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.

AIMS:Cardiac excitability and refractoriness are largely determined by the function and number of inward rectifier K⁺ channels (Kir2.1-2.3), which are differentially expressed in the atria and ventricles, and Nav1.5 channels. We have focused on how Nav1.5 and Kir2.x function within a macromolecular complex by elucidating the molecular determinants that govern Nav1.5/Kir2.x reciprocal modulation.METHODS AND RESULTS:The results demonstrate that there is an unexpected 'internal' PDZ-like binding domain located at the N-terminus of the Nav1.5 channel that mediates its binding to α1-syntrophin. Nav1.5 N-terminal domain, by itself (the 132 aa peptide) (Nter), exerts a 'chaperone-like' effect that increases sodium (I(Na)) and inward rectifier potassium (I(K1)) currents by enhancing the expression of Nav1.5, Kir2.1, and Kir2.2 channels as demonstrated in Chinese hamster ovary (CHO) cells and in rat cardiomyocytes. Site-directed mutagenesis analysis demonstrates that the Nter chaperone-like effect is determined by Serine 20. Nav1.5-Kir2.x reciprocal positive interactions depend on a specific C-terminal PDZ-binding domain sequence (SEI), which is present in Kir2.1 and Kir2.2 channels but not in Kir2.3. Therefore, in human atrial myocytes, the presence of Kir2.3 isoforms precludes reciprocal I(K1)-INa density modulation. Moreover, results in rat and human atrial myocytes demonstrate that binding to α1-syntrophin is necessary for the Nav1.5-Kir2.x-positive reciprocal modulation.CONCLUSIONS:The results demonstrate the critical role of the N-terminal domain of Nav1.5 channels in Nav1.5-Kir2.x-reciprocal interactions and suggest that the molecular mechanisms controlling atrial and ventricular cellular excitability may be different.

AIMS Atrial fibrillation (AF) produces rapid changes in the electrical properties of the atria (electrical remodelling) that promote its own recurrence. In chronic AF (CAF) patients, up-regulation of the slow delayed rectifier K(+) current (IKs) and down-regulation of the voltage-gated Ca(2+) current (ICa,L) are hallmarks of electrical remodelling and critically contribute to the abbreviation of action potential duration and atrial refractory period. Recent evidences suggested that Pitx2c, a bicoid-related homeodomain transcription factor involved in directing cardiac asymmetric morphogenesis, could play a role in atrial remodelling. However, its effects on IKs and ICa,L are unknown. METHODS AND RESULTS Real-time quantitative polymerase chain reaction analysis showed that Pitx2c mRNA expression was significantly higher in human atrial myocytes from CAF patients than those from sinus rhythm patients. The expression of Pitx2c was positively and negatively correlated with IKs and ICa,L densities, respectively. Expression of Pitx2c in HL-1 cells increased IKs density and reduced ICa,L density. Luciferase assays demonstrated that Pitx2c increased transcriptional activity of KCNQ1 and KCNE1 genes. Conversely, its effects on ICa,L could be mediated by the atrial natriuretic peptide. CONCLUSION Our results demonstrated for the first time that CAF increases Pitx2c expression in isolated human atrial myocytes and suggested that this transcription factor could contribute to the CAF-induced IKs increase and ICa,L reduction observed in humans.

BACKGROUND The human cardiac action potential in atrial and ventricular cells is initiated by a fast-activating, fast-inactivating sodium current generated by the SCN5A/Nav1.5 channel in association with its fi1/5CN1B subunit. The role of Nav1.5 in the etiology of many cardiac diseases strongly suggests that proper regulation of cell biology and function of the channel is critical for normal cardiac function. Hence, numerous recent studies have focused on the regulatory mechanisms of Nav1.5 biosynthetic and degradation processes as well as its subcellular localization.OBJECTIVE The purpose of this study was to investigate the role of microRNAs in the Scn5a/Nav1.5 posttranscriptional regulation.METHODS Quantitative polymerase chain reaction, immunohistochemical and electrophysiological measurements of distinct microRNA gain-of-function experiments in cardiomyocytes for the assessment of Scn5a expression.RESULTS Functional studies of HL-1 cardiomyocytes and luciferase assays in fibroblasts demonstrate that Scn5a is directly (miR-98, miR106, miR-200, and miR-219) and indirectly (miR-125 and miR-153) regulated by multiple microRNAs displaying distinct time-dependent profiles. Cotransfection experiments demonstrated that miR-219 and miR-200 have independent opposite effects on Scn5a expression modulation. Of all the microRNAs studied, only miR-219 increases Scn5a expression levels, leading to altered contraction rhythm of HL-1 cardiomyocytes. Electrophysiological analyses in HL-1 cells revealed that miR-219 increases the sodium current. In vivo administration of miR-219 does not alter normal cardiac rhythm, but abolishes some of the effects of flecainide intoxication in mice, particularly QRS prolongation.CONCLUSION This study demonstrates the involvement of multiple microRNAs in the regulation of Scn5a. Particularly, miR-219 increases Scn5a/Nav1.5 transcript and protein expression. Our data suggest that microRNAs, such as miR-219, constitute a promising therapeutical tool to treat sodium cardiac arrhythmias.

Background— Atrial fibrillation is characterized by progressive atrial structural and electrical changes (atrial remodeling) that favor arrhythmia recurrence and maintenance. Reduction of L-type Ca 2+ current ( I Ca,L ) density is a hallmark of the electrical remodeling. Alterations in atrial microRNAs could contribute to the protein changes underlying atrial fibrillation–induced atrial electrical remodeling. This study was undertaken to compare miR-21 levels in isolated myocytes from atrial appendages obtained from patients in sinus rhythm and with chronic atrial fibrillation (CAF) and to determine whether L-type Ca 2+ channel subunits are targets for miR-21. Methods and Results— Quantitative polymerase chain reaction analysis showed that miR-21 was expressed in human atrial myocytes from patients in sinus rhythm and that its expression was significantly greater in CAF myocytes. There was an inverse correlation between miR-21 and the mRNA of the α1c subunit of the calcium channel (CACNA1C) expression and I Ca,L density. Computational analyses predicted that CACNA1C and the mRNA of the β2 subunit of the calcium channel (CACNB2) could be potential targets for miR-21. Luciferase reporter assays demonstrated that miR-21 produced a concentration-dependent decrease in the luciferase activity in Chinese Hamster Ovary cells transfected with CACNA1C and CACNB2 3′ untranslated region regions. miR-21 transfection in HL-1 cells produced changes in I Ca,L properties qualitatively similar to those produced by CAF (ie, a marked reduction of I Ca,L density and shift of the inactivation curves to more depolarized potentials). Conclusions— Our results demonstrated that CAF increases miR-21 expression in enzymatically isolated human atrial myocytes. Moreover, it decreases I Ca,L density by downregulating Ca 2+ channel subunits expression. These results suggested that this microRNA could participate in the CAF-induced I Ca,L downregulation and in the action potential duration shortening that maintains the arrhythmia.

AIMS We hypothesize that some drugs, besides flecainide, increase the inward rectifier current (IK1) generated by Kir2.1 homotetramers (IKir2.1) and thus, exhibit pro- and/or antiarrhythmic effects particularly at the ventricular level. To test this hypothesis, we analysed the effects of propafenone, atenolol, dronedarone, and timolol on Kir2.x channels. METHODS AND RESULTS Currents were recorded with the patch-clamp technique using whole-cell, inside-out, and cell-attached configurations. Propafenone (0.1 nM-1 µM) did not modify either IK1 recorded in human right atrial myocytes or the current generated by homo- or heterotetramers of Kir2.2 and 2.3 channels recorded in transiently transfected Chinese hamster ovary cells. On the other hand, propafenone increased IKir2.1 (EC50 = 12.0 ± 3.0 nM) as a consequence of its interaction with Cys311, an effect which decreased inward rectification of the current. Propafenone significantly increased mean open time and opening frequency at all the voltages tested, resulting in a significant increase of the mean open probability of the channel. Timolol, which interacted with Cys311, was also able to increase IKir2.1. On the contrary, neither atenolol nor dronedarone modified IKir2.1. Molecular modelling of the Kir2.1-drugs interaction allowed identification of the pharmacophore of drugs that increase IKir2.1. CONCLUSIONS Kir2.1 channels exhibit a binding site determined by Cys311 that is responsible for drug-induced IKir2.1 increase. Drug binding decreases channel affinity for polyamines and current rectification, and can be a mechanism of drug-induced pro- and antiarrhythmic effects not considered until now.

Purpose: Human cardiac inward rectifying K+ current (IK1) is mainly carried by Kir2.1 channels in ventricular cells, whereas the relative contribution of Kir2.2 and Kir2.3 seems to be greater in atrial cells. IK1 plays a key role in the control of resting membrane potential and action potential duration and, thus, excitability and refractoriness. It has been described that an increase in the expression of sodium channels (Nav1.5) increases Kir2.1 expression, a modulation that involves synapse-associated protein 97 (SAP97). SAP97 binds to the PDZ domain of the channels whose sequence in Kir2.1 and 2.2 channels is Ser-Glu-Ile, while in Kir2.3 is Ser-Ala-Ile. Thus, we analyzed whether Kir2.1 and Kir2.3 channel activities are modulated differentially by the presence of Nav1.5. Methods: Kir2.x currents (IKir2.x) were recorded by using the patch-clamp technique in CHO cells transiently transfected with wild-type (WT) and mutant Kir2.x and Nav1.5 channels (1:1 ratio). Results: In the presence of WT Nav1.5, IKir2.1 density was significantly increased compared to that in the absence of Nav1.5, both at potentials negative (-199±22 vs -121±12 pA/pF at -120 mV, n>24, P<0.01) and positive (19.7±4.5 vs 8.9±1.2 pA/pF at -50 mV) to the K+ reversal potential. Conversely, cotransfection of Nav1.5 neither with Kir2.3 channels alone nor with Kir2.1 plus Kir2.3 channels did modify IKir2.3 or IKir2.1/2.3 density (n>16). Importantly, when Glu426 of Kir2.1 was replaced by Ala (p.E426A), the increase induced by coexpression of Nav1.5 was abolished at all membrane potentials. Coexpression of Nav1.5 with p.A444E Kir2.3 channels markedly increased IKir2.3 density (-44.0±5.8 pA/pF at -120 mV, n>12, P<0.01). The increase of IKir2.1 and p.A444E IKir2.3 density was not observed when they were cotransfected with two Brugada syndrome-associated SCN5A mutations (p.D1690N and p.D1816VfsX7) that severely impair Nav1.5 channel trafficking. Conclusions: Only expression of Kir2.1 homotetramers is increased by their coexpression with Nav1.5 channels. The specificity of the effect is determined by Glu426 in the PDZ domain of Kir2.1 and could lead to differential electrophysiological effects on atrial and ventricular cells on patients carrying SCN5A mutations.

Human cardiac inward rectifier current (IK1) is generated by Kir2.x channels. Inhibition of IK1 could offer a useful antiarrhythmic strategy against fibrillatory arrhythmias. Therefore, elucidation of Kir2.x channels pharmacology, which still remains elusive, is mandatory. We characterized the electrophysiological and molecular basis of the inhibition produced by the antiarrhythmic propafenone of the current generated by Kir2.x channels (IKir2.x) and the IK1 recorded in human atrial myocytes. Wild type and mutated human Kir2.x channels were transiently transfected in CHO and HEK-293 cells. Macroscopic and single-channel currents were recorded using the patch-clamp technique. At concentrations >1μM propafenone inhibited IKir2.x the order of potency being Kir2.3∼IK1>Kir2.2>Kir2.1 channels. Blockade was irrespective of the extracellular K+ concentration whereas markedly increased when the intracellular K+ concentration was decreased. Propafenone decreased inward rectification since at potentials positive to the K+ equilibrium potential propafenone-induced block decreased in a voltage-dependent manner. Importantly, propafenone favored the occurrence of subconductance levels in Kir2.x channels and decreased phosphatidylinositol 4,5-bisphosphate (PIP2)-channel affinity. Blind docking and site-directed mutagenesis experiments demonstrated that propafenone bound Kir2.x channels at the cytoplasmic domain, close to, but not in the pore itself, the binding site involving two conserved Arg residues (residues 228 and 260 in Kir2.1). Our results suggested that propafenone incorporated into the cytoplasmic domain of the channel in such a way that it decreased the net negative charge sensed by K+ ions and polyamines which, in turn, promotes the appearance of subconductance levels and the decrease of PIP2 affinity of the channels.

BACKGROUND We identified 2 compound heterozygous mutations (p.D1690N and p.G1748D) in the SCN5A gene encoding cardiac Na+ channels (Nav1.5) in a proband diagnosed with Brugada syndrome type 1. Furthermore, in the allele encoding the p.D1690N mutation, the p.H558R polymorphism was also detected.OBJECTIVE The purpose of this study was to analyze the functional properties of the mutated channels as well as the putative modulator effects produced by the presence of the polymorphism.METHODS Wild-type and mutated human Nav1.5 channels were expressed in Chinese hamster ovary cells and recorded using whole-cell patch-damp technique.RESULTS Separately, both p.D1690N and p.G1748D mutations produced a marked reduction in peak Na+ current density (>80%), mainly due to their limited trafficking toward the membrane. Furthermore, p.G1748D mutation profoundly affected channel gating. Both p.D1690N and p.G1748D produced a marked dominant negative effect when cotransfected with either wild-type or p.H558R channels. Conversely, p.H558R was able to rescue defective trafficking of p.D1690N channels toward the membrane when both polymorphism and mutation were in the same construct. Surprisingly, cotransfection with p.D1690N, either atone or together with the polymorphism (p.H558R-D1690N), completely restored the profound gating defects exhibited by p.G1748D channels but only slightly rescued their trafficking.CONCLUSION Our results add further support to the hypothesis that Nav1.5 subunits interact among them before trafficking toward the membrane and suggest that a missense mutation can "rescue" the defective gating produced by another missense mutation.

INTRODUCTION: We functionally analyzed a frameshift mutation in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) found in a proband with repeated episodes of ventricular fibrillation who presented bradycardia and paroxysmal atrial fibrillation. Seven relatives also carry the mutation and showed a Brugada syndrome with an incomplete and variable expression. The mutation (p.D1816VfsX7) resulted in a severe truncation (201 residues) of the Nav1.5 C-terminus. METHODS AND RESULTS: Wild-type (WT) and mutated Nav1.5 channels together with hNavβ1 were expressed in CHO cells and currents were recorded at room temperature using the whole-cell patch-clamp. Expression of p.D1816VfsX7 alone resulted in a marked reduction (≈90%) in peak Na(+) current density compared with WT channels. Peak current density generated by p.D1816VfsX7+WT was ≈50% of that generated by WT channels. p.D1816VfsX7 positively shifted activation and inactivation curves, leading to a significant reduction of the window current. The mutation accelerated current activation and reactivation kinetics and increased the fraction of channels developing slow inactivation with prolonged depolarizations. However, late INa was not modified by the mutation. p.D1816VfsX7 produced a marked reduction of channel trafficking toward the membrane that was not restored by decreasing incubation temperature during cell culture or by incubation with 300 μM mexiletine and 5 mM 4-phenylbutirate. CONCLUSION: Despite a severe truncation of the C-terminus, the resulting mutated channels generate currents, albeit with reduced amplitude and altered biophysical properties, confirming the key role of the C-terminal domain in the expression and function of the cardiac Na(+) channel.