Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents.

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1WTversus Kir2.1Δ314-315, a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases.

Cardiac Kir2.1 and NaV1.5 Channels Traffic Together to the Sarcolemma to Control Excitability.

RATIONALE: In cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. OBJECTIVE: The objective of this study was to test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. METHODS AND RESULTS: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1Δ314-315 or Kir2.1R44A/R46A with wild-type (WT) NaV1.5WT in heterologous cells reduced inward sodium current compared with NaV1.5WT alone or coexpressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1Δ314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas coexpression with Kir2.1Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with NaV1.5WT and Kir2.1WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1WT, NaV1.5WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that NaV1.5WT channels interact with AP1 through the NaV1.5Y1810 residue, suggesting that, like for Kir2.1WT, AP1 can mark NaV1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. CONCLUSIONS: The NaV1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of NaV1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

Tbx20 controls the expression of the KCNH2 gene and of hERG channels.

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 (IhERG) 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 IhERG 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.

Constitutive Intracellular Na+ Excess in Purkinje Cells Promotes Arrhythmogenesis at Lower Levels of Stress Than Ventricular Myocytes From Mice With Catecholaminergic Polymorphic Ventricular Tachycardia.

BACKGROUND: In catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac Purkinje cells (PCs) appear more susceptible to Ca(2+) dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2(R4496C+/Cx40eGFP)), we tested whether PC intracellular Ca(2+) ([Ca(2+)]i) dysregulation results from a constitutive [Na(+)]i surplus relative to VMs. METHODS AND RESULTS: Simultaneous optical mapping of voltage and [Ca(2+)]i in CPVT hearts showed that spontaneous Ca(2+) release preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca(2+) imaging, early and delayed afterdepolarizations trailed spontaneous Ca(2+) release and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant ryanodine receptor type 2 channels, sarcoplasmic reticulum Ca(2+) load, measured by caffeine-induced Ca(2+) transients, was lower in CPVT VMs and PCs than respective controls, and sarcoplasmic reticulum fractional release was greater in both CPVT PCs and VMs than respective controls. [Na(+)]i was higher in both control and CPVT PCs than VMs, whereas the density of the Na(+)/Ca(2+) exchanger current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na(+)]i of PCs promoted delayed afterdepolarizations, which were always preceded by spontaneous Ca(2+) release events from hyperactive ryanodine receptor type 2 channels. Increasing [Na(+)]i monotonically increased delayed afterdepolarization frequency. Confocal imaging experiments showed that postpacing Ca(2+) spark frequency was highest in intact CPVT PCs, but such differences were reversed on saponin-induced membrane permeabilization, indicating that differences in [Na(+)]i played a central role. CONCLUSIONS: In CPVT mice, the constitutive [Na(+)]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs.

Dominant frequency increase rate predicts transition from paroxysmal to long-term persistent atrial fibrillation.

BACKGROUND: Little is known about the mechanisms underlying the transition from paroxysmal to persistent atrial fibrillation (AF). In an ovine model of long-standing persistent AF we tested the hypothesis that the rate of electric and structural remodeling, assessed by dominant frequency (DF) changes, determines the time at which AF becomes persistent. METHODS AND RESULTS: Self-sustained AF was induced by atrial tachypacing. Seven sheep were euthanized 11.5±2.3 days after the transition to persistent AF and without reversal to sinus rhythm; 7 sheep were euthanized after 341.3±16.7 days of long-standing persistent AF. Seven sham-operated animals were in sinus rhythm for 1 year. DF was monitored continuously in each group. Real-time polymerase chain reaction, Western blotting, patch clamping, and histological analyses were used to determine the changes in functional ion channel expression and structural remodeling. Atrial dilatation, mitral valve regurgitation, myocyte hypertrophy, and atrial fibrosis occurred progressively and became statistically significant after the transition to persistent AF, with no evidence for left ventricular dysfunction. DF increased progressively during the paroxysmal-to-persistent AF transition and stabilized when AF became persistent. Importantly, the rate of DF increase correlated strongly with the time to persistent AF. Significant action potential duration abbreviation, secondary to functional ion channel protein expression changes (CaV1.2, NaV1.5, and KV4.2 decrease; Kir2.3 increase), was already present at the transition and persisted for 1 year of follow up. CONCLUSIONS: In the sheep model of long-standing persistent AF, the rate of DF increase predicts the time at which AF stabilizes and becomes persistent, reflecting changes in action potential duration and densities of sodium, L-type calcium, and inward rectifier currents.

Protein assemblies of sodium and inward rectifier potassium channels control cardiac excitability and arrhythmogenesis.

The understanding of how cardiac ion channels function in the normal and the diseased heart has greatly increased over the last four decades thanks to the advent of patch-clamp technology and, more recently, the emergence of genetics, as well as cellular and molecular cardiology. However, our knowledge of how these membrane-embedded proteins physically interact with each other within macromolecular complexes remains incomplete. This review focuses on how the main cardiac inward sodium channel (NaV1.5) and the strong inward rectifier potassium channel (Kir2.1) function within macromolecular complexes to control cardiac excitability. It has become increasingly clear that these two important ion channel proteins physically interact with multiple other protein partners and with each other from early stages of protein trafficking and targeting through membrane anchoring, recycling, and degradation. Recent findings include compartmentalized regulation of NaV1.5 channel expression and function through a PDZ (postsynaptic density protein, Drosophila disc large tumor suppressor, and zonula occludens-1 protein) domain-binding motif, and interaction of caveolin-3 with Kir2.1 and ankyrin-G as a molecular platform for NaV1.5 signaling. At the cardiomyocyte membrane, NaV1.5 and Kir2.1 interact through at least two distinct PDZ domain-scaffolding proteins (synapse-associated protein-97 and α1-syntrophin), thus modulating reciprocally their cell-surface expression at two different microdomains. Emerging evidence also shows that inheritable mutations in plakophilin-2, ankyrin-G, dystrophin, syntrophin, synapse-associated protein-97, and caveolin-3, among others, modify functional expression and/or localization in the cardiac cell of NaV1.5, Kir2.1 or both to give rise to arrhythmogenic diseases. Unveiling the mechanistic underpinnings of macromolecular interactions should increase our understanding of inherited and acquired arrhythmogenic cardiac diseases and may lead to advances in therapy.

Paradoxical SERCA dysregulation contributes to atrial fibrillation in a model of diet-induced obesity.

Obesity is a major risk factor for atrial fibrillation (AF) the most common serious cardiac arrhythmia, but the molecular mechanisms underlying diet-induced AF remain unclear. In this study, we subjected mice to a chronic high-fat diet and acute sympathetic activation ('two-hit' model) to study the mechanisms by which diet-induced obesity promotes AF. Surface electrocardiography revealed that diet-induced obesity and sympathetic activation synergize during intracardiac tachypacing to induce AF. At the cellular level, diet-induced obesity and acute adrenergic stimulation facilitate the formation of delayed afterdepolarizations in atrial myocytes, implicating altered Ca 2+ dynamics as the underlying cause of AF. We found that diet-induced obesity does not alter the expression of major Ca 2+ -handling proteins in atria, including the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA), a major component of beat-to-beat Ca 2+ cycling in the heart. Paradoxically, obesity reduces phospholamban phosphorylation, suggesting decreased SERCA activity, yet atrial myocytes from obese mice showed a significantly increased Ca 2+ transient amplitude and SERCA-mediated Ca 2+ uptake. Adrenergic stimulation further increases the Ca 2+ transient amplitude but does not affect Ca 2+ reuptake in atrial myocytes from obese mice. Transcriptomics analysis showed that a high-fat diet prompts upregulation of neuronatin, a protein that has been implicated in obesity and is known to stimulate SERCA activity. We propose a mechanism in which obesity primes SERCA for paradoxical activation, and adrenergic stimulation facilitates AF conversion through a Ca 2+ -induced Ca 2+ release gain in atrial myocytes. Overall, this study links obesity, altered Ca 2+ signaling, and AF, and targeting this mechanism may prove effective for treating obesity-induced AF.

Decreased FAM13B Expression Increases Atrial Fibrillation Susceptibility by Regulating Sodium Current and Calcium Handling.

A specific genetic variant associated with atrial fibrillation risk, rs17171731, was identified as a regulatory variant responsible for controlling FAM13B expression. The atrial fibrillation risk allele decreases FAM13B expression, whose knockdown alters the expression of many genes in stem cell-derived cardiomyocytes, including SCN2B, and led to pro-arrhythmogenic changes in the late sodium current and Ca2+ cycling. Fam13b knockout mice had increased P-wave and QT interval duration and were more susceptible to pacing-induced arrhythmias vs control mice. FAM13B expression, its regulation, and downstream effects are potential targets for investigation of patient-specific therapeutics.

Molecular mechanisms of chloroquine inhibition of heterologously expressed Kir6.2/SUR2A channels.

Chloroquine and related compounds can inhibit inwardly rectifying potassium channels by multiple potential mechanisms, including pore block and allosteric effects on channel gating. Motivated by reports that chloroquine inhibition of cardiac ATP-sensitive inward rectifier K(+) current (I(KATP)) is antifibrillatory in rabbit ventricle, we investigated the mechanism of chloroquine inhibition of ATP-sensitive potassium (K(ATP)) channels (Kir6.2/SUR2A) expressed in human embryonic kidney 293 cells, using inside-out patch-clamp recordings. We found that chloroquine inhibits the Kir6.2/SUR2A channel by interacting with at least two different sites and by two mechanisms of action. A fast-onset effect is observed at depolarized membrane voltages and enhanced by the N160D mutation in the central cavity, probably reflecting direct channel block resulting from the drug entering the channel pore from the cytoplasmic side. Conversely, a slow-onset, voltage-independent inhibition of I(KATP) is regulated by chloroquine interaction with a different site and probably involves disruption of interactions between Kir6.2/SUR2A and phosphatidylinositol 4,5-bisphosphate. Our findings reveal multiple mechanisms of K(ATP) channel inhibition by chloroquine, highlighting the numerous convergent regulatory mechanisms of these ligand-dependent ion channels.

Paclitaxel mitigates structural alterations and cardiac conduction system defects in a mouse model of Hutchinson-Gilford progeria syndrome.

AIMS: Hutchinson-Gilford progeria syndrome (HGPS) is an ultrarare laminopathy caused by expression of progerin, a lamin A variant, also present at low levels in non-HGPS individuals. HGPS patients age and die prematurely, predominantly from cardiovascular complications. Progerin-induced cardiac repolarization defects have been described previously, although the underlying mechanisms are unknown. METHODS AND RESULTS: We conducted studies in heart tissue from progerin-expressing LmnaG609G/G609G (G609G) mice, including microscopy, intracellular calcium dynamics, patch-clamping, in vivo magnetic resonance imaging, and electrocardiography. G609G mouse cardiomyocytes showed tubulin-cytoskeleton disorganization, t-tubular system disruption, sarcomere shortening, altered excitation-contraction coupling, and reductions in ventricular thickening and cardiac index. G609G mice exhibited severe bradycardia, and significant alterations of atrio-ventricular conduction and repolarization. Most importantly, 50% of G609G mice had altered heart rate variability, and sinoatrial block, both significant signs of premature cardiac aging. G609G cardiomyocytes had electrophysiological alterations, which resulted in an elevated action potential plateau and early afterdepolarization bursting, reflecting slower sodium current inactivation and long Ca+2 transient duration, which may also help explain the mild QT prolongation in some HGPS patients. Chronic treatment with low-dose paclitaxel ameliorated structural and functional alterations in G609G hearts. CONCLUSIONS: Our results demonstrate that tubulin-cytoskeleton disorganization in progerin-expressing cardiomyocytes causes structural, cardiac conduction, and excitation-contraction coupling defects, all of which can be partially corrected by chronic treatment with low dose paclitaxel.

SNTA1 gene rescues ion channel function and is antiarrhythmic in cardiomyocytes derived from induced pluripotent stem cells from muscular dystrophy patients.

Background: Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients. Methods: To test whether dystrophin mutations lead to defective cardiac NaV1.5-Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays. Results: Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers. Conclusions: We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5-Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5-Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias. Funding: Supported by National Institutes of Health R01 HL122352 grant; 'la Caixa' Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.

Mechanisms for Kir channel inhibition by quinacrine: acute pore block of Kir2.x channels and interference in PIP2 interaction with Kir2.x and Kir6.2 channels.

Cardiac inward rectifier potassium currents determine the resting membrane potential and contribute repolarization capacity during phase 3 repolarization. Quinacrine is a cationic amphiphilic drug. In this work, the effects of quinacrine were studied on cardiac Kir channels expressed in HEK 293 cells and on the inward rectifier potassium currents, I(K1) and I(KATP), in cardiac myocytes. We found that quinacrine differentially inhibited Kir channels, Kir6.2 ∼ Kir2.3 > Kir2.1. In addition, we found in cardiac myocytes that quinacrine inhibited I(KATP) > I(K1). We presented evidence that quinacrine displays a double action towards strong inward rectifier Kir2.x channels, i.e., direct pore block and interference in phosphatidylinositol 4,5-bisphosphate, PIP(2)-Kir channel interaction. Pore block is evident in Kir2.1 and 2.3 channels as rapid block; channel block involves residues E224 and E299 facing the cytoplasmic pore of Kir2.1. The interference of the drug with the interaction of Kir2.x and Kir6.2/SUR2A channels and PIP(2) is suggested from four sources of evidence: (1) Slow onset of current block when quinacrine is applied from either the inside or the outside of the channel. (2) Mutation of Kir2.3(I213L) and mutation of Kir6.2(C166S) increase their affinity for PIP(2) and lowers its sensitivity for quinacrine. (3) Mutations of Kir2.1(L222I and K182Q) which decreased its affinity for PIP(2) increased its sensitivity for quinacrine. (4) Co-application of quinacrine with PIP(2) lowers quinacrine-mediated current inhibition. In conclusion, our data demonstrate how an old drug provides insight into a dual a blocking mechanism of Kir carried inward rectifier channels.

Specific residues of the cytoplasmic domains of cardiac inward rectifier potassium channels are effective antifibrillatory targets.

Atrial and ventricular tachyarrhythmias can be perpetuated by up-regulation of inward rectifier potassium channels. Thus, it may be beneficial to block inward rectifier channels under conditions in which their function becomes arrhythmogenic (e.g., inherited gain-of-function mutation channelopathies, ischemia, and chronic and vagally mediated atrial fibrillation). We hypothesize that the antimalarial quinoline chloroquine exerts potent antiarrhythmic effects by interacting with the cytoplasmic domains of Kir2.1 (I(K1)), Kir3.1 (I(KACh)), or Kir6.2 (I(KATP)) and reducing inward rectifier potassium currents. In isolated hearts of three different mammalian species, intracoronary chloroquine perfusion reduced fibrillatory frequency (atrial or ventricular), and effectively terminated the arrhythmia with resumption of sinus rhythm. In patch-clamp experiments chloroquine blocked I(K1), I(KACh), and I(KATP). Comparative molecular modeling and ligand docking of chloroquine in the intracellular domains of Kir2.1, Kir3.1, and Kir6.2 suggested that chloroquine blocks or reduces potassium flow by interacting with negatively charged amino acids facing the ion permeation vestibule of the channel in question. These results open a novel path toward discovering antiarrhythmic pharmacophores that target specific residues of the cytoplasmic domain of inward rectifier potassium channels.

The antimalarial drug mefloquine inhibits cardiac inward rectifier K+ channels: evidence for interference in PIP2-channel interaction.

The antimalarial drug mefloquine was found to inhibit the KATP channel by an unknown mechanism. Because mefloquine is a Cationic amphiphilic drug and is known to insert into lipid bilayers, we postulate that mefloquine interferes with the interaction between PIP2 and Kir channels resulting in channel inhibition. We studied the inhibitory effects of mefloquine on Kir2.1, Kir2.3, Kir2.3(I213L), and Kir6.2/SUR2A channels expressed in HEK-293 cells, and on IK1 and IKATP from feline cardiac myocytes. The order of mefloquine inhibition was Kir6.2/SUR2A ≈ Kir2.3 (IC50 ≈ 2 µM) > Kir2.1 (IC50 > 30 µM). Similar results were obtained in cardiac myocytes. The Kir2.3(I213L) mutant, which enhances the strength of interaction with PIP2 (compared to WT), was significantly less sensitive (IC50 = 9 µM). In inside-out patches, continuous application of PIP2 strikingly prevented the mefloquine inhibition. Our results support the idea that mefloquine interferes with PIP2-Kir channels interactions.

Long QT syndrome - Bench to bedside.

Long QT syndrome (LQTS) is a cardiovascular disorder characterized by an abnormality in cardiac repolarization leading to a prolonged QT interval and T-wave irregularities on the surface electrocardiogram. It is commonly associated with syncope, seizures, susceptibility to torsades de pointes, and risk for sudden death. LQTS is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. The availability of therapy for this lethal disease emphasizes the importance of early and accurate diagnosis. Additionally, understanding of the molecular mechanisms underlying LQTS could help to optimize genotype-specific treatments to prevent deaths in LQTS patients. In this review, we briefly summarize current knowledge regarding molecular underpinning of LQTS, in particular focusing on LQT1, LQT2, and LQT3, and discuss novel strategies to study ion channel dysfunction and drug-specific therapies in LQT1, LQT2, and LQT3 syndromes.

In vitro model of ischemic heart failure using human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used extensively to model inherited heart diseases, but hiPSC-CM models of ischemic heart disease are lacking. Here, our objective was to generate an hiPSC-CM model of ischemic heart disease. To this end, hiPSCs were differentiated into functional hiPSC-CMs and then purified using either a simulated ischemia media or by using magnetic antibody-based purification targeting the nonmyocyte population for depletion from the cell population. Flow cytometry analysis confirmed that each purification approach generated hiPSC-CM cultures that had more than 94% cTnT+ cells. After purification, hiPSC-CMs were replated as confluent syncytial monolayers for electrophysiological phenotype analysis and protein expression by Western blotting. The phenotype of metabolic stress-selected hiPSC-CM monolayers recapitulated many of the functional and structural hallmarks of ischemic CMs, including elevated diastolic calcium, diminished calcium transient amplitude, prolonged action potential duration, depolarized resting membrane potential, hypersensitivity to chemotherapy-induced cardiotoxicity, depolarized mitochondrial membrane potential, depressed SERCA2a expression, reduced maximal oxygen consumption rate, and abnormal response to ß1-adrenergic receptor stimulation. These findings indicate that metabolic selection of hiPSC-CMs generated cell populations with phenotype similar to what is well known to occur in the setting of ischemic heart failure and thus provide a opportunity for study of human ischemic heart disease.

Human influenza A virus causes myocardial and cardiac-specific conduction system infections associated with early inflammation and premature death.

AIMS: Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo, cellular and molecular alterations. METHODS AND RESULTS: We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase. CONCLUSION: Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death.

Tamoxifen inhibits cardiac ATP-sensitive and acetylcholine-activated K+ currents in part by interfering with phosphatidylinositol 4,5-bisphosphate-channel interaction.

Tamoxifen inhibits transmembrane currents of the Kir2.x inward rectifier potassium channels by interfering with the interaction of the channels with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). We tested the hypothesis that Kir channels with low affinity for PIP(2), like the adenosine triphosphate (ATP)-sensitive K(+) channel (K(ATP)) and acetylcholine (ACh)-activated K(+) channel (K(ACh)), have at least the same sensitivity to tamoxifen as Kir2.3. We investigated the effects of tamoxifen (0.1 - 10 microM) on Kir6.2/SUR2A (K(ATP)) and Kir3.1/3.4 (K(ACh)) channels expressed in HEK-293 cells and ATP-sensitive K(+) current (I(KATP)) and ACh-activated K(+) current (I(KACh)) in feline atrial myocytes. The onset of tamoxifen inhibition of both I(KATP) and I(KACh) was slow (T(1/2) approximately 3.5 min) and concentration-dependent but voltage-independent. The time course and degree of inhibition was independent of external or internal drug application. Tamoxifen interacts with the pore forming subunit, Kir6.2, rather than with the SUR subunit. The inhibitory potency of tamoxifen on the Kir6.2/SUR2A channel was decreased by the mutation (C166S) on Kir6.2 and in the continuous presence of PIP(2). In atrial myocytes, the mechanism and potency of the effects of tamoxifen on K(ATP) and K(ACh) channels were comparable to those in HEK-293 cells. These data suggest that, similar to its effects on Kir2.x currents, tamoxifen inhibits K(ATP) and K(ACh) currents by interfering with the interaction between the channel and PIP(2).

Human perinatal stem cell derived extracellular matrix enables rapid maturation of hiPSC-CM structural and functional phenotypes.

The immature phenotype of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is a major limitation to the use of these valuable cells for pre-clinical toxicity testing and for disease modeling. Here we tested the hypothesis that human perinatal stem cell derived extracellular matrix (ECM) promotes hiPSC-CM maturation to a greater extent than mouse cell derived ECM. We refer to the human ECM as Matrix Plus (Matrix Plus) and compare effects to commercially available mouse ECM (Matrigel). hiPSC-CMs cultured on Matrix Plus mature functionally and structurally seven days after thaw from cryopreservation. Mature hiPSC-CMs showed rod-shaped morphology, highly organized sarcomeres, elevated cTnI expression and mitochondrial distribution and function like adult cardiomyocytes. Matrix Plus also promoted mature hiPSC-CM electrophysiological function and monolayers' response to hERG ion channel specific blocker was Torsades de Pointes (TdP) reentrant arrhythmia activations in 100% of tested monolayers. Importantly, Matrix Plus enabled high throughput cardiotoxicity screening using mature human cardiomyocytes with validation utilizing reference compounds recommended for the evolving Comprehensive In Vitro Proarrhythmia Assay (CiPA) coordinated by the Health and Environmental Sciences Institute (HESI). Matrix Plus offers a solution to the commonly encountered problem of hiPSC-CM immaturity that has hindered implementation of these human based cell assays for pre-clinical drug discovery.

Molecular and Electrophysiological Analyses of ATP2B4 Gene Variants in Bilateral Adrenal Hyperaldosteronism.

Primary aldosteronism (PA) is the most common cause of secondary hypertension with a high prevalence among patients with resistant hypertension. Despite the recent discovery of somatic variants in aldosterone-producing adenoma (APA)-associated PA, causes for PA due to bilateral aldosterone production (bilateral hyperaldosteronism; BHA) remain unknown. Herein, we identified rare gene variants in ATP2B4, in a cohort of patients with BHA. ATP2B4 belongs to the same family of Ca-ATPases as ATP2B3, which is involved in the pathogenesis of APA. Endogenous ATP2B4 expression was characterized in adrenal tissue, and the gene variants were functionally analyzed for effects on aldosterone synthase (CYP11B2) expression, steroid production in basal and agonist-stimulated conditions, and for changes in biophysical properties of channel properties. Knockdown of ATP2B4 in HAC15 exhibited reduced angiotensin II stimulation in one of four shRNA clones. Stable HAC15 cell lines with doxycycline (dox) - inducible wild-type and variant forms of ATP2B4 - were generated, and dox-induced upregulation of ATP2B4 mRNA and protein was confirmed. However, ATP2B4 variants did not alter basal or agonist-stimulated CYP11B2 expression. Whole-cell recordings in HAC15 cells indicated robust endogenous ATP2B4 conductance in native cells but reduced conductance with overexpressed WT and variant ATP2B4. The previously defined PA-causing ATP2B3 variant served as a positive control and exhibited elevated CYP11B2 mRNA. In conclusion, while this study did not confirm a pathogenic role for ATP2B4 variants in BHA, we describe the sequencing analysis for familial and sporadic BHA and outline a template for the thorough in vitro characterization of gene variants.