Long QT Syndrome modelled with human induced pluripotent stem cells (hiPSc)

Cardiac arrhythmias arise when the myocardium is electrically unstable. They represent a life-threatening phenomena with more than 300,000 people dying every year for arrhythmic sudden cardiac death (SCD) only in the United States. One of the most important and well described arrhythmic condition is the Long QT Syndrome (LQTS). LQTS is an heterogeneous disorder of myocardial repolarization characterized by a prolongation of QT interval on the electrocardiogram and clinically manifested with syncopal episodes and SCD. Mutations in 13 genes regulating cardiac action potential (AP) have been found directly responsible for LQTS. In addition, genes regulating Ca2+-handling are found associated with a LQTS phenotype. Furthermore, many modifier genes were identified as responsible in shaping the phenotype of LQTS mutations. The most recent experimental model available for in vitro studies of cardiac arrhythmias is represented by human induced pluripotent stem cells (hiPSC). The subsequent differentiation generates functional cardiomyocytes (hiPSC-CMs) with the same genotype of patients. The aims of this study were: 1) to characterized the functional features underlying an asymptomatic or symptomatic phenotype derived from the same KCNQ1 mutation; 2) to assess the functional effect of a new de novo mutation in CALM1. Functional studies were performed on hiPSC-CMs with the patch clamp technique in voltage- and current-clamp configurations. In addition, an in silico IK1 conductance was injected with dynamic-clamp to provide for the low expression of native IK1, which is characteristic of immature cells. Field potential duration (FPD) was assessed by 256-electrodes MultiElectrode Array in hiPSC-CMs embryoid bodies. The first study was focused on the mutation Y111C on KCNQ1 gene, encoding for Kv7.1 responsible for the repolarizing current IKs. Two relatives with the same mutation were involved: the symptomatic (S) son, with a markedly prolonged corrected QT interval (QTc), and the asymptomatic (AS) father, with a borderline QTc. Control, AS and S hiPSC-CMs lines were generated. Results show that: 1) Y111C mutation induced a prolongation in AP duration (APD) and FPD in S but not in AS cells. 2) IKs density was diminished in S and a partial rescue was evidenced in AS. 3) A different IKs density was evidenced between AS and S hiPSC-CMs when transfected with wild type KCNQ1. These results suggest a likely trafficking defect of Kv7.1 to cell membrane; in addition, we hypothesized the presence of a rescue mechanism, likely a modifier gene, involving protein folding and degradation in AS, blunting the effect of Y111C mutation. The second study was focused on F142L mutation in CALM1 gene, which has been recently found directly related to a very severe form of LQTS. CALM1 encodes for calmodulin, an ubiquitous Ca2+-binding protein mainly involved in the regulation of the electrical activity of cardiomyocytes. Two hiPSC-CMs lines were generated from healthy donors and F142L patient. Results show that F142L mutation caused: 1) a prolongation in APD and FPD; 2) a strong reduction in Ca2+-current (ICaL) Ca2+-dependent inactivation. 3) an incomplete ICaL steady-state inactivation with an increased window current. 4) an increased rate of arrhythmogenic events under β-adrenergic stimulation. We can conclude that F142L mutation is the responsible of the severe phenotype evidenced in patients by strongly impairing ICaL biophysical properties. Overall, hiPSC-CMs totally recapitulate the clinical phenotype and allows the study of cardiac arrhythmias of genetic origin with an high-level translational relevance. Furthermore, they represent an excellent starting point for the generation of mutation-specific and patient-specific pharmacological therapies.