Biology subjects

Jouve, C.

Publications and source records attributed to Jouve, C..

7 recordsLinked to original sources

bioRxiv2026

Desmin p.R406W mutation is associated with arrhythmias through structural and electrophysiological remodeling

Background and AimsMutations in the desmin (DES) gene cause a variety of cardiomyopathies associated with arrhythmias, yet the electrophysiological consequences of these variants remain largely uncharacterized. The aim of this study was to investigate the pathogenic mechanisms of the de novo DES p.R406W variant, which was identified in a 9-year-old patient who suffered from severe ventricular arrhythmias and sudden cardiac death without overt structural heart disease. MethodsHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the DES p.R406W variant (including the patients line) were compared to isogenic controls. Action potentials (AP) of hiPSC-CMs were recorded using patch-clamp. Furthermore, 3D engineered heart tissues (EHTs) were generated from hiPSC-CMs and their APs were recorded with sharp microelectrodes. Analytical techniques also included transmission electron microscopy (TEM) and integrated transcriptomic and proteomic profiling. Finally, a heterozygous knock-in (KI) mouse model carrying the Des p.R405W ortholog was evaluated through surface ECG, echocardiography and ex vivo cardiac optical mapping. ResultsThe DES p.R406W mutation prolonged AP duration in IM-R406W hiPSC-CMs and EHTs vs Control ones. Multi-omics analysis of EHTs revealed a dysregulation of genes and proteins involved in contractile function, cell adhesion, and electrical activity. TEM imaging revealed changes in Z-disc architecture in mutant tissues. Twenty-week-old Des p.R405W KI mice exhibited ventricular conduction slowing (prolonged QRS) and a high susceptibility to ventricular tachyarrhythmias, likely due to reentrant mechanisms. Mild hypertrophy was also observed, but only in females. ConclusionThe DES p.R406W variant is highly pathogenic, causing electrical and structural remodeling of the myocardium. This study highlights the effectiveness of hiPSC-CMs and EHTs in recapitulating the clinical phenotype of desminopathy, providing a platform for investigating the mechanisms of early-onset cardiac arrhythmias and SCD.

pathology↗

bioRxiv2025

Mavacamten improves energy balance in a pre-clinical model of RASopathy-associated hypertrophic cardiomyopathy

Background and aimDevelopmental disorders caused by activating mutations in the RAS-MAPK pathway account for nearly 20% of hypertrophic cardiomyopathy (HCM) cases in paediatric patients. Compared to sarcomeric HCM, RAS-HCM presents a higher risk of obstruction and hospitalisation. The myosin inhibitor mavacamten has been approved in the European Union for treating adults with obstructive HCM; however, clinical trials have excluded syndromic HCM. Consequently, this study aimed to characterise the functional and energetic disturbances induced by the RASopathy mutation BRAF p.Thr599Arg in cardiomyocytes and to evaluate the effects of mavacamten treatment. MethodsHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a CRISPR-induced BRAF T599R mutation and their isogenic control were employed to model RAS-HCM in vitro. The cell size, contractility, and transcriptomic profile were assessed to determine the phenotype of the cardiomyocytes. Energetics were evaluated using the Mito Stress assay, live ATP levels, and NAD(P)H and FAD+ autofluorescence. ResultsBRAF-mutant cardiomyocytes demonstrated hypertrophy and hypercontractility. Furthermore, energetic profiling revealed increased mitochondrial NAD(P)H and FAD+ pools and an enhanced energetic state in the Mito Stress assay with increased maximal respiratory capacity. However, they also exhibited a significant ATP drop during rapid pacing compared to the control, suggesting that mitochondrial capacity remains insufficient to meet the ATP demand. Mavacamten treatment normalised excessive ATP consumption during acute pacing, suggesting reduced mitochondrial overactivity. ConclusionsBRAF-mutant cardiomyocytes recapitulate the characteristics of HCM in vitro. Mavacamten mitigates dysfunctions and restores energetic balance under stress conditions, indicating it holds potential as a therapeutic option for RASopathy-associated HCM.

physiology↗

bioRxiv2024

Myocardial disarray drives metabolic inefficiency in human cardiomyocytes

Adult cardiomyocytes are embedded within a highly organized myocardial microenvironment that imposes critical geometric cues essential for the alignment and distribution of organelles and the shaping of their unique, rectangular cellular morphology. Despite the association of cardiomyocyte disarray with human heart disease, the functional consequences of this cellular disorganization remain poorly understood. Here, we leveraged micropatterned substrates to promote structural alignment in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), contrasting the effects of mechanical alignment on mitochondrial form and function with hiPSC-CMs cultured under standard unconstrained conditions. Cardiomyocytes cultured under unconstrained conditions exhibited misaligned sarcomeres and a perinuclear mitochondrial distribution while micropatterned hiPSC-CMs developed linear myofibrils and reconfigured sarcomere and mitochondrial organization, which increased mitochondrial respiration without augmenting mitochondrial mass. Notably, micropatterned hiPSC-CMs exhibited an increased number of mitochondrial-associated membranes, as determined by proximity ligation assays and transmission electron microscopy, suggesting enhanced interactions between the sarcoplasmic reticulum and mitochondria. Together, these findings demonstrate that mitochondrial-sarcoplasmic architecture and geometry are critical spatial features that ensure bioenergetic efficiency of cardiomyocytes. This work underscores the importance of cellular organization in cardiomyocyte metabolism and function, providing insights into the pathophysiology of cardiac diseases marked by cellular disarray.

cell biology↗

bioRxiv2024

The p.H222P lamin A/C mutation induces heart failure via impaired mitochondrial calcium uptake in human cardiac laminopathy

BackgroundMutations in the LMNA gene, which encodes lamin A/C, cause a variety of diseases known as laminopathies. Some mutations are particularly associated with the occurrence of dilated cardiomyopathy and heart failure, but the genotype-phenotype relationship and underlying mechanisms are unclear. Here, we used induced pluripotent stem cells (hiPSCs) from a patient carrying a LMNA point mutation (c.665A>C, p.His222Pro) to investigate the mechanisms leading to contractile dysfunction. MethodsLMNA p.H222P mutant and a CRISPR/Cas9 corrected isogenic control hiPSCs clones were differentiated into cardiomyocytes (hiPSC-CMs). Immunofluorescence staining was performed on hiPSC-CMs to quantify their sarcomere organization (SarcOrgScore) using a Matlab code. Ring-shaped cardiac 3D organoids were generated to compare the contractile properties of the two clones. Calcium transients in mutant and corrected hiPSC-CMs were measured by live confocal imaging. Mitochondrial respiration parameters were measured by Seahorse. ResultshiPSC-CMs were generated from the LMNA mutant and the corrected hiPSCs with no difference in the differentiation yield (proportion of troponin-positive cells: 95.0% for LMNA p.H222P vs. 95.1% for Ctrl-iso1, p=0.726). hiPSC-CMs displayed well-formed sarcomeres and their organization was similar between the two cell lines. However, cardiac 3D organoids generated with LMNA p.H222P hiPSC-CMs showed an impaired contractility compared to control organoids. Calcium transient recordings in LMNA p.H222P mutant cardiomyocytes showed a significantly higher calcium transient amplitude with a significantly slower calcium re-uptake. Transcriptomic analyses suggested a global mitochondrial dysfunction and in particular an impaired mitochondrial calcium uptake with a significantly decreased expression of the mitochondrial calcium uniporter (MCU). This decrease in MCU expression was confirmed by western blot and was accompanied by an increased MICU1:MCU, as well as an increased PDH Ser232 and PDH Ser300 phosphorylation, indicating a decreased mitochondrial calcium uptake in the LMNA mutant hiPSC-CMs. Measurement of mitochondrial respiration showed lower basal and maximal respiration in LMNA p.H222P hiPSC-CMs. Consistently, the ATP levels were significantly lower in LMNA p.H222P hiPSC-CMs as compared to isogenic controls. ConclusionsLMNA p.H222P mutant hiPSC-CMs exhibit contractile dysfunction associated with mitochondrial dysfunction with impaired MCU complex activity, decreased mitochondrial calcium homeostasis and reduced mitochondrial energy production. NOVELTY AND SIGNIFICANCEO_ST_ABSWhat is known?C_ST_ABS- Mutations in LMNA, which encodes the nuclear lamins A/C, cause a variety of diseases (called laminopathies), which can involve the cardiac muscle leading to dilated cardiomyopathy and systolic heart failure. - The pathological mechanisms linking the nuclear envelope abnormalities induced by LMNA mutations to the development of a reduced cardiac muscle contractility are not well understood. What new information does this article contribute?- LMNA mutant cardiomyocytes have a profound mitochondrial dysfunction with impaired MCU complex activity, decreased mitochondrial calcium homeostasis, and reduced mitochondrial energy production. - Our study uncovers an unappreciated pathophysiological mechanism and opens new possibilities by suggesting MCU activators as a novel therapeutic for patients with LMNA cardiomyopathy.

pathology↗

bioRxiv2023

Regulatory mechanisms in multiple vascular diseases locus LRP1 involve repression by SNAIL and extracellular matrix remodeling

BackgroundGenome-wide association studies (GWAS) implicate common genetic variations in the low-density lipoprotein receptor-related protein 1 locus (LRP1) in risk for multiple vascular diseases and traits. However, the underlying biological mechanisms are unknown. MethodsFine mapping analyses included Bayesian colocalization to identify the most likely causal variant. Human induced pluripotent stem cells (iPSC) were genome-edited using CRISPR-Cas9 to delete or modify candidate enhancer regions, and generate LRP1 knockout cell lines (KO). Cells were differentiated into smooth muscle cells (SMCs) through a mesodermal lineage. Transcription regulation was assessed using luciferase reporter assay, transcription factor knockdown and chromatin immunoprecipitation. Phenotype changes in cells were conducted using cellular assays, bulk RNA-sequencing and mass spectrometry. ResultsMulti-trait co-localization analyses pointed at rs11172113 as the most likely causal variant in LRP1 for fibromuscular dysplasia, migraine, pulse pressure and pulmonary function trait. We found rs11172113-T allele to associate with higher LRP1 expression. Genomic deletion in iPSC-derived SMCs supported rs11172113 to locate in an enhancer region regulating LRP1 expression. We found transcription factors MECP2 and SNAIL to repress LRP1 expression through an allele-specific mechanism, involving SNAIL interaction with disease risk allele. LRP1 KO decreased iPSC-derived SMCs proliferation and migration. Differentially expressed genes were enriched for collagen-containing extracellular matrix, connective tissue and lung development. LRP1 KO showed potentiated canonical TGF-{beta} signaling through enhanced phosphorylation of SMAD2/3. Analyses of protein content of decellularized extracts indicated partial extracellular matrix (ECM) remodeling involving enhanced secretion of CYR61, a known LRP1 ligand involved in vascular integrity and TIMP3, implicated in extracellular matrix maintenance and also known to interact with LRP1. ConclusionsOur findings support allele specific LRP1 gene repression by the endothelial-to-mesenchymal transition regulator SNAIL. We propose decreased LRP1 expression in SMCs to remodel the ECM enhanced by TGF- {beta} as a potential mechanism of this pleiotropic locus for vascular diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/539992v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@11926a8org.highwire.dtl.DTLVardef@390a37org.highwire.dtl.DTLVardef@cdad4dorg.highwire.dtl.DTLVardef@12cbf92_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell derived cardiomyocytes

We developed a 96-well plate assay which allows fast, reproducible and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (PEG) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25{+/-}1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4{+/-}0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural read-outs.

bioengineering↗

bioRxiv2022

Genomic, transcriptomic and proteomic depiction of iPSC-derived smooth muscle cells as emerging cellular models for arterial diseases

BackgroundVascular smooth muscle cells (VSMCs) plasticity is a central mechanism in cardiovascular health and disease. We aimed at providing deep cellular phenotyping, epigenomic and proteomic depiction of SMCs derived from induced pluripotent stem cells (iPSCs) and evaluating their potential as cellular models in the context of complex genetic arterial diseases. MethodsWe differentiated 3 human iPSC lines using either RepSox (R-SMCs) or PDGF-BB and TGF-{beta} (TP-SMCs), during the second half of a 24-days-long protocol. In addition to cellular assays, we performed RNA-Seq and assay for transposase accessible chromatin (ATAC)-Seq at 6 time-points of differentiation. The extracellular matrix content (matrisome) generated by iPSCs derived SMCs was analyzed using mass spectrometry. ResultsBoth iPSCs differentiation protocols generated SMCs with positive expression of SMC markers. TP-SMCs exhibited greater capacity of proliferation, migration and lower calcium release in response to contractile stimuli compared to R-SMCs. RNA-Seq data showed that genes involved in the contractile function of arteries were highly expressed in R-SMCs compared to TP-SMCs or primary SMCs. Matrisome analyses supported an overexpression of proteins involved in wound repair in TP-SMCs and a higher secretion of basal membrane constituents by R-SMCs. Open chromatin regions of R-SMCs and TP-SMCs were significantly enriched for variants associated with coronary artery disease and blood pressure, while only TP-SMCs were enriched for variants associated with peripheral artery disease. ConclusionsOur study portrayed two iPSCs derived SMCs models presenting complementary cellular phenotypes of high relevance to SMC plasticity. In combination with genome-editing tools, our data supports high relevance of the use of these cellular models to the study of complex regulatory mechanisms at genetic risk loci involved in several arterial diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/490058v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f1002corg.highwire.dtl.DTLVardef@1426fa7org.highwire.dtl.DTLVardef@b05b56org.highwire.dtl.DTLVardef@3c6e88_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗