A key role of TRPC channels in the regulation of electromechanical activity of the developing heart

Aims It is well established that dysfunction of voltage-dependent ion channels results in arrhythmias and conduction disturbances in the foetal and adult heart. However, the involvement of voltage-insensitive cationic TRPC (transient receptor potential canonical) channels remains unclear. We assessed the hypothesis that TRPC channels play a crucial role in the spontaneous activity of the developing heart. Methods and results TRPC isoforms were investigated in isolated hearts obtained from 4-day-old chick embryos. Using RT-PCR, western blotting and co-immunoprecipitation, we report for the first time that TRPC1, 3, 4, 5, 6, and 7 isoforms are expressed at the mRNA and protein levels and that they can form a macromolecular complex with the α1C subunit of the L-type voltage-gated calcium channel (Cav1.2) in atria and ventricle. Using ex vivo electrocardiograms, electrograms of isolated atria and ventricle and ventricular mechanograms, we found that inhibition of TRPC channels by SKF-96365 leads to negative chrono-, dromo-, and inotropic effects, prolongs the QT interval, and provokes first- and second-degree atrioventricular blocks. Pyr3, a specific antagonist of TRPC3, affected essentially atrioventricular conduction. On the other hand, specific blockade of the L-type calcium channel with nifedipine rapidly stopped ventricular contractile activity without affecting rhythmic electrical activity. Conclusions These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesi

Influx cationique dépendant des canaux TRPCs dans les cellules musculaires squelettiques (régulation par le complexe dystrophine/alpha1-syntrophine et par la voie PLC)

La dystrophine est une protéine du cytosquelette normalement exprimée sous la membrane des cellules musculaires squelettiques. L'absence de cette protéine dans la Dystrophie Musculaire de Duchenne (DMD) entraîne la nécrose des fibres musculaires, résultant entre autres d'une dérégulation des mouvements calciques à travers le sarcolemme et par conséquent d'une augmentation du calcium libre dans le myoplasme. A l'heure actuelle, le lien entre l'absence de dystrophine et l'altération calcique n'est toujours pas établi et l'objectif de ce travail a été de le mettre en évidence. Les expériences ont été principalement réalisées sur les lignées cellulaires SolC1 déficientes en dystrophine et SolD6 exprimant la mini-dystrophine ainsi que sur des cultures primaires de souris normales et de souris mdx, modèle animal de la DMD. Notre étude démontre que, dans les cellules déficientes en dystrophine, les entrées calciques activées par la déplétion en calcium du réticulum sarcoplasmique, sont considérablement augmentées. Par la technique de siRNA, nous avons pu identifier les canaux TRPC1 et TRPC4 par où transitent les influx cationiques dans les myotubes SolD6. Nous avons également décrit pour la première fois un lien moléculaire entre TRPC1/TRPC4 et la dystrophine, l a1-syntrophine et le domaine PDZ de cette dernière. Ce complexe a1-syntrophine/TRPCs est réduit dans les cellules déficientes en dystrophine car l'expression de l'a1-syntrophine au sarcolemme est diminuée. Nous suggérons qu'une régulation normale des entrées de calcium à travers TRPC1/TRPC4 dépend de l'association entre ces canaux non dépendants du potentiel et l'a1-syntrophine. En effet, la surexpression de l'a1-syntrophine dans les cellules déficientes en dystrophine rétablit l'entrée de calcium. Inversement, des expériences avec des siRNAs dirigés contre l'a1-syntrophine entrainent une augmentation des influx cationiques dans les cellules exprimant la mini-dystrophine à des niveaux proches des influx mesurés dans les cellules déficientes en dystrophine. En plus de son rôle de protéine d'échafaudage, l'a1-syntrophine serait donc cruciale pour réguler l'activité des canaux TRPC1/TRPC4 dans le muscle squelettique. D'autre part, nous avons pu mettre en évidence par des traitements pharmacologiques que l'influx cationique exacerbé des cellules SolC1 déficientes en dystrophine est dépendant de la voie PLC/PKC. Dans ces myotubes, l absence de la dystrophine et/ou de l'a1-syntrophine entrainent à travers TRPC1 des entrées accrues de calcium, potentialisées par la voie PLC/PKC. Ce travail de thèse a mis clairement en évidence un influx cationique dépendant des canaux TRPCs et régulé par l'a1-syntrophine dans la cellule musculaire squelettique. L'absence de cette dernière au sarcolemme pourrait conférer une nouvelle sensibilité au canal TRPC1 entrainant alors sa suractivation et une entrée incontrôlée de calcium dans le cytoplasme des cellules déficientes en dystrophine.The dystrophin is a cytoskeleton protein normally expressed underneath the sarcolemma of skeletal muscle. The lack of this protein in Duchenne Muscular Dystrophy leads to muscle necrosis and to increased intracellular free calcium in the cytoplasm. Actually, the link between calcium mishandling and the absence of dystrophin is not well established and the aim of my study is to demonstrate it. Our works showed that cationic influx activated by calcium depletion of sarcoplasmic reticulum is strongly increased. We identified TRPC1 and TRPC4 channels supporting cationic influx in myotubes expressing mini-dystrophin. We also described for the first time a molecular link between dystrophin and TRPC1/TRPC4 channels, the alpha1-syntrophin. We suggested that normal regulation of syntrophin overexpression leads to reduction of abnormal cationic influx in dystrophin-deficient myotubes. Conversely, alpha1-syntrophin repression leads to increased cationic entry in myotubes expressing mini-dystrophin. The presence at normal level of this protein appears to be crucial for normal regulation of TRPC1/TRPC4 channels in skeletal muscle. On the other hand, we demonstrated an increased cationic influx supported by TRPC in dystrophin-deficient myotubes, which seems to be potentiated by PLC/PKC pathwayPOITIERS-BU Sciences (861942102) / SudocSudocFranceF

Response by Benitah et al to Letter Regarding Article, “Orai1 Channel Inhibition Preserves Left Ventricular Systolic Function and Normal Ca 2+ Handling After Pressure Overload”

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Excitation-contraction coupling and relaxation alteration in right ventricular remodelling caused by pulmonary arterial hypertension

International audiencePulmonary arterial hypertension is a progressive and lethal cardiopulmonary disease. The rise in right ventricular afterload leads to right ventricular hypertrophy and failure. Right ventricular failure is the most important prognostic factor for morbidity and mortality in pulmonary arterial hypertension or pulmonary hypertension caused by left heart diseases. Surprisingly, the right ventricle is not targeted by pulmonary arterial hypertension-specific therapies. The current profound lack of basic understanding of pulmonary arterial hypertension-related right ventricular remodelling can explain, at least in part, this paradox. The physiology and haemodynamic function of the right ventricle in the normal state differ considerably from those of the left ventricle, and the known mechanisms of left ventricular dysfunction cannot be generalized to right ventricular dysfunction. Ion channel activities and calcium homeostasis tightly regulate cardiac function, and their dysfunction contributes to the pathogenesis of cardiac diseases. This review focuses on the ion channels (potassium, calcium) and intracellular calcium handling remodelling involved in right ventricular hypertrophy and dysfunction caused by pulmonary arterial hypertension

Targeting Orai1-Mediated Store-Operated Ca2+ Entry in Heart Failure

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Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels

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The SOCE Machinery: An Unbalanced Knowledge between Left and Right Ventricular Pathophysiology

Right ventricular failure (RVF) is the most important prognostic factor for morbidity and mortality in pulmonary arterial hypertension (PAH) or pulmonary hypertension (PH) caused by left heart diseases. However, right ventricle (RV) remodeling is understudied and not targeted by specific therapies. This can be partly explained by the lack of basic knowledge of RV remodeling. Since the physiology and hemodynamic function of the RV differ from those of the left ventricle (LV), the mechanisms of LV dysfunction cannot be generalized to that of the RV, albeit a knowledge of these being helpful to understanding RV remodeling and dysfunction. Store-operated Ca2+ entry (SOCE) has recently emerged to participate in the LV cardiomyocyte Ca2+ homeostasis and as a critical player in Ca2+ mishandling in a pathological context. In this paper, we highlight the current knowledge on the SOCE contribution to the LV and RV dysfunctions, as SOCE molecules are present in both compartments. he relative lack of studies on RV dysfunction indicates the necessity of further investigations, a significant challenge over the coming years

Specific Upregulation of TRPC1 and TRPC5 Channels by Mineralocorticoid Pathway in Adult Rat Ventricular Cardiomyocytes

International audienceWhereas cardiac TRPC (transient receptor potential canonical) channels and the associated store-operated Ca2+ entry (SOCE) are abnormally elevated during cardiac hypertrophy and heart failure, the mechanism of this upregulation is not fully elucidated but might be related to the activation of the mineralocorticoid pathway. Using a combination of biochemical, Ca2+ imaging, and electrophysiological techniques, we determined the effect of 24-h aldosterone treatment on the TRPCs/Orai-dependent SOCE in adult rat ventricular cardiomyocytes (ARVMs). The 24-h aldosterone treatment (from 100 nM to 1 µM) enhanced depletion-induced Ca2+ entry in ARVMs, as assessed by a faster reduction of Fura-2 fluorescence decay upon the addition of Mn2+ and increased Fluo-4/AM fluorescence following Ca2+ store depletion. These effects were prevented by co-treatment with a specific mineralocorticoid receptor (MR) antagonist, RU-28318, and they are associated with the enhanced depletion-induced N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2)-sensitive macroscopic current recorded by patch-clamp experiments. Molecular screening by qRT-PCR and Western blot showed a specific upregulation of TRPC1, TRPC5, and STIM1 expression at the messenger RNA (mRNA) and protein levels upon 24-h aldosterone treatment of ARVMs, corroborated by immunostaining. Our study provides evidence that the mineralocorticoid pathway specifically promotes TRPC1/TRPC5-mediated SOCE in adult rat cardiomyocytes