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The liver plays a key role in the metabolism of lipoproteins, controlling both production and catabolism. To accelerate the development of new lipid‐lowering therapies in humans, it is essential to have a relevant in vitro study model available. The current hepatocyte‐like cells (HLCs) models derived from hiPSC can be used to model many genetically driven diseases but require further improvement to better recapitulate the complexity of liver functions. Here, we aimed to improve the maturation of HLCs using a three‐dimensional (3D) approach using Biomimesys®, a hyaluronic acid‐based hydroscaffold in which hiPSCs may directly form aggregates and differentiate toward a functional liver organoid model. After a 28‐day differentiation 3D protocol, we showed that many hepatic genes were upregulated in the 3D model (liver organoids) in comparison with the 2D model (HLCs). Liver organoids, grown on Biomimesys®, exhibited an autonomous cell organization, were composed of different cell types and displayed enhanced cytochromes P450 activities compared to HLCs. Regarding the functional capacities of these organoids, we showed that they were able to accumulate lipids (hepatic steatosis), internalize low‐density lipoprotein and secrete apolipoprotein B. Interestingly, we showed for the first time that this model was also able to produce apolipoprotein (a), the apolipoprotein (a) specific of Lp(a). This innovative hiPSC‐derived liver organoid model may serve as a relevant model for studying human lipopoprotein metabolism, including Lp(a). [ABSTRACT FROM AUTHOR]

Human heart development is governed by transcription factor (TF) networks controlling dynamic and temporal gene expression alterations. Therefore, to comprehensively characterize these transcriptional regulations, day-to-day transcriptomic profiles were generated throughout the directed cardiac differentiation, starting from three distinct human- induced pluripotent stem cell lines from healthy donors (32 days). We applied an expression-based correlation score to the chronological expression profiles of the TF genes, and clustered them into 12 sequential gene expression waves. We then identified a regulatory network of more than 23,000 activation and inhibition links between 216 TFs. Within this network, we observed previously unknown inferred transcriptional activations linking IRX3 and IRX5 TFs to three master cardiac TFs: GATA4, NKX2-5 and TBX5. Luciferase and co-immunoprecipitation assays demonstrated that these five TFs could (1) activate each other's expression; (2) interact physically as multiprotein complexes; and (3) together, finely regulate the expression of SCN5A, encoding the major cardiac sodium channel. Altogether, these results unveiled thousands of interactions between TFs, generating multiple robust hypotheses governing human cardiac development. [ABSTRACT FROM AUTHOR]

Mutations in the DES gene, which encodes the intermediate filament desmin, lead to desminopathy, a rare disease characterized by skeletal muscle weakness and different forms of cardiomyopathies associated with cardiac conduction defects and arrhythmias. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying the DES p.R406W mutation, and employed CRISPR/Cas9 to rectify the mutation in the patient's hiPSC line and introduced the mutation in an hiPSC line from a control individual unrelated to the patient. These hiPSC lines represent useful models for delving into the mechanisms of desminopathy and developing new therapeutic approaches. [ABSTRACT FROM AUTHOR]

Recent genetic data suggest that abnormal cardiac development participates to the pathogenesis of Brugada Syndrome (BrS), a rare inherited arrhythmia responsible for sudden cardiac death in young adults. In vitro cardiac differentiation of human induced pluripotent stem cells (hiPSCs) mimics cardiac development at the cellular level up to a prenatal stage. This study aims at defining whether BrS impairs cardiac differentiation of hiPSCs. Single-cell transcriptomic data were generated at day 30 of in vitro cardiac differentiation of hiPSCs from 2 control subjects (n = 6139 cells) and 2 BrS patients (n = 13756 cells). These transcriptomic data have been annotated using public data obtained from human heart samples at 7.5 post conception weeks (PCW). In addition, bulk 3′RNA-seq data were generated in triplicate for 3 control hiPSC lines and 3 BrS-patient hiPSC lines at each day of the 30-day cardiac-directed differentiation protocol. Single-cell data revealed (1) differences in cell type distribution, with BrS-hiPSCs and control-hiPSCs cardiac-directed differentiation generating 9.8% and 32.85% cardiomyocytes and 16.6% and 2.75% smooth muscle cells, respectively, and (2) cardiomyocytes transcriptomic alterations associated with biological processes of cardiac development, morphogenesis and conduction in BrS. Based on the finding, showing that BrS is associated with alterations in cardiac differentiation, we used the bulk transcriptomic kinetic data to analyze temporal transcriptomic alterations during cardiac-directed differentiation. Differential gene expression analysis unveiled (1) gene expression signatures specific to each BrS patients and (2) expression alterations that were common to all 3 BrS-hiPSCs lines as compared with control lines. Interestingly, the misexpressed genes were associated to the cell differentiation process (GO: 0030154) and their expression kinetics were altered during the cardiac differentiation process. By generating and analyzing single-cell and bulk transcriptomic data during cardiac-directed differentiation of hiPSCs, this study strongly suggests that transcriptomic remodeling occurs as early as the cardiac development for BrS patients. [ABSTRACT FROM AUTHOR]

Aims Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function. Methods and results Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. Conclusion Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases. [ABSTRACT FROM AUTHOR]

Global cellular electrophysiological phenotype was then evaluated with action potential (AP) recordings, but no AP basal parameters specifically segregated BrS hiPSC-CMs, and spontaneous beating frequencies did not differ between all cell lines (Figure S4). Brugada syndrome (BrS) is an inherited arrhythmic disease predisposing to sudden cardiac death (SCD), characterized by a typical electrocardiogram pattern that includes a J point elevation with a coved type ST segment.1 BrS is a complex genetic disease in which ~20% of patients carry rare variants in I SCN5A i gene, whereas the others remain genetically unresolved.2 Despite this genetic complexity, we hypothesize that a common cellular phenotypic trait is at the root of this specific BrS ECG pattern. Importantly, the expression of I SCN5A i , the main BrS culprit gene identified to date,4 remained unchanged, excluding I SCN5A i expression levels as a hallmark for BrS hiPSC-CM phenotype. Early afterdepolarizations (EADs) were observed in 39-70% of all six BrS ventricular-like hiPSC-CMs versus only in 4% and 4.7% of Ctrl and non-BrS hiPSC-CMs, respectively (Figure 3B, Figure S5). [Extracted from the article]

Aims The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. Methods and results Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD , the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H- RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. Conclusion This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances. Open in new tab Download slide Open in new tab Download slide [ABSTRACT FROM AUTHOR]

Mutations in voltagegated Naα channels have been linked to several channelopathies leading to a wide variety of diseases including cardiac arrhythmias, epilepsy, and myotonia. We have previously demonstrated that voltage- gated Na± channel (Nav)1.5 trafficking-deficient mutant channels could lead to a dominant negative effect by impairing trafficking of the wild-type (WT) channel. We also reported that voltage-gated Na± channels associate as dimers with coupled gating properties. Here, we hypothesized that the dominant negative effect of mutant Na± channels could also occur through coupled gating. This was tested using cell surface biotinylation and single channel recordings to measure the gating probability and coupled gating of the dimers. As previously reported, coexpression of Nav1.5-L325R with WT channels led to a dominant negative effect, as reflected by a 75% reduction in current density. Surprisingly, cell surface biotinylation showed that Nav1.5- L325R mutant is capable of trafficking, with 40% of Nav1.5-L325R reaching the cell surface when expressed alone. Importantly, even though a dominant negative effect on the Na± current is observed when WT and Nav1.5-L325R are expressed together, the total Nav channel cell surface expression was not significantly altered compared with WT channels alone. Thus, the trafficking deficiency could not explain the 75% decrease in inward Na± current. Interestingly, single channel recordings showed that Nav1.5-L325R exerted a dominant negative effect on the WT channel at the gating level. Both coupled gating and gating probability of WT:L325R dimers were drastically impaired. We conclude that dominant negative suppression exerted by Nav1.5 mutants can also be caused by impairing the WT gating probability, a mechanism resulting from the dimerization and coupled gating of voltage-gated Na± channel α-subunits. [ABSTRACT FROM AUTHOR]

Elevated circulating lipoprotein(a) (Lp(a)) is a genetically determined risk factor for coronary artery disease and aortic valve stenosis (Tsimikas, 2017). Importantly, the LPA gene, which encodes the apolipoprotein(a) (protein-component of Lp(a)), is missing in most species, and human liver cell-lines do not secrete Lp(a). There is a need for the development of human in vitro models suitable for investigating biological mechanisms involved in Lp(a) metabolism. We here generated and characterized iPSCs from a patient with extremely high Lp(a) plasma levels genetically determined (Coassin et al., 2022). This unique cellular model offers great opportunities and new perspectives for investigations on biological mechanisms involved in Lp(a) metabolism. [ABSTRACT FROM AUTHOR]

Hemorrhagic shock (HS) is the consequence of an important loss in blood volume leading to an insufficient delivery in oxygen. HS causes metabolic changes, multi-organ dysfunction and inflammation. The acute kidney injury related to HS is associated with hyperkalemia mainly through an alteration of Na/K ATPase pump. In a previous O-GlcNAcylomic study, we have identified Na/K ATPase as potentially O-GlcNAcylated protein. O-GlcNAcylation is a post-translational modification with a putative beneficial effect in shock situation. This study aims at evaluating if Na/K ATPase is O-GlcNAcylated and its potential impact during the early phase of hemorrhagic shock. Wistar rats were subjected to a HS protocol induced via exsanguination and then randomly treated or not with NButGT (10 mg/kg), a O-GlcNAcase inhibitor, to increase O-GlcNAc levels. Blood pressure and heart rate were monitored throughout the experiment. Blood was collected from the animals to perform a complete blood count and a blood gas analysis. Kidney was immediately frozen for histological and biochemical studies. Immunoprecipitation and Wheat Germ Agglutinin (WGA) based lectin affinity gel electrophoresis were used to study the O-GlcNAcylation of Na/K ATPase. NButGT increased by 2 O-GlcNAc levels in heart and kidney. This increase in O-GlcNAc restored mean arterial pressure (MAP) (P < 0.01) and bicarbonates (P < 0.01). In treated group, kalemia (Sham: 4.2 ± 0.1; HS: 4.7 ± 0.1; NButGT: 4.3 ± 0.1 mmol/L; P < 0.05) and natremia (Sham: 141.5 ± 0.4; HS: 139.8 ± 0.5; NButGT: 141.9 ± 0.4 mmol/L; P < 0.01) were restored. The study of the O-GlcNAcylation of the Na/K ATPase by immunoprecipitation and WGA gel validated its O-GlcNAcylation. Histological analysis of kidney revealed that HS reduced NA/K ATPase expression during HS and treatment by NButGT restored it (Sham: 15.1 ± 1.1; HS: 10.3 ± 0.6; NButGT: 13.78 ± 1.1; % area; P < 0.05). Our results demonstrate that increase O-GlcNAcylation during hemorrhagic shock restored MAP and reduced markers of metabolic acidosis. In our study, animal treatment with NButGT to increase O-GlcNAc levels, restored natremia and kalemia. This effect is associated with an increase in Na/K ATPase expression. To our knowledge, our study validates the O-GlcNAcylation of the Na/K ATPase. With regard to our results, now it will be relevant to study the effect of O-GlcNAcylation on the activity and localisation. [ABSTRACT FROM AUTHOR]

Aims: Loss-of-function mutations in SCN5A, the gene encoding Nav1.5 channel, have been associated with inherited progressive cardiac conduction disease (PCCD). We have proposed that Scri5o heterozygous l+/-) mice, which are characterized by ventricular fibrotic remodelling with ageing, represent a model for PCCD. Our objectives were to identify the molecular pathway involved in fibrosis development and prevent its activation. Conclusion: Methods and results: Our study shows that myocardial interstitial fibrosis occurred in Scn.5ö+/- mice only after 45 weeks of age. Fibrosis was triggered by transforming growth factor β (TGF-β) pathway activation. Younger Scn5o+/- mice were characterized by a higher connexin 43 expression than wild-type (WT) mice. After the age of 45 weeks, connexin 43 expression decreased in both W T and Scn5a+/- mice, although the decrease was larger in Scn5o+/- mice. Chronic inhibition of cardiac sodium current with flecainide (50mg/l+/- mice from the age of 45 weel+/- mouse. [ABSTRACT FROM AUTHOR]