serum procollagen type III N-terminal peptide seen during progression to persistent AF, and also mitigated atrial dilatation, myocyte hypertrophy, fibrosis, and the expected increase in dominant frequency of excitation. Atria of GMCT-treated animals had significantly less TGF-β1-Smad2/3 signaling pathway activation and expression of α-smooth muscle actin and collagen than saline-treated animals. Ex-vivo hearts from GMCT-treated animals had significantly longer action potential durations and fewer rotors and wavebreaks during AF, and myocytes had lower functional expression of inward rectifier K+ channel (Kir2.3) than saline-treated animals. Importantly, GMCT increased the probability of spontaneous AF termination, decreased AF inducibility and reduced overall AF burden. Conclusions Inhibiting Gal-3 during AF progression might be useful as an adjuvant treatment to improve outcomes of catheter ablation for persistent AF. Gal-3 inhibition may be a potential new upstream therapy for prevention of AF progression.

Long QT syndrome (LQTS) exhibits great phenotype variability among family members carrying the same mutation, which can be partially attributed to genetic factors. We functionally analyzed the KCNH2 (encoding for Kv11.1 or hERG channels) and TBX20 (encoding for the transcription factor Tbx20) variants found by next-generation sequencing in two siblings with LQTS in a Spanish family of African ancestry. Affected relatives harbor a heterozygous mutation in KCNH2 that encodes for p.T152HfsX180 Kv11.1 (hERG). This peptide, by itself, failed to generate any current when transfected into Chinese hamster ovary (CHO) cells but, surprisingly, exerted "chaperone-like" effects over native hERG channels in both CHO cells and mouse atrial-derived HL-1 cells. Therefore, heterozygous transfection of native (WT) and p.T152HfsX180 hERG channels generated a current that was indistinguishable from that generated by WT channels alone. Some affected relatives also harbor the p.R311C mutation in Tbx20. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Tbx20 enhanced human KCNH2 gene expression and hERG currents (I hERG ) and shortened action-potential duration (APD). However, Tbx20 did not modify the expression or activity of any other channel involved in ventricular repolarization. Conversely, p.R311C Tbx20 did not increase KCNH2 expression in hiPSC-CMs, which led to decreased I hERG and increased APD. Our results suggest that Tbx20 controls the expression of hERG channels responsible for the rapid component of the delayed rectifier current. On the contrary, p.R311C Tbx20 specifically disables the Tbx20 protranscriptional activity over KCNH2. Therefore, TBX20 can be considered a KCNH2-modifying gene.is characterized by abnormal prolongation of the QT interval of the electrocardiogram (ECG) and is due to delayed ventricular repolarization. LQTS increases the occurrence of ventricular tachyarrhythmias, particularly torsade de pointes, leading to recurrent syncope, seizures, ventricular fibrillation, and sudden cardiac death (SCD) (1). At least 15 genes have been reported in autosomal-dominant forms of LQTS (1). However, mutations in KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3) represent the most frequent forms of LQTS (∼90%) (1, 2).KCNH2 encodes Kv11.1, or hERG, channels, which generate the rapid component of the delayed rectifier current (I Kr ) responsible for ventricular repolarization in humans (3). In a Spanish family of African ancestry suffering LQTS, we identified a frameshift and a missense mutation in KCNH2 that were assumed to be the diseasecausing mutations. However, in some family members, we also identified a missense mutation in TBX20 coding for the transcription factor Tbx20, which is necessary in early stages of heart development (4). Importantly, results in flies and mice demonstrated that Tbx20 is also required for maintaining adult heart function (5, 6).Here we have tested the KCNH2 and TBX20 mutations to establish whether they can account for prolongation of repolarization. Our results dem...

Background Mutations in SCN2B, encoding voltage-gated sodium channel (VGSC) β2 subunits, are associated with human cardiac arrhythmias, including atrial fibrillation and Brugada syndrome. Because of this, we propose that β2 subunits play critical roles in the establishment or maintenance of normal cardiac electrical activity in vivo. Methods and Results To understand the pathophysiological roles of β2 in the heart, we investigated the cardiac phenotype of Scn2b null mice. We observed reduced sodium and potassium current density in ventricular myocytes, as well as conduction slowing in the right ventricular outflow tract region. Functional re-entry, resulting from the interplay between slowed conduction, prolonged repolarization, and increased incidence of premature ventricular complexes, was found to underlie the mechanism of spontaneous polymorphic ventricular tachycardia. Scn5a transcript levels were similar in Scn2b null and wild type ventricles, as were levels of Nav1.5 protein, suggesting that similar to previous work in neurons, the major function of β2 subunits in the ventricle is to chaperone VGSC α subunits to the plasma membrane. Interestingly, Scn2b deletion resulted in region-specific effects in the heart. Scn2b null atria had normal levels of sodium current density compared to wild type. Scn2b null hearts were more susceptible to atrial fibrillation, had increased levels of fibrosis, and higher repolarization dispersion than WT littermates. Conclusions Genetic deletion of Scn2b in mice results in ventricular and atrial arrhythmias, consistent with reported SCN2B mutations in human patients.

During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in protein kinase A (PKA)–mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent “hyperphosphorylation” of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation–contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch–clamp recordings and confocal Ca2+ imaging during β-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (ICa) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during β-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.

Previous work shows that transforming growth factor-β1 (TGF-β1) promotes several heart alterations, including atrial fibrillation (AF). In this work, we hypothesized that these effects might be associated with a potential modulation of Na(+) and K(+) channels. Atrial myocytes were cultured 1-2 days under either control conditions, or the presence of TGF-β1. Subsequently, Na(+) (I(Na)) and K(+) (I(K)) currents were investigated under whole-cell patch-clamp conditions. Three K(+) currents were isolated: inward rectifier (I(Kin)), outward transitory (I(to)), and outward sustained (I(Ksus)). Interestingly, TGF-β1 decreased (50%) the densities of I(Kin) and I(Ksus) but not of I(to). In addition, the growth factor reduced by 80% the amount of I(Na) available at -80 mV. This effect was due to a significant reduction (30%) in the maximum I(Na) recruited at very negative potentials or I(max), as well as to an increased fraction of inactivated Na(+) channels. The latter effect was, in turn, associated to a -7 mV shift in V(1/2) of inactivation. TGF-β1 also reduced by 60% the maximum amount of intramembrane charge movement of Na(+) channels or Q(max), but did not affect the corresponding voltage dependence of activation. This suggests that TGF-β1 promotes loss of Na(+) channels from the plasma membrane. Moreover, TGF-β1 also reduced (50%) the expression of the principal subunit of Na(+) channels, as indicated by western blot analysis. Thus, TGF-β1 inhibits the expression of Na(+) channels, as well as the activity of K(+) channels that give rise to I(Ksus) and I(Kin). These results may contribute to explaining the previously observed proarrhythmic effects of TGF-β1.