Kevin J. Sampson

Mutation of an A-kinase-anchoring protein causes long-QT syndrome

A-kinase anchoring proteins (AKAPs) recruit signaling molecules and present them to downstream targets to achieve efficient spatial and temporal control of their phosphorylation state. In the heart, sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD), mediated by β-adrenergic receptor (βAR) activation, requires assembly of AKAP9 (Yotiao) with the IKs potassium channel α subunit (KCNQ1). KCNQ1 mutations that disrupt this complex cause type 1 long-QT syndrome (LQT1), one of the potentially lethal heritable arrhythmia syndromes. Here, we report identification of (i) regions on Yotiao critical to its binding to KCNQ1 and (ii) a single putative LQTS-causing mutation (S1570L) in AKAP9 (Yotiao) localized to the KCNQ1 binding domain in 1/50 (2%) subjects with a clinically robust phenotype for LQTS but absent in 1,320 reference alleles. The inherited S1570L mutation reduces the interaction between KCNQ1 and Yotiao, reduces the cAMP-induced phosphorylation of the channel, eliminates the functional response of the IKs channel to cAMP, and prolongs the action potential in a computational model of the ventricular cardiocyte. These reconstituted cellular consequences of the inherited S1570L-Yotiao mutation are consistent with delayed repolarization of the ventricular action potential observed in the affected siblings. Thus, we have demonstrated a link between genetic perturbations in AKAP and human disease in general and AKAP9 and LQTS in particular.

Molecular basis of ranolazine block of LQT-3 mutant sodium channels: evidence for site of action

1 We studied the effects of ranolazine, an antianginal agent with promise as an antiarrhythmic drug, on wild‐type (WT) and long QT syndrome variant 3 (LQT‐3) mutant Na+ channels expressed in human embryonic kidney (HEK) 293 cells and knock‐in mouse cardiomyocytes and used site‐directed mutagenesis to probe the site of action of the drug. 2 We find preferential ranolazine block of sustained vs peak Na+ channel current for LQT‐3 mutant (ΔKPQ and Y1795C) channels (IC50=15 vs 135 μM) with similar results obtained in HEK 293 cells and knock‐in myocytes. 3 Ranolazine block of both peak and sustained Na+ channel current is significantly reduced by mutation (F1760A) of a single residue previously shown to contribute critically to the binding site for local anesthetic (LA) molecules in the Na+ channel. 4 Ranolazine significantly decreases action potential duration (APD) at 50 and 90% repolarization by 23±5 and 27±3%, respectively, in ΔKPQ mouse ventricular myocytes but has little effect on APD of WT myocytes. 5 Computational modeling of human cardiac myocyte electrical activity that incorporates our voltage‐clamp data predicts marked ranolazine‐induced APD shortening in cells expressing LQT‐3 mutant channels. 6 Our results demonstrate for the first time the utility of ranolazine as a blocker of sustained Na+ channel activity induced by inherited mutations that cause human disease and further, that these effects are very likely due to interactions of ranolazine with the receptor site for LA molecules in the sodium channel.

Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics

Understanding the basis for differential responses to drug therapies remains a challenge despite advances in genetics and genomics. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to investigate the pharmacology of disease processes in therapeutically and genetically relevant primary cell types in vitro and to interweave clinical and basic molecular data. We report here the derivation of iPSCs from a long QT syndrome patient with complex genetics. The proband was found to have a de novo SCN5A LQT-3 mutation (F1473C) and a polymorphism (K897T) in KCNH2, the gene for LQT-2. Analysis of the biophysics and molecular pharmacology of ion channels expressed in cardiomyocytes (CMs) differentiated from these iPSCs (iPSC-CMs) demonstrates a primary LQT-3 (Na+ channel) defect responsible for the arrhythmias not influenced by the KCNH2 polymorphism. The F1473C mutation occurs in the channel inactivation gate and enhances late Na+ channel current (INaL) that is carried by channels that fail to inactivate completely and conduct increased inward current during prolonged depolarization, resulting in delayed repolarization, a prolonged QT interval, and increased risk of fatal arrhythmia. We find a very pronounced rate dependence of INaL such that increasing the pacing rate markedly reduces INaL and, in addition, increases its inhibition by the Na+ channel blocker mexiletine. These rate-dependent properties and drug interactions, unique to the proband’s iPSC-CMs, correlate with improved management of arrhythmias in the patient and provide support for this approach in developing patient-specific clinical regimens.

Autonomic Control of Cardiac Action Potentials. Role of Potassium Channel Kinetics in Response to Sympathetic Stimulation

IKs, the slowly activating component of the delayed rectifier current, plays a major role in repolarization of the cardiac action potential (AP). Genetic mutations in the &agr;- (KCNQ1) and &bgr;- (KCNE1) subunits of IKs underlie Long QT Syndrome type 1 and 5 (LQT-1 and LQT-5), respectively, and predispose carriers to the development of polymorphic ventricular arrhythmias and sudden cardiac death. &bgr;-adrenergic stimulation increases IKs and results in rate dependent AP shortening, a control system that can be disrupted by some mutations linked to LQT-1 and LQT-5. The mechanisms by which IKs regulates action potential duration (APD) during &bgr;-adrenergic stimulation at different heart rates are not known, nor are the consequences of mutation induced disruption of this regulation. Here we develop a complementary experimental and theoretical approach to address these questions. We reconstituted IKs in CHO cells (ie, KCNQ1 coexpressed with KCNE1 and the adaptator protein Yotiao) and quantitatively examined the effects of &bgr;-adrenergic stimulation on channel kinetics. We then developed theoretical models of IKs in the absence and presence of &bgr;-adrenergic stimulation. We simulated the effects of sympathetic stimulation on channel activation (speeding) and deactivation (slowing) kinetics on the whole cell action potential under different pacing conditions. The model suggests these kinetic effects are critically important in rate-dependent control of action potential duration. We also investigate the effects of two LQT-5 mutations that alter kinetics and impair sympathetic stimulation of IKs and show the likely mechanism by which they lead to tachyarrhythmias and indicate a distinct role of IKS kinetics in this electrical dysfunction. The full text of this article is available online at http://circres.ahajournals.org.

KCNE1 alters the voltage sensor movements necessary to open the KCNQ1 channel gate

The delayed rectifier IKs potassium channel, formed by coassembly of α- (KCNQ1) and β- (KCNE1) subunits, is essential for cardiac function. Although KCNE1 is necessary to reproduce the functional properties of the native IKs channel, the mechanism(s) through which KCNE1 modulates KCNQ1 is unknown. Here we report measurements of voltage sensor movements in KCNQ1 and KCNQ1/KCNE1 channels using voltage clamp fluorometry. KCNQ1 channels exhibit indistinguishable voltage dependence of fluorescence and current signals, suggesting a one-to-one relationship between voltage sensor movement and channel opening. KCNE1 coexpression dramatically separates the voltage dependence of KCNQ1/KCNE1 current and fluorescence, suggesting an imposed requirement for movements of multiple voltage sensors before KCNQ1/KCNE1 channel opening. This work provides insight into the mechanism by which KCNE1 modulates the IKs channel and presents a mechanism for distinct β-subunit regulation of ion channel proteins.

Altered Na+ Channels Promote Pause-Induced Spontaneous Diastolic Activity in Long QT Syndrome Type 3 Myocytes

Long QT syndrome (LQTS) type 3 (LQT3), typified by the ΔKPQ mutation (LQT3 mutation in which amino acid residues 1505 to 1507 [KPQ] are deleted), is caused by increased sodium entry during the action potential plateau resulting from mutation-altered inactivation of the Nav1.5 channel. Although rare, LQT3 is the most lethal of common LQTS variants. Here we tested the hypothesis that cellular electrical dysfunction, caused not only by action potential prolongation but also by mutation-altered Na+ entry, distinguishes LQT3 from other LQTS variants and may contribute to its distinct lethality. We compared cellular electrical activity in myocytes isolated from mice heterozygous for the ΔKPQ mutation (ΔKPQ) and myocytes from wild-type littermates. Current-clamp pause protocols induced rate-dependent spontaneous diastolic activity (delayed after depolarizations) in 6 of 7 ΔKPQ, but no wild-type, myocytes (n=11) tested. Voltage-clamp pause protocols that independently control depolarization duration and interpulse interval identified a distinct contribution of both depolarization duration and mutant Na+ channel activity to the generation of Cai2+-dependent diastolic transient inward current. This was found at rates and depolarization durations relevant both to the mouse model and to LQT3 patients. Flecainide, which preferentially inhibits mutation-altered late Na+ current and is used to treat LQT3 patients, suppresses transient inward current formation in voltage-clamped ΔKPQ myocytes. Our results demonstrate a marked contribution of mutation-altered Na+ entry to the incidence of pause-dependent spontaneous diastolic activity in ΔKPQ myocytes and suggest that altered Na+ entry may contribute to the elevated lethality of LQT3 versus other LQTS variants.

A computational model of Purkinje fibre single cell electrophysiology: implications for the long QT syndrome

Computer modelling has emerged as a particularly useful tool in understanding the physiology and pathophysiology of cardiac tissues. Models of ventricular, atrial and nodal tissue have evolved and include detailed ion channel kinetics and intercellular Ca2+ handling. Purkinje fibre cells play a central role in the electrophysiology of the heart and in the genesis of cardiac arrhythmias. In this study, a new computational model has been constructed that incorporates the major membrane currents that have been isolated in recent experiments using Purkinje fibre cells. The model, which integrates mathematical models of human ion channels based on detailed biophysical studies of their kinetic and voltage‐dependent properties, recapitulates distinct electrophysiological characteristics unique to Purkinje fibre cells compared to neighbouring ventricular myocytes. These characteristics include automaticity, hyperpolarized voltage range of the action potential plateau potential, and prolonged action potential duration. Simulations of selective ion channel blockade reproduce responses to pharmacological challenges characteristic of isolated Purkinje fibres in vitro, and importantly, the model predicts that Purkinje fibre cells are prone to severe arrhythmogenic activity in patients harbouring long QT syndrome 3 but much less so for other common forms of long QT. This new Purkinje cellular model can be a useful tool to study tissue‐specific drug interactions and the effects of disease‐related ion channel dysfunction on the cardiac conduction system.

Allosteric gating mechanism underlies the flexible gating of KCNQ1 potassium channels

KCNQ1 (Kv7.1) is a unique member of the superfamily of voltage-gated K+ channels in that it displays a remarkable range of gating behaviors tuned by coassembly with different β subunits of the KCNE family of proteins. To better understand the basis for the biophysical diversity of KCNQ1 channels, we here investigate the basis of KCNQ1 gating in the absence of β subunits using voltage-clamp fluorometry (VCF). In our previous study, we found the kinetics and voltage dependence of voltage-sensor movements are very similar to those of the channel gate, as if multiple voltage-sensor movements are not required to precede gate opening. Here, we have tested two different hypotheses to explain KCNQ1 gating: (i) KCNQ1 voltage sensors undergo a single concerted movement that leads to channel opening, or (ii) individual voltage-sensor movements lead to channel opening before all voltage sensors have moved. Here, we find that KCNQ1 voltage sensors move relatively independently, but that the channel can conduct before all voltage sensors have activated. We explore a KCNQ1 point mutation that causes some channels to transition to the open state even in the absence of voltage-sensor movement. To interpret these results, we adopt an allosteric gating scheme wherein KCNQ1 is able to transition to the open state after zero to four voltage-sensor movements. This model allows for widely varying gating behavior, depending on the relative strength of the opening transition, and suggests how KCNQ1 could be controlled by coassembly with different KCNE family members.

A Novel and Lethal De Novo LQT-3 Mutation in a Newborn with Distinct Molecular Pharmacology and Therapeutic Response

Background SCN5A encodes the α-subunit (Nav1.5) of the principle Na+ channel in the human heart. Genetic lesions in SCN5A can cause congenital long QT syndrome (LQTS) variant 3 (LQT-3) in adults by disrupting inactivation of the Nav1.5 channel. Pharmacological targeting of mutation-altered Na+ channels has proven promising in developing a gene-specific therapeutic strategy to manage specifically this LQTS variant. SCN5A mutations that cause similar channel dysfunction may also contribute to sudden infant death syndrome (SIDS) and other arrhythmias in newborns, but the prevalence, impact, and therapeutic management of SCN5A mutations may be distinct in infants compared with adults. Methods and Results Here, in a multidisciplinary approach, we report a de novo SCN5A mutation (F1473C) discovered in a newborn presenting with extreme QT prolongation and differential responses to the Na+ channel blockers flecainide and mexiletine. Our goal was to determine the Na+ channel phenotype caused by this severe mutation and to determine whether distinct effects of different Na+ channel blockers on mutant channel activity provide a mechanistic understanding of the distinct therapeutic responsiveness of the mutation carrier. Sequence analysis of the proband revealed the novel missense SCN5A mutation (F1473C) and a common variant in KCNH2 (K897T). Patch clamp analysis of HEK 293 cells transiently transfected with wild-type or mutant Na+ channels revealed significant changes in channel biophysics, all contributing to the probands phenotype as predicted by in silico modeling. Furthermore, subtle differences in drug action were detected in correcting mutant channel activity that, together with both the known genetic background and age of the patient, contribute to the distinct therapeutic responses observed clinically. Significance The results of our study provide further evidence of the grave vulnerability of newborns to Na+ channel defects and suggest that both genetic background and age are particularly important in developing a mutation-specific therapeutic personalized approach to manage disorders in the young.

Adrenergic regulation of a key cardiac potassium channel can contribute to atrial fibrillation: evidence from an IKs transgenic mouse

Inherited gain‐of‐function mutations of genes coding for subunits of the heart slow potassium (IKs) channel can cause familial atrial fibrillation (AF). Here we consider a potentially more prevalent mechanism and hypothesize that β‐adrenergic receptor (β‐AR)‐mediated regulation of the IKs channel, a natural gain‐of‐function pathway, can also lead to AF. Using a transgenic IKs channel mouse model, we studied the role of the channel and its regulation by β‐AR stimulation on atrial arrhythmias. In vivo administration of isoprenaline (isoproterenol) predisposes IKs channel transgenic mice but not wild‐type (WT) littermates that lack IKs to prolonged atrial arrhythmias. Patch‐clamp analysis demonstrated expression and isoprenaline‐mediated regulation of IKs in atrial myocytes from transgenic but not WT littermates. Furthermore, computational modelling revealed that β‐AR stimulation‐dependent accumulation of open IKs channels accounts for the pro‐arrhythmic substrate. Our results provide evidence that β‐AR‐regulated IKs channels can play a role in AF and imply that specific IKs deregulation, perhaps through disruption of the IKs macromolecular complex necessary for β‐AR‐mediated IKs channel regulation, may be a novel therapeutic strategy for treating this most common arrhythmia.