0235 : In vivo overexpression of a cardiac sodium channel mutant in mice

Loss-of-function mutations in the cardiac Na+ channel α-subunit gene, SCN5A, cause Brugada syndrome (BrS), a hereditary disease characterized by ventricular fibrillation and sudden cardiac death. We previously evidenced, in HEK cells, the dominant-negative effect of the R104W BrS mutation in Nav1.5, inducing the retention of the wild-type (WT) channel and the proteasomal degradation of the mutant protein. To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAV).We used a dual AAV vector strategy combining viral DNA recombination and trans-splicing. One-week old mice were injected with two AAV serotypes capsid 9: one, packaging the cardiac specific troponin-T promoter, the 5’ half of hSCN5A, the 5’ donor site of a synthetic intron and a highly recombinogenic sequence; and another, packaging the same recombinogenic sequence, the 3’ acceptor site of the synthetic intron, the 3’ half of hSCN5A, the gfp gene as a reporter, and the SV40 polyA signal. Six weeks after injection, the hSCN5A full-gene expression and the percentage of transduced cardiac cells were assessed by qPCR, western blot (WB) analysis and immunohistochemistry on transduced heart tissues. The Na+ current was recorded by the patchclamp technique in isolated cardiomyocytes.Both WT and mutant human Nav1.5 transcripts and proteins were observed by RT-qPCR, WB and immunohistochemistry on injected-mice heart tissues. Patch-clamp recordings in WT-channel injected mice evidenced a two-fold increase of the Na+ current. In contrast, the cardiac Na+ current of R104Winjected mice was impaired (i.e. the current density was decreased by 45% and the activation was shifted by -4mV).Our data suggest that the trans-splicing and viral DNA recombination strategy using AAV9 serotype and a cardiac-specific promoter is successful to overexpress WT or mutant Na+ channels in mouse hearts. This approach allowed us to modulate the cardiac Na+ current in adult mice

Intracardiac electrophysiology to characterize susceptibility to ventricular arrhythmias in murine models

Introduction: Sudden cardiac death (SCD) and ventricular fibrillation are rare but severe complications of many cardiovascular diseases and represent a major health issue worldwide. Although the primary causes are often acute or chronic coronary diseases, genetic conditions, such as inherited channelopathies or non-ischemic cardiomyopathies are leading causes of SCD among the young. However, relevant experimental models to study the underlying mechanisms of arrhythmias and develop new therapies are still needed. The number of genetically engineered mouse models with cardiac phenotype is growing, making electrophysiological studies in mice essential tools to study arrhythmogenicity and arrhythmia mechanisms and to test novel treatments. Recently, intracardiac catheterization via the jugular vein was described to induce and record ventricular arrhythmias in living anesthetized mice. Several strategies have been reported, developed in healthy wild-type animals and based on aggressive right ventricular stimulation.Methods: Here, we report a protocol based on programmed electrical stimulation (PES) performed in clinical practice in patients with cardiac rhythm disorders, adapted to two transgenic mice models of arrhythmia - Brugada syndrome and cardiolaminopathy.Results: We show that this progressive protocol, based on a limited number of right ventricular extrastimuli, enables to reveal different rhythmic phenotypes between control and diseased mice. In this study, we provide detailed information on PES in mice, including catheter positioning, stimulation protocols, intracardiac and surface ECG interpretation and we reveal a higher susceptibility of two mouse lines to experience triggered ventricular arrhythmias, when compared to control mice.Discussion: Overall, this technique allows to characterize arrhythmias and provides results in phenotyping 2 arrhythmogenic-disease murine models

Actin-microtubule cytoskeletal interplay mediated by MRTF-A/SRF signaling promotes dilated cardiomyopathy caused by LMNA mutations

Publisher Copyright: © 2022, The Author(s).Mutations in the lamin A/C gene (LMNA) cause dilated cardiomyopathy associated with increased activity of ERK1/2 in the heart. We recently showed that ERK1/2 phosphorylates cofilin-1 on threonine 25 (phospho(T25)-cofilin-1) that in turn disassembles the actin cytoskeleton. Here, we show that in muscle cells carrying a cardiomyopathy-causing LMNA mutation, phospho(T25)-cofilin-1 binds to myocardin-related transcription factor A (MRTF-A) in the cytoplasm, thus preventing the stimulation of serum response factor (SRF) in the nucleus. Inhibiting the MRTF-A/SRF axis leads to decreased α-tubulin acetylation by reducing the expression of ATAT1 gene encoding α-tubulin acetyltransferase 1. Hence, tubulin acetylation is decreased in cardiomyocytes derived from male patients with LMNA mutations and in heart and isolated cardiomyocytes from Lmnap.H222P/H222P male mice. In Atat1 knockout mice, deficient for acetylated α-tubulin, we observe left ventricular dilation and mislocalization of Connexin 43 (Cx43) in heart. Increasing α-tubulin acetylation levels in Lmnap.H222P/H222P mice with tubastatin A treatment restores the proper localization of Cx43 and improves cardiac function. In summary, we show for the first time an actin-microtubule cytoskeletal interplay mediated by cofilin-1 and MRTF-A/SRF, promoting the dilated cardiomyopathy caused by LMNA mutations. Our findings suggest that modulating α-tubulin acetylation levels is a feasible strategy for improving cardiac function.Peer reviewe

J Clin Invest

Arterial cardiovascular events are the leading cause of death in patients with JAK2V617F myeloproliferative neoplasms (MPN). However, their mechanisms are poorly understood. The high prevalence of myocardial infarction without significant coronary stenosis or atherosclerosis in patients with MPN suggests that vascular function is altered. Consequences of JAK2V617F mutation on vascular reactivity are unknown. We observe here increased responses to vasoconstrictors in arteries from Jak2V617F mice, resulting from disturbed endothelial nitric oxide pathway and increased endothelial oxidative stress. This response was reproduced in wild-type mice by circulating microvesicles isolated from patients carrying JAK2V617F and by erythrocyte-derived microvesicles from transgenic mice. Microvesicles of other cellular origins had no effect. This effect was observed ex vivo on isolated aortas, but also in vivo on femoral arteries. Proteomic analysis of microvesicles derived from JAK2V617F erythrocytes identified increased expression of myeloperoxidase as the likely mechanism accounting for microvesicles effect. Myeloperoxidase inhibition in microvesicles derived from JAK2V617F erythrocytes supressed their effect on oxidative stress. Antioxidants, such as simvastatin and N-acetyl-cysteine, improved arterial dysfunction in Jak2V617F mice. In conclusion, JAK2V617F MPN are characterized by exacerbated vasoconstrictor responses resulting from increased endothelial oxidative stress caused by circulating erythrocyte-derived microvesicles. Simvastatin appears as promising therapeutic strategy in this setting

Etude des mécanismes adaptateurs neuro-hormonaux et rénaux induits par différents traitements diurétiques au cours de l'insuffisance cardiaque et de l'hypertension artérielle expérimentales

L'objectif de ces travaux est l'optimisation du traitement diurétique au cours de l'hypertension artérielle (HTA) et de l'insuffisance cardiaque (IC). Plusieurs types de diurétiques ont été évalués afin de préciser les mécanismes pouvant interférer à long terme avec leur effet : interactions avec la stimulation neuro-hormonale et phénomènes intra-rénaux d'adaptation secondaire à l'augmentation induite de la natriurèse. Dans l'IC, induite chez le rat par ligature coronaire : étude de la modulation des effets de l'indapamide sur le système rénine angiotensine aldostérone (SRAA) par le blocage par un inhibiteur de l'enzyme de conversion (périndopril). Puis étude de l'efficacité et du degré de mise en jeu du SRAA lors d'une administration chronique et aiguë d'un antagoniste des récepteurs AI de l'adénosine (A1AdoR) et du furosémide ayant un mécanisme d'action différent, et de les associer pour conserver une activité thérapeutique optimale lors d'un traitement prolongé. Comparaison des effets diurétiques, natriurétiques et hémodynamiques et éventuellement déterminer l'efficacité de leur association. Puis nous avons évalué les effets de l'A1AdoR, du captopril, du furosémide et de leur association sur la survie des animaux. Dans l'HTA, nous avons utilisé le rat SHR. Nous avons étudié la stimulation neuro-hormonale, le remodelage cardiovasculaire, la réactivité cardiaque et vasculaire lors de l'association bisoprolol-hydrochlorotiazide à faibles doses. Le système neuro-hormonal joue un rôle important dans le remodelage cardiovasculaire, et la combinaison de diurétiques à mécanismes d'action différents représente une voie pharmacologique et thérapeutique à explorer.The objective of this work is the optimization of the diuretic treatment during arterial hypertension (HTA) and heart failure (HF). Several types of diuretic were evaluated in order to precise the mechanisms able to interfere on the long term with their effects : interactions with neurohormonal stimulation and intra-renal phenomena of secondary adaptation to the increase induced by the natriuresis. In HF, induced in rat by coronary ligation : study of the modulation of the effects of indapamide on the renin-angiotensin-aldosterone system (RAAS) blocked by an angiotensin-converting enzyme inhibitor (perindopril). Then, study of the effectiveness and the degree of setting concerned the RAAS at the time of a chronic and acute administration of A1 adenosine receptor antagonist (A1AdoR) and furosemide having a different mechanism of action, and to associate them to preserve an optimal therapeutic activity during a long term treatment. Comparison of diuretics, natriuretics and hemodynamics effects and eventually determine the effectiveness of their association. Then we evaluated the effects of A1AdoR, captopril, furosemide and their association on animal's survival. In HTA, we used SHR rats. We studied neurohormonal stimulation, cardiovascular remodelling, and the cardia and vascular reactivity in combination with low doses of bisoprolol-hydrochlorotiazide. The neurohormonal system plays an important part in cardiovascular remodelling and diuretics combination with different mechanisms of action represents a pharmacological and therapeutic way to explore.PARIS-BIUP (751062107) / SudocSudocFranceF

Le montage : «recoller les morceaux»

Lepeutrec Françoise, Coussein Sophie, Mougenot Nathalie. Le montage : «recoller les morceaux». In: Diplômées, n°216, 2006. La création d images : des métiers de femme ? pp. 17-21

Effect of genetic background on the cardiac phenotype in a mouse model of Emery-Dreifuss muscular dystrophy

International audienceA-type lamins gene (LMNA) mutations cause an autosomal dominant inherited form of Emery-Dreifuss muscular dystrophy (EDMD). EDMD is characterized by slowly progressive muscle weakness and wasting and dilated cardiomyopathy, often leading to heart failure-related disability. EDMD is highly penetrant with poor prognosis and there is currently no specific therapy available. Clinical variability ranges from early onset with severe presentation in childhood to late onset with slow progression in adulthood. Genetic background is a well-known factor that significantly affects phenotype in several mouse models of human diseases. This phenotypic variability is attributed, at least in part, to genetic modifiers that regulate the disease process. To characterize the phenotype of A-type lamins mutation on different genetic background, we created and phenotyped C57BL/6JRj-Lmna H222P/H222P mice (C57 Lmna p.H222P) and compared them with the 129S2/SvPasCrl-Lmna H222P/H222P mice (129 Lmna p.H222P). These mouse strains were compared with their respective control strains at multiple time points between 3 and 10 months of age. Both contractile and electrical cardiac muscle functions, as well as survival were characterized. We found that 129 Lmna p.H222P mice showed significantly reduced body weight and reduced cardiac function earlier than in the C57 Lmna p.H222P mice. We also revealed that only 129 Lmna p.H222P mice developed heart arrhythmias. The 129 Lmna p.H222P model with an earlier onset and more pronounced cardiac phenotype may be more useful for evaluating therapies that target cardiac muscle function, and heart arrhythmias

0151 : In vivo modulation of the cardiac Na+ current using AAVs

International audienc