Effects of Amiodarone and N-desethylamiodarone on Cardiac Voltage-Gated Sodium Channels

Amiodarone (AMD) is a potent antiarrhythmic drug with high efficacy for treating atrial fibrillation and tachycardia. The pharmacologic profile of AMD is complex. AMD possesses biophysical characteristics of all of class I, II, III, and IV agents. Despite its adverse side effects, AMD remains the most commonly prescribed antiarrhythmic drug. AMD was described to prolong the QT interval and can lead to torsades de pointes. Our goal was to study the effects of AMD on peak and late sodium currents (INa,P and INa,L) and determine whether these effects change as AMD is metabolized into N-Desethylamiodarone (DES). We hypothesized that AMD and DES block both INa,P and INa,L with similar profiles due to structural similarities. Given the inherent small amounts of INa,L in NaV1.5, we screened AMD and DES against the Long QT-3-causing mutation, ∆KPQ, to better detect any drug-mediated effect on INa,L. Our results show that AMD and DES do not affect WT or ∆KPQ activation; however, both drugs altered the apparent valence of steady-state fast-inactivation. In addition, AMD and DES preferentially block ∆KPQ peak conductance compared to WT. Both compounds significantly increase INa,L and window currents. We conclude that both compounds have pro-arrhythmic effects on NaV1.5, especially ∆KPQ; however, DES seems to have a greater pro-arrhythmic effect than AMD

A Mixed Periodic Paralysis & Myotonia Mutant, P1158S, Imparts pH-Sensitivity in Skeletal Muscle Voltage-gated Sodium Channels

Skeletal muscle channelopathies, many of which are inherited as autosomal dominant mutations, include myotonia and periodic paralysis. Myotonia is defined by a delayed relaxation after muscular contraction, whereas periodic paralysis is defined by episodic attacks of weakness. One sub-type of periodic paralysis, known as hypokalemic periodic paralysis (hypoPP), is associated with low potassium levels. Interestingly, the P1158S missense mutant, located in the third domain S4-S5 linker of the “skeletal muscle”, Nav1.4, has been implicated in causing both myotonia and hypoPP. A common trigger for these conditions is physical activity. We previously reported that Nav1.4 is relatively insensitive to changes in extracellular pH compared to Nav1.2 and Nav1.5. Given that intense exercise is often accompanied by blood acidosis, we decided to test whether changes in pH would push gating in P1158S towards either phenotype. Our results suggest that, unlike in WT-Nav1.4, low pH depolarizes the voltage-dependence of activation and steady-state fast inactivation, decreases current density, and increases late currents in P1185S. Thus, P1185S turns the normally pH-insensitive Nav1.4 into a proton-sensitive channel. Using action potential modeling we predict a pH-to-phenotype correlation in patients with P1158S. We conclude that activities which alter blood pH may trigger the noted phenotypes in P1158S patients

Mechanisms Underlying the Antiarrhythmic Effect of ARumenamide-787 in Experimental Models of the J Wave Syndromes and Hypothermia

BACKGROUND: Brugada (BrS) and early repolarization syndromes (ERS), the so-called J wave syndromes (JWS), are associated with life-threatening ventricular arrhythmias. Pharmacologic approaches to therapy are currently limited. In this study, we examine the effects of ARumenamide-787 (AR-787) to suppress the electrocardiographic and arrhythmic manifestations of JWS and hypothermia. METHODS: We studied the effects of AR-787 on INa and IKr in HEK-293 cells stably expressing the α- and β1-subunits of the cardiac (NaV1.5) sodium channel and hERG channel, respectively. In addition, we studied its effect on Ito, INa and ICa in dissociated canine ventricular myocytes along with action potentials and ECG from coronary-perfused right (RV) and left (LV) ventricular wedge preparations. The Ito agonist, NS5806 (5-10 μM), ICa blocker, verapamil (2.5 μM), and INa blocker, ajmaline (2.5 μM), were used to mimic the genetic defects associated with JWS and to induce the electrocardiographic and arrhythmic manifestations of JWS (prominent J waves/ST segment elevation, phase 2 reentry and polymorphic VT/VF) in canine ventricular wedge preparations. RESULTS: AR-787 (1, 10 and 50 μM) exerted pleiotropic effects on cardiac ion channels. The predominant effect was inhibition of the transient outward current (Ito) and enhancement of the sodium channel current (INa), with lesser effects to inhibit IKr and augment calcium channel current (ICa). AR-787 diminished the electrocardiographic J wave and prevented and/or suppressed all arrhythmic activity in canine RV and LV experimental models of BrS, ERS and hypothermia. CONCLUSIONS: Our findings point to AR-787 as promising candidate for the pharmacologic treatment of JWS and hypothermia

SCN5A mutations in 442 neonates and children: genotype-phenotype correlation and identification of higher-risk subgroups.

Aims To clarify the clinical characteristics and outcomes of children with SCN5A-mediated disease and to improve their risk stratification. Methods and results A multicentre, international, retrospective cohort study was conducted in 25 tertiary hospitals in 13 countries between 1990 and 2015. All patients ≤16 years of age diagnosed with a genetically confirmed SCN5A mutation were included in the analysis. There was no restriction made based on their clinical diagnosis. A total of 442 children {55.7% boys, 40.3% probands, median age: 8.0 [interquartile range (IQR) 9.5] years} from 350 families were included; 67.9% were asymptomatic at diagnosis. Four main phenotypes were identified: isolated progressive cardiac conduction disorders (25.6%), overlap phenotype (15.6%), isolated long QT syndrome type 3 (10.6%), and isolated Brugada syndrome type 1 (1.8%); 44.3% had a negative electrocardiogram phenotype. During a median follow-up of 5.9 (IQR 5.9) years, 272 cardiac events (CEs) occurred in 139 (31.5%) patients. Patients whose mutation localized in the C-terminus had a lower risk. Compound genotype, both gain- and loss-of-function SCN5A mutation, age ≤1 year at diagnosis in probands and age ≤1 year at diagnosis in non-probands were independent predictors of CE. Conclusion In this large paediatric cohort of SCN5A mutation-positive subjects, cardiac conduction disorders were the most prevalent phenotype; CEs occurred in about one-third of genotype-positive children, and several independent risk factors were identified, including age ≤1 year at diagnosis, compound mutation, and mutation with both gain- and loss-of-function

Exome sequencing and the management of neurometabolic disorders

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.)

Exome Sequencing and the Management of Neurometabolic Disorders

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.)

Physiological and pharmacological switches combine to uniquely modulate the most common cardiac sodium channel mutant, E1784K

The SCN5a gene encodes the cardiac voltage-gated sodium channel (NaV1.5) mainly expressed in cardiac muscle cells. The inward sodium current (INa) conducted by NaV1.5 triggers depolarization in the cardiac action potential. Mutations in SCN5a predominantly give rise to Long-QT syndrome 3 (LQT3), Brugada syndrome 1 (BrS1), and their overlapping phenotypes (mixed syndrome). The most common SCN5a mutation, expressed as E1784K in the NaV1.5 C-terminal domain (CTD), mainly displays LQT3 and sometimes mixed syndromes. E1784K causes mixed channel defects by decreasing the inward peak INa and increasing late INa, thought to underlie BrS1 and LQT3 pathogeneses, respectively. Very little is known, however, on how physiological and pharmacological switches modulate E1784K channel properties. These triggers may often govern phenotypes in SCN5a mutation carriers. The goal of my thesis is to study how exercise-related physiological triggers and pharmacological agents modulate E1784K ion channel properties. I used the whole-cell patch clamp technique to study elevated temperature, elevated cytosolic calcium, and their combined effects with ranolazine, on E1784K. Ranolazine is an antianginal drug with preferential selectivity for blocking late INa versus peak INa. My main results show that E1784K is uniquely altered by the triggers studied, compared to other NaV1.5 mutants: (1) Elevated temperature augments late INa in E1784K. (2) Elevated cytosolic calcium, which correlates with exercise-ameliorated LQT3, effectively blocks late INa in most NaV1.5 mutants. However, E1784K is resistant to the native calcium-induced block on late INa. (3) When temperature and cytosolic calcium are combined, they decrease ranolazine efficacy to suppress late INa in E1784K. The calcium-sensitivity in E1784K is clearly affected due to the mutant-induced instability in the CTD, which may cause a steric clash between the channel and ranolazine. To predict E1784K effects on arrhythmogenesis, I simulated a dynamic action potential model to account for the frequency-dependent elevations in cytosolic calcium. Alternans is observed at high heart rates in E1784K and is exacerbated by febrile temperatures and ranolazine. This work demonstrates the importance of personalized medicine since NaV1.5 mutants like E1784K display unique sensitivity to physiological triggers that potentially govern antiarrhythmic efficacy

Repurposing drugs to treat cardiovascular disease in the era of precision medicine

Drug repurposing is the use of a given therapeutic agent for indications other than that for which it was originally designed or intended. The concept is appealing because of potentially lower development costs and shorter timelines than are needed to produce a new drug. To date, drug repurposing for cardiovascular indications has been opportunistic and driven by knowledge of disease mechanisms or serendipitous observation rather than by systematic endeavours to match an existing drug to a new indication. Innovations in two areas of personalized medicine — computational approaches to associate drug effects with disease signatures and predictive model systems to screen drugs for disease-modifying activities — support efforts that together create an efficient pipeline to systematically repurpose drugs to treat cardiovascular disease. Furthermore, new experimental strategies that guide the medicinal chemistry re-engineering of drugs could improve repurposing efforts by tailoring a medicine to its new indication. In this Review, we summarize the historical approach to repurposing and discuss the technological advances that have created a new landscape of opportunities