Generation of a patient-specific iPSC cell line with cardiac arrhythmias and dilated cardiomyopathy (CBRCULi016-A), an isogenic control (CBRCULi016-A-1), and a paternal control (CBRCULi017-A).

Dilated cardiomyopathy (DCM) is a prevalent cause of heart failure. We generated induced pluripotent stem cell (iPSC) lines from a DCM patient carrying a mutation in the SCN5A gene, with his healthy father serving as a control. Notably, we employed CRISPR-Cas9 to rectify the mutation in the patient's iPSC line. The resulting iPSC lines expressed pluripotency markers, underwent differentiation into all three embryonic germ layers, maintained a normal karyotype, and lacked reprogramming viral vectors. These iPSC lines serve as a model for delving into the mechanisms of DCM and hold promise for the development of personalized therapeutic approaches.

Biophysical properties of NaV1.5 channels from atrial-like and ventricular-like cardiomyocytes derived from human induced pluripotent stem cells.

Generating atrial-like cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial for modeling and treating atrial-related diseases, such as atrial arrythmias including atrial fibrillations. However, it is essential to obtain a comprehensive understanding of the electrophysiological properties of these cells. The objective of the present study was to investigate the molecular, electrical, and biophysical properties of several ion channels, especially NaV1.5 channels, in atrial hiPSC cardiomyocytes. Atrial cardiomyocytes were obtained by the differentiation of hiPSCs treated with retinoic acid (RA). The quality of the atrial specification was assessed by qPCR, immunocytofluorescence, and western blotting. The electrophysiological properties of action potentials (APs), Ca2+ dynamics, K+ and Na+ currents were investigated using patch-clamp and optical mapping approaches. We evaluated mRNA transcript and protein expressions to show that atrial cardiomyocytes expressed higher atrial- and sinoatrial-specific markers (MYL7, CACNA1D) and lower ventricular-specific markers (MYL2, CACNA1C, GJA1) than ventricular cardiomyocytes. The amplitude, duration, and steady-state phase of APs in atrial cardiomyocytes decreased, and had a shape similar to that of mature atrial cardiomyocytes. Interestingly, NaV1.5 channels in atrial cardiomyocytes exhibited lower mRNA transcripts and protein expression, which could explain the lower current densities recorded by patch-clamp. Moreover, Na+ currents exhibited differences in activation and inactivation parameters. These differences could be explained by an increase in SCN2B regulatory subunit expression and a decrease in SCN1B and SCN4B regulatory subunit expressions. Our results show that a RA treatment made it possible to obtain atrial cardiomyocytes and investigate differences in NaV1.5 channel properties between ventricular- and atrial-like cells.

Cardiac involvement in patient-specific induced pluripotent stem cells of myotonic dystrophy type 1: unveiling the impact of voltage-gated sodium channels.

Myotonic dystrophy type 1 (DM1) is a genetic disorder that causes muscle weakness and myotonia. In DM1 patients, cardiac electrical manifestations include conduction defects and atrial fibrillation. DM1 results in the expansion of a CTG transcribed into CUG-containing transcripts that accumulate in the nucleus as RNA foci and alter the activity of several splicing regulators. The underlying pathological mechanism involves two key RNA-binding proteins (MBNL and CELF) with expanded CUG repeats that sequester MBNL and alter the activity of CELF resulting in spliceopathy and abnormal electrical activity. In the present study, we identified two DM1 patients with heart conduction abnormalities and characterized their hiPSC lines. Two differentiation protocols were used to investigate both the ventricular and the atrial electrophysiological aspects of DM1 and unveil the impact of the mutation on voltage-gated ion channels, electrical activity, and calcium homeostasis in DM1 cardiomyocytes derived from hiPSCs. Our analysis revealed the presence of molecular hallmarks of DM1, including the accumulation of RNA foci and sequestration of MBNL1 in DM1 hiPSC-CMs. We also observed mis-splicing of SCN5A and haploinsufficiency of DMPK. Furthermore, we conducted separate characterizations of atrial and ventricular electrical activity, conduction properties, and calcium homeostasis. Both DM1 cell lines exhibited reduced density of sodium and calcium currents, prolonged action potential duration, slower conduction velocity, and impaired calcium transient propagation in both ventricular and atrial cardiomyocytes. Notably, arrhythmogenic events were recorded, including both ventricular and atrial arrhythmias were observed in the two DM1 cell lines. These findings enhance our comprehension of the molecular mechanisms underlying DM1 and provide valuable insights into the pathophysiology of ventricular and atrial involvement.

Optical Mapping of Cardiomyocytes in Monolayer Derived from Induced Pluripotent Stem Cells.

Optical mapping is a powerful imaging technique widely adopted to measure membrane potential changes and intracellular Ca2+ variations in excitable tissues using voltage-sensitive dyes and Ca2+ indicators, respectively. This powerful tool has rapidly become indispensable in the field of cardiac electrophysiology for studying depolarization wave propagation, estimating the conduction velocity of electrical impulses, and measuring Ca2+ dynamics in cardiac cells and tissues. In addition, mapping these electrophysiological parameters is important for understanding cardiac arrhythmia mechanisms. In this review, we delve into the fundamentals of cardiac optical mapping technology and its applications when applied to hiPSC-derived cardiomyocytes and discuss related advantages and challenges. We also provide a detailed description of the processing and analysis of optical mapping data, which is a crucial step in the study of cardiac diseases and arrhythmia mechanisms for extracting and comparing relevant electrophysiological parameters.

NaV1.5 knockout in iPSCs: a novel approach to study NaV1.5 variants in a human cardiomyocyte environment.

Cardiomyocytes derived from patient-specific induced pluripotent stem cells (iPSC-CMs) successfully reproduce the mechanisms of several channelopathies. However, this approach involve cell reprogramming from somatic tissue biopsies or genomic editing in healthy iPSCs for every mutation found and to be investigated. We aim to knockout (KO) NaV1.5, the cardiac sodium channel, in a healthy human iPSC line, characterize the model and then, use it to express variants of NaV1.5. We develop a homozygous NaV1.5 KO iPSC line able to differentiate into cardiomyocytes with CRISPR/Cas9 tool. The NaV1.5 KO iPSC-CMs exhibited an organized contractile apparatus, spontaneous contractile activity, and electrophysiological recordings confirmed the major reduction in total Na+ currents. The action potentials (APs) exhibited a reduction in their amplitude and in their maximal rate of rise. Voltage optical mapping recordings revealed that the conduction velocity Ca2+ transient waves propagation velocities were slow. A wild-type (WT) NaV1.5 channel expressed by transient transfection in the KO iPSC-CMs restored Na+ channel expression and AP properties. The expression of NaV1.5/delQKP, a long QT type 3 (LQT3) variant, in the NaV1.5 KO iPSC-CMs showed that dysfunctional Na+ channels exhibited a persistent Na+ current and caused prolonged AP duration that led to arrhythmic events, characteristics of LQT3.

iPSC-derived cardiomyocytes from patients with myotonic dystrophy type 1 have abnormal ion channel functions and slower conduction velocities.

Cardiac complications such as electrical abnormalities including conduction delays and arrhythmias are the main cause of death in individuals with Myotonic Dystrophy type 1 (DM1). We developed a disease model using iPSC-derived cardiomyocytes (iPSC-CMs) from a healthy individual and two DM1 patients with different CTG repeats lengths and clinical history (DM1-1300 and DM1-300). We confirmed the presence of toxic RNA foci and mis-spliced MBNL1/2 transcripts in DM1 iPSC-CMs. In DM1-1300, we identified a switch in the cardiac sodium channel SCN5A from the adult to the neonatal isoform. The down-regulation of adult SCN5A isoforms is consistent with a shift in the sodium current activation to depolarized potentials observed in DM1-1300. L-type calcium current density was higher in iPSC-CMs from DM1-1300, which is correlated with the overexpression of the CaV1.2 transcript and proteins. Importantly, INa and ICaL dysfunctions resulted in prolonged action potentials duration, slower velocities, and decreased overshoots. Optical mapping analysis revealed a slower conduction velocity in DM1-1300 iPSC-CM monolayers. In conclusion, our data revealed two distinct ions channels perturbations in DM1 iPSC-CM from the patient with cardiac dysfunction, one affecting Na+ channels and one affecting Ca2+ channels. Both have an impact on cardiac APs and ultimately on heart conduction.