Atrial arrhythmogenicity of KCNJ2 mutations in short QT syndrome:Insights from virtual human atria

Gain-of-function mutations in KCNJ2-encoded Kir2.1 channels underlie variant 3 (SQT3) of the short QT syndrome, which is associated with atrial fibrillation (AF). Using biophysically-detailed human atria computer models, this study investigated the mechanistic link between SQT3 mutations and atrial arrhythmogenesis, and potential ion channel targets for treatment of SQT3. A contemporary model of the human atrial action potential (AP) was modified to recapitulate functional changes in IK1 due to heterozygous and homozygous forms of the D172N and E299V Kir2.1 mutations. Wild-type (WT) and mutant formulations were incorporated into multi-scale homogeneous and heterogeneous tissue models. Effects of mutations on AP duration (APD), conduction velocity (CV), effective refractory period (ERP), tissue excitation threshold and their rate-dependence, as well as the wavelength of re-entry (WL) were quantified. The D172N and E299V Kir2.1 mutations produced distinct effects on IK1 and APD shortening. Both mutations decreased WL for re-entry through a reduction in ERP and CV. Stability of re-entrant excitation waves in 2D and 3D tissue models was mediated by changes to tissue excitability and dispersion of APD in mutation conditions. Combined block of IK1 and IKr was effective in terminating re-entry associated with heterozygous D172N conditions, whereas IKr block alone may be a safer alternative for the E299V mutation. Combined inhibition of IKr and IKur produced a synergistic anti-arrhythmic effect in both forms of SQT3. In conclusion, this study provides mechanistic insights into atrial proarrhythmia with SQT3 Kir2.1 mutations and highlights possible pharmacological strategies for management of SQT3-linked AF

Atrial arrhythmogenicity of KCNJ2 mutations in short QT syndrome: Insights from virtual human atria

Gain-of-function mutations in KCNJ2-encoded Kir2.1 channels underlie variant 3 (SQT3) of the short QT syndrome, which is associated with atrial fibrillation (AF). Using biophysically-detailed human atria computer models, this study investigated the mechanistic link between SQT3 mutations and atrial arrhythmogenesis, and potential ion channel targets for treatment of SQT3. A contemporary model of the human atrial action potential (AP) was modified to recapitulate functional changes in IK1 due to heterozygous and homozygous forms of the D172N and E299V Kir2.1 mutations. Wild-type (WT) and mutant formulations were incorporated into multi-scale homogeneous and heterogeneous tissue models. Effects of mutations on AP duration (APD), conduction velocity (CV), effective refractory period (ERP), tissue excitation threshold and their rate-dependence, as well as the wavelength of re-entry (WL) were quantified. The D172N and E299V Kir2.1 mutations produced distinct effects on IK1 and APD shortening. Both mutations decreased WL for re-entry through a reduction in ERP and CV. Stability of re-entrant excitation waves in 2D and 3D tissue models was mediated by changes to tissue excitability and dispersion of APD in mutation conditions. Combined block of IK1 and IKr was effective in terminating re-entry associated with heterozygous D172N conditions, whereas IKr block alone may be a safer alternative for the E299V mutation. Combined inhibition of IKr and IKur produced a synergistic anti-arrhythmic effect in both forms of SQT3. In conclusion, this study provides mechanistic insights into atrial proarrhythmia with SQT3 Kir2.1 mutations and highlights possible pharmacological strategies for management of SQT3-linked AF

SARS-CoV-2, COVID-19 and inherited arrhythmia syndromes.

Ever since the first case was reported at the end of 2019, the SARS-COV-2 virus and associated lung disease COVID-19 has spread throughout the world and has become a pandemic. In particular, the high transmission rate of the virus has made it a threat to public health globally. Currently, there is no proven effective therapy against the virus, and the impact on other diseases is also uncertain, especially inherited arrhythmia syndrome. Arrhythmogenic effect of COVID-19 can be expected, potentially contributing to disease outcome. This may be of importance for patients with an increased risk for cardiac arrhythmias, either secondary to acquired conditions or co-morbidities or consequent to inherited syndromes. Management of patients with inherited arrhythmia syndromes such as Long QT syndrome, Brugada syndrome, Short QT syndrome and Catecholaminergic Polymorphic Ventricular Tachycardia in the setting of the COVID-19 pandemic may prove particularly challenging. Depending on the inherited defect involved, these patients may be susceptible to pro-arrhythmic effects of COVID-19-related issues such as fever, stress, electrolyte disturbances and use of antiviral drugs. We here describe the potential COVID-19 associated risks and therapeutic considerations for patients with distinct inherited arrhythmia syndromes and provide recommendations, pending local possibilities, for their monitoring and management during this pandemic

Effects of amiodarone on short QT syndrome variant 3 in human ventricles: a simulation study.

Background Short QT syndrome (SQTS) is a newly identified clinical disorder associated with atrial and/or ventricular arrhythmias and increased risk of sudden cardiac death (SCD). The SQTS variant 3 is linked to D172N mutation to the KCNJ2 gene that causes a gain-of-function to the inward rectifier potassium channel current (I K1), which shortens the ventricular action potential duration (APD) and effective refractory period (ERP). Pro-arrhythmogenic effects of SQTS have been characterized, but less is known about the possible pharmacological treatment of SQTS. Therefore, in this study, we used computational modeling to assess the effects of amiodarone, class III anti-arrhythmic agent, on human ventricular electrophysiology in SQT3. Methods The ten Tusscher et al. model for the human ventricular action potentials (APs) was modified to incorporate I K1 formulations based on experimental data of Kir2.1 channels (including WT, WT-D172N and D172N conditions). The modified cell model was then implemented to construct one-dimensional (1D) and 2D tissue models. The blocking effects of amiodarone on ionic currents were modeled using IC50 and Hill coefficient values from literatures. Effects of amiodarone on APD, ERP and pseudo-ECG traces were computed. Effects of the drug on the temporal and spatial vulnerability of ventricular tissue to genesis and maintenance of re-entry were measured, as well as on the dynamic behavior of re-entry. Results Amiodarone prolonged the ventricular cell APD and decreased the maximal voltage heterogeneity (δV) among three difference cells types across transmural ventricular wall, leading to a decreased transmural heterogeneity of APD along a 1D model of ventricular transmural strand. Amiodarone increased cellular ERP, prolonged QT interval and decreased the T-wave amplitude. It reduced tissue’s temporal susceptibility to the initiation of re-entry and increased the minimum substrate size necessary to sustain re-entry in the 2D tissue. Conclusions At the therapeutic-relevant concentration of amiodarone, the APD and ERP at the single cell level were increased significantly. The QT interval in pseudo-ECG was prolonged and the re-entry in tissue was prevented. This study provides further evidence that amiodarone may be a potential pharmacological agent for preventing arrhythmogenesis for SQT3 patients

Functional and pharmacological characterization of an S5 domain hERG mutation associated with short QT syndrome

Congenital short QT syndrome (SQTS) is a repolarization disorder characterized by abbreviated QT intervals, atrial and ventricular arrhythmias and a risk of sudden death. This study characterized a missense mutation (I560T) in the S5 domain of the hERG K+ channel that has been associated with variant 1 of the SQTS. Whole cell patch clamp recordings of wild-type (WT) and I560T hERG current (IhERG) were made at 37 °C from hERG expressing HEK 293 cells, and the structural context of the mutation was investigated using a recently reported cryo-EM structure of hERG. Under conventional voltage clamp, the I560T mutation increased IhERG amplitude without altering the voltage-dependence of activation, although it accelerated activation time-course and also slowed deactivation time-course at some voltages. The voltage dependence of IhERG inactivation was positively shifted (by ∼24 mV) and the time-course of inactivation was slowed by the I560T mutation. There was also a modest decrease in K+ over Na+ ion selectivity with the I560T mutation. Under action potential (AP) voltage clamp, the net charge carried by hERG was significantly increased during ventricular, Purkinje fibre and atrial APs, with maximal IhERG also occurring earlier during the plateau phase of ventricular and Purkinje fibre APs. The I560T mutation exerted only a modest effect on quinidine sensitivity of IhERG: the IC50 for mutant IhERG was 2.3 fold that for WT IhERG under conventional voltage clamp. Under AP voltage clamp the inhibitory effect of 1 μM quinidine was largely retained for I560T hERG and the timing of peak I560T IhERG was altered towards that of the WT channel. In both the open channel structure and a closed hERG channel model based on the closely-related EAG structure, I560T side-chains were oriented towards membrane lipid and away from adjacent domains of the channel, contrasting with previous predictions based on homology modelling. In summary, the I560T mutation produces multiple effects on hERG channel operation that result in a gain-of-function that is expected to abbreviate ventricular, atrial and Purkinje fibre repolarization. Quinidine is likely to be of value in offsetting the increase in IhERG and altered IhERG timing during ventricular APs in SQTS with this mutation

Investigation of the Effects of the Short QT Syndrome D172N Kir2.1 Mutation on Ventricular Action Potential Profile Using Dynamic Clamp

The congenital short QT syndrome (SQTS) is a cardiac condition that leads to abbreviated ventricular repolarization and an increased susceptibility to arrhythmia and sudden death. The SQT3 form of the syndrome is due to mutations to the KCNJ2 gene that encodes Kir2.1, a critical component of channels underlying cardiac inwardly rectifying K(+) current, I(K1). The first reported SQT3 KCNJ2 mutation gives rise to the D172N Kir2.1 mutation, the consequences of which have been studied on recombinant channels in vitro and in ventricular cell and tissue simulations. The aim of this study was to establish the effects of the D172N mutation on ventricular repolarization through real-time replacement of I(K1) using the dynamic clamp technique. Whole-cell patch-clamp recordings were made from adult guinea-pig left ventricular myocytes at physiological temperature. Action potentials (APs) were elicited at 1 Hz. Intrinsic I(K1) was inhibited with a low concentration (50 µM) of Ba(2+) ions, which led to AP prolongation and triangulation, accompanied by a ∼6 mV depolarization of resting membrane potential. Application of synthetic I(K1) through dynamic clamp restored AP duration, shape and resting potential. Replacement of wild-type (WT) I(K1) with heterozygotic (WT-D172N) or homozygotic (D172N) mutant formulations under dynamic clamp significantly abbreviated AP duration (APD(90)) and accelerated maximal AP repolarization velocity, with no significant hyperpolarization of resting potential. Across stimulation frequencies from 0.5 to 3 Hz, the relationship between APD(90) and cycle length was downward shifted, reflecting AP abbreviation at all stimulation frequencies tested. In further AP measurements at 1 Hz from hiPSC cardiomyocytes, the D172N mutation produced similar effects on APD and repolarization velocity; however, resting potential was moderately hyperpolarized by application of mutant I(K1) to these cells. Overall, the results of this study support the major changes in ventricular cell AP repolarization with the D172N predicted from prior AP modelling and highlight the potential utility of using adult ventricular cardiomyocytes for dynamic clamp exploration of functional consequences of Kir2.1 mutations

The hERG1 (KV11.1) potassium channel : its modulation and the functional characterisation of genetic variants

The human ether á-go-go related gene (hERG1 or KCNH2) encodes the pore forming subunit of the cardiac delayed rectifier potassium (IKr) channel. Its unique kinetics result in a resurgent current crucial for the repolarisation of the cardiac action potential and a capability to suppress premature excitation. hERG1 is widely expressed with roles e.g. in neuronal firing, intestinal and uterine contractility, and insulin secretion. Furthermore overexpression and ectopic expression of hERG1 occurs in cancer where it is involved in proliferation, migration, chemotherapy resistance etc. The long QT syndrome (LQTS) often presents as sudden cardiac death in children and young adults. LQTS is characterised by electrocardiogram abnormalities with arrhythmia that can lead to palpitations, syncope, seizure, cardiac arrest and death. Underlying the congenital form of LQTS are mutations in ion channel proteins (including hERG1, the loss-of-function of which gives rise to LQT2) and their interacting proteins. Carriers of a particular mutation may be symptomatic (to varying extents) or asymptomatic, with the deleterious effects only emerging due to the presence of other factors. This is analogous to drug-induced LQTS where arrhythmia may occur in 1 of 120,000 users of certain non-cardiac drugs. Virtually all drug-induced LQTS is caused by inhibition of hERG1. Consequently in the field of safety pharmacology the hERG1 channel has for the last 20 years and continues to have a huge impact as the primary in vitro predictor of the proarrhythmic risk for a drug. Various aspects of the hERG1 channel are investigated in the studies presented in this thesis. The effect of prucalopride, a gastrointestinal prokinetic drug, on hERG1 was examined. Prucalopride exhibited rapid state and concentration dependent inhibition of hERG1 however, at therapeutic concentrations block is insignificant (hERG safety margin of ≥300). This in vitro prediction has translated to the clinical studies of this drug and the market. The heterogeneous phenotype associated with LQTS may arise from genetic modifiers such polymorphisms and mutations. Heterologous expression of the prevalent hERG1 K897T polymorphism identified a reduced hERG1 current density as the primary difference from wild-type, a result of decreased protein expression. Additionally a slowing of deactivation and alteration of inactivation was evident. Also studied but using induced pluripotent stem cell (iPSC) derived cardiomyocytes was hERG1 R176W. Unlike previous LQT2 iPSC models the origin here was a relatively asymptomatic individual. The phenotypic characteristics of LQT2 were however still reproduced in vitro (i.e. a decrease in IKr and action potential prolongation) though as a milder version. Finally the effect of ceramide, a sphingolipid which accumulates in heart failure and is involved in lipotoxicity, on hERG1 was investigated. Ceramide was found to reduce hERG1 current in a time dependent manner through tagging (ubiquitination) of the cell surface protein for internalisation and targeting to lysosomes.HERG (human ether-a-go-go related gene, hERG1 tai KCNH2) geenin ilmentämä proteiini tuottaa kaliumkanavan, joka vastaa sydänsolujen sähköfysiologisissa mittauksissa kuvattua IKr virtaa. HERG kaliumkanava aukeaa sydämen aktiopotentiaalin repolarisaatiovaiheessa ja estää näin ennenaikaisen aktiopotentiaalin laukeamisen. hERG1 proteiinia ilmentyy sydämen lisäksi useissa muissa kudoksissa mm. hermosoluissa, suolessa, kohdussa ja haiman insuliinia tuottavissa soluissa. hERG1 ilmentyy lisäksi useissa eri syöpäsoluissa, joissa se osallistuu syöpäsolujen jakautumisen säätelyyn, etäpesäkkeiden syntymiseen ja kemoterapiaresistenssin muodostumiseen. Pitkä QT-oireyhtymä on harvinainen perinnöllinen sairaus, joka ilmentyy lasten ja nuorten aikuisten sydänperäisenä äkkikuolemana. Pitkä QT-oireyhtymä voidaan tunnistaa sydänsähkökäyrästä kääntyvien kärkien kammiotakykardiana. Pitkä QT-oireyhtymän oireita ovat tajuttomuus, epilepsia, sydänpysähdys ja äkkikuolema. Geneettisten tutkimusten ansiosta tiedetään että perinnöllisen pitkä-QT oireyhtymän alaluokka LQT2 johtuu joko HERG geenin mutaatioista tai sen kanssa vuorovaikuttavien proteiinien mutaatioista. HERG mutaatioiden kantajat voivat olla täysin oireettomia mutta haitalliset henkeä uhkaavat vaikutukset saattavat tulla esille muiden tekijöiden vaikutuksesta. Joidenkin lääkkeiden erittäin harvinaisena haittavaikutuksena ilmentyvä rytmihäiriö on analoginen pitkä QT-oireyhtymän kanssa. Lääkeen aiheuttaman rytmihäiriön esiintyvyys on 1:120 000 tiettyjen muiden kuin sydänlääkkeiden lääkkeiden käyttäjistä. Käytännössä lähes kaikki lääkkeiden aiheuttamat sydämen rytmihäiriöt aiheutuvat HERG kaliumkanavan salpauksesta. Sen takia ei ole sattumaa että lääketeollisuuden varhainen lääketurvallisuutta ennustava tutkimus on siirtynyt laaja-alaisesti käyttämään HERG kaliumkanavan sähköfysiologisia mittauksia entistäkin turvallisempien lääkemolekyylien seulomiseksi ja kehittämiseksi lääkkeeksi asti. Tässä väitöskirjassa on tutkittu hERG1 kaliumkanavaa useasta eri lähtökohdasta käsin. Ensimäisessä julkaisussa kyettiin osoittamaan sähköfysiologisin in vitro mittauksin että ummetuksen hoitoon kehitetty lääke prucalopride salpasi hERG1 kaliumkanavaa vasta yli 300-kertaa suuremmissa pitoisuuksissa kuin mitä tarvitaan lääkkeen kliinisen tehon esille saamiseksi. Näin ollen in vitro mittauksin kyettiin ennustamaan että lääkkeellä on äärimmäisen pieni riski aihuttaa sydänperäisiä oireita, joka on sittemmin näytetty toteen kliinisissä tutkimuksissa. Toisessa julkaisussa tutkittiin pitkä-QT oireyhtymään liittyvää vaihtelevaa sähköfysiologista ilmiasua, joka voi aiheutua joko HERG geenin mutaatioista tai monimuotoisuudesta. HERG geenin K897T monimuotoinen versio vähensi solumallissa HERG proteiinin määrää verrattuna valtamutaatioon. Lisäksi monimuotoisen ionikanavan aukeamisen ja sulkeutumisen nopeus erosi valtamutaatiosta. Kolmannessa julkaisussa tutkittiin uudelleenohjelmoidussa sydänkantasolumallissa (iPS kardiomyosyytti) HERG proteiinin R176W monimuotoista versiota, joka oli peräisin melko oireettomalta henkilöltä. Pitkä QT-oireyhtymän ilmiasu (sydänsolun IKr virran väheneminen ja aktiopotentiaalin keston piteneminen) tuli esille sykkivien uudelleenohjelmoitujen sydänkantasolujen sähköfysiologisissa mittauksissa. Viimeisessä julkaisussa tutkittiin miten sydämen vajaatoiminnan aikana sydänlihakseen kertyvä sfingolipidi keramidi vaikuttaa HERG kaliumkanavan toimintaan. Osoittautui että keramidi vähensi HERG ionivirtaa ajasta riippuvasti, jolloin solukalvolla oleva HERG proteiini leimattiin ubikitiinillä solun sisälle lysosomiin hajotettavaksi