Youakim Saliba1, Mathilde Keck2, Alexandre Marchand2, Fabrice Atassi2, Aude Ouillé3, Olivier Cazorla3, Mohamed Trebak4, Catherine Pavoine2, Alain Lacampagne3, Jean-Sébastien Hulot5, Nassim Farès6, Jérémy Fauconnier3, Anne-Marie Lompré7. 1. Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1166, ICAN, F-75005 Paris, France INSERM, UMR_S 1166, ICAN, F-75005 Paris, France Laboratoire de Recherche en Physiologie et Physiopathologie, Pôle Technologie Santé, Faculté de Médecine, Université Saint Joseph, Beyrouth, Lebanon. 2. Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1166, ICAN, F-75005 Paris, France INSERM, UMR_S 1166, ICAN, F-75005 Paris, France. 3. Université Montpellier 1 et 2, Inserm U1046, Montpellier, France. 4. SUNY College of Nanoscale Science and Engineering, Albany, NY, USA. 5. Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1166, ICAN, F-75005 Paris, France INSERM, UMR_S 1166, ICAN, F-75005 Paris, France Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 6. Laboratoire de Recherche en Physiologie et Physiopathologie, Pôle Technologie Santé, Faculté de Médecine, Université Saint Joseph, Beyrouth, Lebanon. 7. Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1166, ICAN, F-75005 Paris, France INSERM, UMR_S 1166, ICAN, F-75005 Paris, France anne-marie.lompre@upmc.fr.
Abstract
AIMS: Stromal interaction molecule 1 (STIM1) has been shown to control a calcium (Ca(2+)) influx pathway that emerges during the hypertrophic remodelling of cardiomyocytes. Our aim was to determine the interaction of Orai1 and Orai3 with STIM1 and their role in the constitutive store-independent and the store-operated, STIM1-dependent, Ca(2+) influx in cardiomyocytes. METHODS AND RESULTS: We characterized the expression profile of Orai proteins and their interaction with STIM1 in both normal and hypertrophied adult rat ventricular cardiomyocytes. Orai1 and 3 protein levels were unaltered during the hypertrophic process and both proteins co-immunoprecipitated with STIM1. The level of STIM1 and Orai1 were significantly greater in the macromolecular complex precipitated by the Orai3 antibody in hypertrophied cardiomyocytes. We then used a non-viral method to deliver Cy3-tagged siRNAs in vivo to adult ventricular cardiomyocytes and silence Orai channel candidates. Cardiomyocytes were subsequently isolated then the voltage-independent, i.e. store-independent and store-operated Ca(2+) entries were measured on Fura-2 AM loaded Cy3-labelled and control isolated cardiomyocytes. The whole cell patch-clamp technique was used to measure Orai-mediated currents. Specific Orai1 and Orai3 knockdown established Orai3, but not Orai1, as the critical partner of STIM1 carrying these voltage-independent Ca(2+) entries in the adult hypertrophied cardiomyocytes. Orai3 also drove an arachidonic acid-activated inward current. CONCLUSION: Cardiac Orai3 is the essential partner of STIM1 and drives voltage-independent Ca(2+) entries in adult cardiomyocytes. Arachidonic acid-activated currents, which are supported by Orai3, are present in adult cardiomyocytes and increased during hypertrophy. Published on behalf of the European Society of Cardiology. All rights reserved.
AIMS: Stromal interaction molecule 1 (STIM1) has been shown to control a calcium (Ca(2+)) influx pathway that emerges during the hypertrophic remodelling of cardiomyocytes. Our aim was to determine the interaction of Orai1 and Orai3 with STIM1 and their role in the constitutive store-independent and the store-operated, STIM1-dependent, Ca(2+) influx in cardiomyocytes. METHODS AND RESULTS: We characterized the expression profile of Orai proteins and their interaction with STIM1 in both normal and hypertrophied adult rat ventricular cardiomyocytes. Orai1 and 3 protein levels were unaltered during the hypertrophic process and both proteins co-immunoprecipitated with STIM1. The level of STIM1 and Orai1 were significantly greater in the macromolecular complex precipitated by the Orai3 antibody in hypertrophied cardiomyocytes. We then used a non-viral method to deliver Cy3-tagged siRNAs in vivo to adult ventricular cardiomyocytes and silence Orai channel candidates. Cardiomyocytes were subsequently isolated then the voltage-independent, i.e. store-independent and store-operated Ca(2+) entries were measured on Fura-2 AM loaded Cy3-labelled and control isolated cardiomyocytes. The whole cell patch-clamp technique was used to measure Orai-mediated currents. Specific Orai1 and Orai3 knockdown established Orai3, but not Orai1, as the critical partner of STIM1 carrying these voltage-independent Ca(2+) entries in the adult hypertrophied cardiomyocytes. Orai3 also drove an arachidonic acid-activated inward current. CONCLUSION: Cardiac Orai3 is the essential partner of STIM1 and drives voltage-independent Ca(2+) entries in adult cardiomyocytes. Arachidonic acid-activated currents, which are supported by Orai3, are present in adult cardiomyocytes and increased during hypertrophy. Published on behalf of the European Society of Cardiology. All rights reserved.
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