Biophysical Characterization of a Novel SCN5A Mutation Associated With an Atypical Phenotype of Atrial and Ventricular Arrhythmias and Sudden Death

Background: Sudden cardiac death (SCD) is an unexpected death that occurs within an hour of the onset of symptoms. Hereditary primary electrical disorders account for up to 1/3 of all SCD cases in younger individuals and include conditions such as catecholaminergic polymorphic ventricular tachycardia (CPVT). These disorders are caused by mutations in the genes encoding cardiac ion channels, hence they are known as cardiac channelopathies. We identified a novel variant, T1857I, in the C-terminus of Nav1.5 (SCN5A) linked to a family with a CPVT-like phenotype characterized by atrial tachy-arrhythmias and polymorphic ventricular ectopy occurring at rest and with adrenergic stimulation, and a strong family history of SCD. Objective: Our goal was to functionally characterize the novel Nav1.5 variant and determine a possible link between channel gating and clinical phenotype. Methods: We first used electrocardiogram recordings to visualize the patient cardiac electrical properties. Then, we performed voltage-clamp of transiently transfected CHO cells. Lastly, we used the ventricular/atrial models to visualize gating defects on cardiac excitability. Results: Voltage-dependences of both activation and inactivation were right-shifted, the overlap between activation and inactivation predicted increased window currents, the recovery from fast inactivation was slowed, there was no significant difference in late currents, and there was no difference in use-dependent inactivation. The O’Hara-Rudy model suggests ventricular after depolarizations and atrial Grandi-based model suggests a slight prolongation of atrial action potential duration. Conclusion: We conclude that T1857I likely causes a net gain-of-function in Nav1.5 gating, which may in turn lead to ventricular after depolarization, predisposing carriers to tachy-arrhythmias

Modulating human iPSC-CM electrophysiology for in vitro drug screening

Every year drugs are retracted from the market due to serious side effects, of which 45% negatively influences heart rhythm. This can be prevented with proper testing during drug development to determine the safety for the human heart. However, in drug development mostly animals are used to study the effects of drugs, yet an animal heart responds differently to drugs than a human heart. A human alternative is necessary to correctly predict the safety of new drugs and decrease the amount of tests in animals. This transition has been boosted by the development of stem cell-derived heart muscle cells (iPSC-CMs): cells from the skin that have been transformed to stem cells and subsequently to heart muscle cells. iPSC-CMs harbor one major disadvantage: they do not resemble human adult heart muscle cells electrically and are hyperactive, which decreases the accuracy of their drug response. In this thesis we applied various techniques to modulate the electrical characteristics of iPSC-CMs to create a better model for animal-free drug screening. We succeeded in decreasing the hyperactivity of iPSC-CMs and observed a more accurate drug response. We observed variation between iPSC-CMs in what approach they needed to decrease the hyperactivity, which needed to be tailored for each individual cell. Because of this variation it is difficult to pick one strategy which works best for all iPSC-CMs. The variation between iPSC-CMs is a worldwide problem; besides, guidelines for the production and use of iPSC-CMs are currently lacking, which hampers the transition to test animal-free greatly

Modulating human iPSC-CM electrophysiology for in vitro drug screening

Every year drugs are retracted from the market due to serious side effects, of which 45% negatively influences heart rhythm. This can be prevented with proper testing during drug development to determine the safety for the human heart. However, in drug development mostly animals are used to study the effects of drugs, yet an animal heart responds differently to drugs than a human heart. A human alternative is necessary to correctly predict the safety of new drugs and decrease the amount of tests in animals. This transition has been boosted by the development of stem cell-derived heart muscle cells (iPSC-CMs): cells from the skin that have been transformed to stem cells and subsequently to heart muscle cells. iPSC-CMs harbor one major disadvantage: they do not resemble human adult heart muscle cells electrically and are hyperactive, which decreases the accuracy of their drug response. In this thesis we applied various techniques to modulate the electrical characteristics of iPSC-CMs to create a better model for animal-free drug screening. We succeeded in decreasing the hyperactivity of iPSC-CMs and observed a more accurate drug response. We observed variation between iPSC-CMs in what approach they needed to decrease the hyperactivity, which needed to be tailored for each individual cell. Because of this variation it is difficult to pick one strategy which works best for all iPSC-CMs. The variation between iPSC-CMs is a worldwide problem; besides, guidelines for the production and use of iPSC-CMs are currently lacking, which hampers the transition to test animal-free greatly

The immature electrophysiological phenotype of iPSC-CMs still hampers in vitro drug screening : Special focus on IK1

Preclinical drug screens are not based on human physiology, possibly complicating predictions on cardiotoxicity. Drug screening can be humanised with in vitro assays using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, in contrast to adult ventricular cardiomyocytes, iPSC-CMs beat spontaneously due to presence of the pacemaking current If and reduced densities of the hyperpolarising current IK1. In adult cardiomyocytes, IK1 finalises repolarisation by stabilising the resting membrane potential while also maintaining excitability. The reduced IK1 density contributes to proarrhythmic traits in iPSC-CMs, which leads to an electrophysiological phenotype that might bias drug responses. The proarrhythmic traits can be suppressed by increasing IK1 in a balanced manner. We systematically evaluated all studies that report strategies to mature iPSC-CMs and found that only few studies report IK1 current densities. Furthermore, these studies did not succeed in establishing sufficient IK1 levels as they either added too little or too much IK1. We conclude that reduced densities of IK1 remain a major flaw in iPSC-CMs, which hampers their use for in vitro drug screening

Required GK1 to Suppress Automaticity of iPSC-CMs Depends Strongly on IK1 Model Structure

Human-induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) are a virtually endless source of human cardiomyocytes that may become a great tool for safety pharmacology; however, their electrical phenotype is immature: they show spontaneous action potentials (APs) and an unstable and depolarized resting membrane potential (RMP) because of lack of IK1. Such immaturity hampers their application in assessing drug safety. The electronic overexpression of IK1 (e.g., through the dynamic clamp (DC) technique) is an option to overcome this deficit. In this computational study, we aim to estimate how much IK1 is needed to bring hiPSC-CMs to a stable and hyperpolarized RMP and which mathematical description of IK1 is most suitable for DC experiments. We compared five mature IK1 formulations (Bett, Dhamoon, Ishihara, O'Hara-Rudy, and ten Tusscher) with the native one (Paci), evaluating the main properties (outward peak, degree of rectification), and we quantified their effects on AP features (RMP, V˙max, APD50, APD90 (AP duration at 50 and 90% of repolarization), and APD50/APD90) after including them in the hiPSC-CM mathematical model by Paci. Then, we automatically identified the critical conductance for IK1 ( GK1, critical), the minimally required amount of IK1 suppressing spontaneous activity. Preconditioning the cell model with depolarizing/hyperpolarizing prepulses allowed us to highlight time dependency of the IK1 formulations. Simulations showed that inclusion of mature IK1 formulations resulted in hyperpolarized RMP and higher V˙max, and observed GK1, critical and the effect on AP duration strongly depended on IK1 formulation. Finally, the Ishihara IK1 led to shorter (-16.3%) and prolonged (+6.5%) APD90 in response to hyperpolarizing and depolarizing prepulses, respectively, whereas other models showed negligible effects. Fine-tuning of GK1 is an important step in DC experiments. Our computational work proposes a procedure to automatically identify how much IK1 current is required to inject to stop the spontaneous activity and suggests the use of the Ishihara IK1 model to perform DC experiments in hiPSC-CMs

A systematic literature review of economic evaluations and cost-of-illness studies of inherited cardiomyopathies

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are commonly inherited heart conditions associated with a high risk of heart failure and sudden cardiac death. To understand the economic and societal disease burden, this study systematically identified and reviewed cost-of-illness (COI) studies and economic evaluations (EEs) of various interventions for HCM and DCM. A literature search was performed in MEDLINE, EMBASE, NHS EED, EconLit and Web of Science to identify COI studies and EEs published between 1 January 2010 and 28 April 2021. The selection of studies and their critical appraisal were performed jointly by two independent researchers. For the quality assessment, the ‘Consensus on Health Economic Criteria’ list was used. Two COI studies and 11 EEs were eligible for inclusion. Cost-effectiveness varied among interventions and depended on the targeted patient population. Both COI studies identified only hospitalisation costs in HCM. The mean study quality was high in EEs but low in COI studies. Most studies excluded costs for patients, caregivers and productivity losses. Overall, knowledge of the societal and economic burden of inherited cardiomyopathies is limited. Future research needs to include quality-adjusted life years and a broader range of costs to provide an information base for optimising care for affected patients