Zeina Reda Al Sayed

Malgré les progrès considérables cellulaire arythmique commun entre des patients réalisés en termes de compréhension des à fonds génétiques différents atteints de mécanismes à l'origine des maladies du rythme syndrome de Brugada (BrS). Nous avons montré cardiaque, ces dernières restent parmi les qu'IRXS participe à la régulation de la problèmes les plus fréquemment rencontrés en conduction ventriculaire rapide en régulant cardiologie et constituent la principale cause de l'expression de SCNSA et des jonctions gap dans décès. Le dysfonctionnement ou la désexpression les cardiomyocytes humains. En ce qui concerne d'un seul canal ionique peuvent être à l'origine de la BrS, indépendamment du contexte génétique plusieurs types d'arythmies en fonction du fond ou de la présence des mutation SCN5A, les génétique du patient. Les cardiomyocytes dérivées hiPS-CMs ventriculaires issues de patients de cellules souches pluripotentes induites de différents atteints de BrS étaient plus sujets aux l'homme (hiPS-CMs) confèrent un outil précieux arythmies qui étaient associees à une et fiable pour mieux comprendre l'arythmie augmentation du sodium tardif. Globalement, les cardiaque. hiPS-CMs fournissent un outil pertinent pour De là, nous avons défié ce modèle pour modéliser l'arythmie cardiaque humaine, en plus caractériser l'arythmie causée par des mutations de elles confèrent un environnement cellulaire facteur de transcription Iroquois, IRX5, et pour pertinent pour étudier de nouvelles fonctions afin explorer la présence d'un phénotype de développer des pistes thérapeutiques.

BACKGROUND: Primary aldosteronism is the most common form of secondary hypertension. The most frequent genetic cause of aldosterone-producing adenomas is somatic mutations in the potassium channel KCNJ5. They affect the ion selectivity of the channel, with sodium influx leading to cell membrane depolarization and activation of calcium signaling, the major trigger for aldosterone biosynthesis. METHODS: To investigate how KCNJ5 mutations lead to the development of aldosterone-producing adenomas, we established an adrenocortical cell model in which sodium entry into the cells can be modulated on demand using chemogenetic tools [H295R-S2 α7-5HT3-R (α7-5HT3 receptor) cells]. We investigated their functional and molecular characteristics with regard to aldosterone biosynthesis and cell proliferation. RESULTS: A clonal cell line with stable expression of the chimeric α7-5HT3-R in H295R-S2 (human adrenocortical carcinoma cell line, Strain 2) cells was obtained. Increased sodium entry through α7-5HT3-R upon stimulation with uPSEM-817 (uPharmacologically Selective Effector Molecule-817) led to cell membrane depolarization, opening of voltage-gated Ca ²⁺ channels, and increased intracellular Ca ²⁺ concentrations, resulting in the stimulation of CYP11B2 expression and increased aldosterone biosynthesis. Increased intracellular sodium influx did not increase proliferation but rather induced apoptosis. RNA sequencing and steroidome analyses revealed unique profiles associated with Na ⁺ entry, with only partial overlap with Ang II (angiotensin II) or potassium-induced changes. CONCLUSIONS: H295R-S2 α7-5HT3-R cells are a new model reproducing the major features of cells harboring KCNJ5 mutations. Increased expression of CYP11B2 and stimulation of the mineralocorticoid biosynthesis pathway are associated with a decrease of cell proliferation and an increase of apoptosis, indicating that additional events may be required for the development of aldosterone-producing adenomas.

BACKGROUND: Drug-induced QT prolongation (diLQT) is a feared side effect that could expose susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG (human ether-a-go-go-related gene) channels that generate the delayed-rectifier potassium current (I _Kr ) and thereby regulate the late repolarization phase. There is an important interindividual susceptibility to develop diLQT, which is of unknown origin but can be reproduced in patient-specific induced pluripotent stem cell–derived cardiomyocytes (iPS-CMs). We aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT. METHODS: We measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest sensitivity (HS) or lowest sensitivity (LS) to sotalol administration in vivo (ie, on the basis of the measure of the maximal change in QT interval 3 hours after administration). Specific small interfering RNAs and CAVIN1-T2A-GFP adenovirus were used to manipulate CAVIN1 expression. RESULTS: Whereas HS and LS iPS-CMs showed similar electrophysiological characteristics at baseline, the late repolarization phase was prolonged and I _Kr significantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. I _Kr reduction was caused by a rapid translocation of hERG channel from the membrane to the cytoskeleton-associated fractions upon sotalol application. CAVIN1 , essential for caveolae biogenesis, was 2× more highly expressed in HS iPS-CMs, and its knockdown by small interfering RNA reduced their sensitivity to sotalol. CAVIN1 overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. We found that treatment with sotalol promoted translocation of the hERG channel from the plasma membrane to the cytoskeleton fractions in a process dependent on CAVIN1 (caveolae associated protein 1) expression. CAVIN1 silencing reduced the number of caveolae at the membrane and abrogated the translocation of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers, such as E4031, vandetanib, and clarithromycin. CONCLUSIONS: Our study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the interindividual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1.

Peripartum cardiomyopathy (PPCM) is an idiopathic form of pregnancy-induced heart failure associated with preeclampsia. Circulating factors in late pregnancy are thought to contribute to both diseases, suggesting a common underlying pathophysiological process. However, what drives this process remains unclear. Using serum proteomics, we identified the senescence-associated secretory phenotype (SASP), a marker of cellular senescence associated with biological aging, as the most highly up-regulated pathway in young women with PPCM or preeclampsia. Placentas from women with preeclampsia displayed multiple markers of amplified senescence and tissue aging, as well as overall increased gene expression of 28 circulating proteins that contributed to SASP pathway enrichment in serum samples from patients with preeclampsia or PPCM. The most highly expressed placental SASP factor, activin A, was associated with cardiac dysfunction or heart failure severity in women with preeclampsia or PPCM. In a murine model of PPCM induced by cardiomyocyte-specific deletion of the gene encoding peroxisome proliferator–activated receptor γ coactivator-1α, inhibiting activin A signaling in the early postpartum period with a monoclonal antibody to the activin type II receptor improved heart function. In addition, attenuating placental senescence with the senolytic compound fisetin in late pregnancy improved cardiac function in these animals. These findings link senescence biology to cardiac dysfunction in pregnancy and help to elucidate the pathogenesis underlying cardiovascular diseases of pregnancy.

Background: Drug-induced QT prolongation (diLQT) is a feared side-effect as exposing susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG channels that generate the repolarizing current (IKr) and thereby regulate the late repolarization phase. There is an important inter-individual susceptibility to develop diLQT which is of unknown origin but can be reproduced in patient-specific iPSC-derived cardiomyocytes (iPS-CMs). Objective: We aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT. Methods: We measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest or lowest sensitivity (HS or LS) to sotalol administration in vivo ( i . e ., based on the measure of the maximal change in QT interval 3 hours after administration). Specific small-interfering RNAs (siRNA) and CAVIN1-T2A-GFP adenovirus were used to manipulate CAVIN1 expression. Results: While HS and LS iPS-CMs showed similar electrophysiological characteristics at the baseline, the late repolarization phase was prolonged, and I _Kr significantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. I _Kr reduction was caused by a rapid translocation of hERG channel from the plasma membrane to the cytoskeleton upon sotalol application. This phenomenon was suppressed by blocking active endocytosis using dynasore. CAVIN1 , essential for caveolae biogenesis, was two-times more expressed in HS iPS-CMs and its knockdown using siRNA decreased their sensitivity to sotalol. CAVIN1 overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. Mechanistically, we found that treatment with sotalol promoted trafficking of the hERG channel from the plasma membrane to the cytoskeleton through caveolae and in a manner dependent on CAVIN1 expression. CAVIN1 silencing reduced the number of caveolae at the membrane and abrogated the internalization of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers such as E4031, vandetanib, and clarithromycin. Conclusions: Our study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the inter-individual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1. CLINICAL PERSPECTIVE: What is new?: - The inter-individual susceptibility underlying diLQT development involves unbridled turnover of cardiac ion channels from the plasma membrane. - This phenomenon is finely tuned by CAVIN1, a protein that is essential for essential for caveolae biogenesis. - Treatment with hERG blocker promoted trafficking of the hERG channel from the plasma membrane to the cytoskeleton through caveolae and in a manner dependent on CAVIN1 expression. What are the clinical implications?: - While congenital long QT is primarily from a genetic origin, the development of drug-induced long QT involves differences in the trafficking machinery of cardiac ion channels. - The prediction of CAVIN1 expression levels could help preventing drug-induced cardiotoxicity.

Introduction: Several drugs can induce QT prolongation by altering cardiac ion currents, notably IKr. The cardiotoxic response to a drug is however extremely variable between individuals, ranging from no impact to life-threatening ventricular arrhythmias. The molecular mechanisms underlying this variability remain unclear. Previously, we reported higher expression levels of Cavin1, an essential protein for caveolae formation, in cardiomyocytes (CM) derived from patients with high susceptibility to develop sotalol-induced long QT. Purpose: We aimed to explore whether Cavin1 could sway the cardiac repolarization response to drugs. Method: We used induced pluripotent stem cells-derived CM (iPS-CM) from two subjects with the highest sensitivity (HS) to sotalol (ie, those with the highest increase in QTc) and two individuals with the lowest sensitivity to sotalol (LS) to perform molecular and electrophysiological investigations. Results: At baseline, no differences in terms of repolarization durations, IKr amplitude, and ion channel expression were noted between the HS- and the LS-iPS-CM. However, the HS-iPS-CM displayed significant prolongation in action potential and field potential (FPD) durations after the application of 30 and 50μM of sotalol. They also displayed higher sensitivity to other drugs targeting hERG (E4031, vandetanib, and clarithromycin). Cavin1 mRNA and protein levels were increased by 2 fold in the HS-iPS-CM as compared to the LS-iPS-CM. Overexpressing Cavin1 in the LS-iPS-CM using adenovirus induced a higher sensitivity to sotalol as only 10μM was sufficient to induce 12% longer FPD. Consistently, the infection with Cavin1 increased the sensitivity of LS-CM to the other hERG blockers. In contrast, siRNA against Cavin1 reduced its expression by 60% in HS iPS-CM and switched their phenotype to a lower sensitivity to sotalol and the other hERG blockers. Interestingly, in line with the shortening of the FPD in LS-iPS-CM, overexpressing Cavin1 increased hERG expression in isolated membrane fraction from HEK cells stably expressing KCNH2. This suggests a role of Cavin1 in modulating hERG trafficking/recycling to the membrane and thereby the number of channels that can be blocked by drugs. Conclusion: Altogether, this work identified Cavin1 as a novel modulator of cardiomyocyte response to hERG blockers and a potential risk factor for diLQT. Further understanding of its contribution may pave the way for personalized drug prescription to avoid undesirable cardiotoxicity. Funding Acknowledgement: Type of funding sources: Foundation. Main funding source(s): Leducq

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.