The generation of control human iPSC lines is important in fundamental research to understand the physiological and physiopathological mechanisms underlying human diseases. We generated and ...characterized two control hiPSC lines from lymphoblastoid cells collected from apparently healthy individuals. These hiPSCs display pluripotency markers, can differentiate into three embryonic germ layers, possess normal karyotypes and colony morphologies, and have no reprogramming viral vectors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Excitation-contraction coupling in muscle cells is initiated by a restricted membrane depolarization delimited within the neuromuscular junction. This targeted depolarization triggers an action ...potential that propagates and induces a global cellular calcium response and a consequent contraction. To date, numerous studies have investigated this excitation-calcium response coupling by using different techniques to depolarize muscle cells. However, none of these techniques mimic the temporal and spatial resolution of membrane depolarization observed in the neuromuscular junction. By using optogenetics in C2C12 muscle cells, we developed a technique to study the calcium response following membrane depolarization induced by photostimulations of membrane surface similar or narrower than the neuromuscular junction area. These stimulations coupled to confocal calcium imaging generate a global cellular calcium response that is the consequence of a membrane depolarization propagation. In this context, this technique provides an interesting, contactless and relatively easy way of investigation of calcium increase/release as well as calcium decrease/re-uptake triggered by a propagated membrane depolarization.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Anomalies in constitutive calcium entry (CCE) have been commonly attributed to cell dysfunction in pathological conditions such as cancer. Calcium influxes of this type rely on channels, such as ...transient receptor potential (TRP) channels, to be constitutively opened and strongly depend on membrane potential and a calcium driving force. We developed an optogenetic approach based on the expression of the halorhodopsin chloride pump to study CCE in non-excitable cells. Using C2C12 cells, we found that halorhodopsin can be used to achieve a finely tuned control of membrane polarization. Escalating the membrane polarization by incremental changes in light led to a concomitant increase in CCE through transient receptor potential vanilloid 2 (TRPV2) channels. Moreover, light-induced calcium entry through TRPV2 channels promoted cell migration. Our study shows for the first time that by modulating CCE and related physiological responses, such as cell motility, halorhodopsin serves as a potentially powerful tool that could open new avenues for the study of CCE and associated cellular behaviors.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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