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

Optogenetic control of membrane potential and calcium homeostasis in skeletal muscle cells : impact on myogenic processes

Le calcium est un second messager qui participe à de nombreux processus cellulaires tels que la prolifération, la migration, la différenciation, l’apoptose et la transmission de messagers neuronaux. Dans le modèle musculaire squelettique, le calcium est un acteur important dans le processus du couplage excitation-contraction. Il est également impliqué dans la myogenèse et dans les processus de réparation. Une dérégulation du calcium dans les cellules musculaires participe à leur dégénérescence comme il a été observé dans la dystrophie musculaire de Duchenne (DMD). L’objectif de cette thèse a été par des approches innovantes d’optogénétique, d’explorer et de moduler des mécanismes fondamentaux dépendants du calcium tels que la migration, la fusion, la différenciation et la contraction dans différents modèles musculaires. 1- Dans un premier temps, l’effet de la stimulation de l’halorhodopsine (eNpHR) sur le contrôle du potentiel de membrane et sur le processus de migration de myoblastes C2C12 a été étudié. La transfection de eNpHR3.0 dans des myoblastes C2C12 a permis de générer des courants sortants diminuant le potentiel de membrane vers des valeurs stabilisées tout le long de la stimulation. Cette polarisation membranaire induit des élévations transitoires de calcium cytosolique, dépendant du canal TRPV2 localisé à la membrane plasmique. Après avoir démontré l’implication de TRPV2 dans les processus migratoires des myoblastes, les entrées de calcium induites par la stimulation lumineuse ont permis d’augmenter la migration TRPV2-dépendante des myoblastes C2C12. 2- Dans un second temps, l’impact de la stimulation par le canal rhodopsine 2 (ChR2) a été évalué pour le contrôle de l’activité calcique et sur les processus de différenciation dans des cultures primaires de myotubes murins. Après avoir caractérisé la signature calcique des cultures primaires de myotubes en différenciation avec la protéine fluorescente sensible au calcium GCaMP, un protocole de simulation optique a été développé pour reproduire la signature calcique spontanée de myotubes différenciés. En premier lieu, le contrôle de l’activité calcique par la stimulation optique de ChR2 a été confirmé au niveau d’une cellule unique ainsi qu’à l’échelle d’une culture entière. Par la suite, l’application d’un protocole de stimulation optique en culture pendant la différenciation a permis de moduler les processus de différenciation tels que la fusion et la contraction des myotubes primaires. 3- La signature calcique de cellules dystrophiques représentatives de la DMD a été explorée dans deux modèles cellulaires différents composés de cultures primaires isolées de souris mdx et de cellules musculaires humaines dérivés d’hiPSCs provenant de patients DMD. Des dérégulations de la signature calcique ont été observées dans ces deux modèles dystrophiques. Une exploration fonctionnelle a été réalisée sur les cellules musculaires dérivées d’hiPSCs au travers de stimulations électriques, pharmacologiques et optiques démontrant leur capacité à développer un phénotype musculaire et confirmant leur intérêt potentiel pour la modélisation de maladies telles que la DMD.Ces travaux ouvrent des perspectives sur l’utilisation de l’optogénétique pour évaluer la fonctionnalité des cellules et pour moduler certains processus cellulaires pour de futures applications thérapeutiques.Calcium is a second messenger which participates in many cellular processes such as proliferation, migration, differentiation, apoptosis and transmission of neuronal messengers. In skeletal muscle, calcium is an important player in the process of excitation-contraction coupling. It is also involved in myogenesis and in repair processes. Deregulation of calcium in muscle cells contributes to their degeneration as observed in Duchenne Muscular Dystrophy (DMD). The objective of this thesis was to explore and modulate basic calcium-dependent mechanisms such as migration, fusion, differentiation and contraction in different muscle models through innovative optogenetic approaches. 1- First, the effect of halorhodopsin (eNpHR) stimulation on the control of membrane potential and on the migration process of C2C12 myoblasts was studied. Transfection of eNpHR3.0 into C2C12 myoblasts generated outward currents decreasing the membrane potential to stable values throughout stimulation. This membrane polarization induces cytosolic calcium transients, dependent on the TRPV2 channel located at the plasma membrane. After demonstrating the involvement of TRPV2 in the migratory process of myoblasts, the light-induced calcium constitutive entries allowed to increase the TRPV2-dependent migration of C2C12 myoblasts. 2- Secondly, the impact of channelrhodopsin 2 (ChR2) stimulation was evaluated for the control of calcium activity and on the differentiation processes in primary cultures of murine myotubes. We characterized the calcium signature of myotube primary cultures with the fluorescent calcium sensitive protein GCaMP. An optical simulation protocol was then developed to reproduce the spontaneous calcium signature of differentiated myotubes. To reach this goal, we first confirmed the control of calcium activity by optical stimulation of ChR2 at the single cell level as well as at the level of a whole culture. Finally, the application of an optical stimulation protocol in culture during differentiation allowed to modulate differentiation processes such as fusion and contraction of primary myotubes. 3- The calcium signature of dystrophic cells representative of DMD was explored in two different cell models: primary cultures isolated from mdx mice and human muscle cells derived from DMD hiPSCs. Changes in calcium signature was observed in these two dystrophic models. A functional exploration was performed on muscle cells derived from hiPSCs through electrical, pharmacological and optical stimulation demonstrating their ability to develop a muscle phenotype and confirming their potential interest in modeling diseases such as DMD.This work opens perspectives on the use of optogenetics to evaluate cell functionality and to modulate some cellular processes for future therapeutic applications

Generation of control iPSC lines CBRCULi008-A and CBRCULi009-A derived from lymphoblastoid cell lines

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

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

Abstract 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

Optogenetic approach for targeted activation of global calcium transients in differentiated C2C12 myotubes

Abstract 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

Fine Tuning of Calcium Constitutive Entry by Optogenetically-Controlled Membrane Polarization: Impact on Cell Migration

International audienceAnomalies 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

In vitro differentiation of W8B2 + human cardiac stem cells: gene expression of ionic channels and spontaneous calcium activity

International audienceBackground: Human cardiac stem cells expressing the W8B2 marker (W8B2 + CSCs) were recently identified and proposed as a new model of multipotent CSCs capable of differentiating into smooth muscle cells, endothelial cells and immature myocytes. Nevertheless, no characterization of ion channel or calcium activity during the differentiation of these stem cells has been reported.Methods: The objectives of this study were thus to analyze (using the TaqMan Low-Density Array technique) the gene profile of W8B2+ CSCs pertaining to the regulation of ion channels, transporters and other players involved in the calcium homeostasis of these cells. We also analyzed spontaneous calcium activity (via the GCaMP calcium probe) during the in vitro differentiation of W8B2+ CSCs into cardiac myocytes.Results: Our results show an entirely different electrophysiological genomic profile between W8B2+ CSCs before and after differentiation. Some specific nodal genes, such as Tbx3, HCN, ICaT, L, KV, and NCX, are overexpressed after this differentiation. In addition, we reveal spontaneous calcium activity or a calcium clock whose kinetics change during the differentiation process. A pharmacological study carried out on differentiated W8B2+ CSCs showed that the NCX exchanger and IP3 stores play a fundamental role in the generation of these calcium oscillations.Conclusions: Taken together, the present results provide important information on ion channel expression and intrinsic calcium dynamics during the differentiation process of stem cells expressing the W8B2 marker