Laura Boulogne , Tanushri Dargar , Camille Brun , Christelle Leon , Christophe Chouabe , Hélène Thibault , Gabriel Bidaux , Laurent Sebbag , Vincent Gache , Ludovic Gomez
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引用次数: 0
Abstract
Introduction
Despite promising preclinical results targeting sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2), clinical trials application remains limited, highlighting the urgency to develop suitable model reflecting clinical physiopathology. Recently, we discovered a new regulatory mechanism of SERCA2 based on serine 663-phosphorylation involved as a key regulator of Ca2+ homeostasis in several cell types (HEK, MEF and isolated mice cardiomyocytes).
Objective
To assess translational potential, we developed a human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) model focused on modulation of SERCA2 activity.
Method
hiPSC-derived cardiomyocytes were infected (6 days, MOI 200 000) with the AAV9-Serca [WT], the AAV9-Serca2[S663A] phosphoresistant or the AAV9-Serca2[S663E] phosphomimetic mutants. For [Ca2+]ERexperiments, basal and refilling slope were measured in hiPSC-CM using D4ER Ca2+ probe. Cytosolic Ca2+ imaging was investigated using Fura2 probe (2 μM). Cell viability was evaluated after 5 h hypoxia (1%O2) and 5 h reoxygenation (H/R).
Results
In opposite to SERCA2[S663E] cells, the evaluation of SERCA2 activity revealed that SERCA2[S663A] hiPSC-CM displayed significantly increased ER Ca2+ refilling rate (+56%) and ER Ca2+ content (+66%) versus SERCA2[WT]. Interestingly, SERCA2[S663A] displayed a significant decreased cell death after H/R stress compared to SERCA2[WT]. Although, measuring of cytosolic Ca2+ transients in beating control hiPSC-CMs, there were no changes (peak amplitude 0.08 μm, area under curve 0.04 μm2) in all mutants due to ineffective infection.
Conclusion
Our results provide an essential regulatory mechanism of Ca2+ homeostasis and cell death, based on the phosphorylation state of SERCA2 in human cell types, notably hiPSC-CM. Although still requiring some adjustments, hiPSC-CM appear to be an essential model for translational research in the field of heart disease.
期刊介绍：
The Journal publishes original peer-reviewed clinical and research articles, epidemiological studies, new methodological clinical approaches, review articles and editorials. Topics covered include coronary artery and valve diseases, interventional and pediatric cardiology, cardiovascular surgery, cardiomyopathy and heart failure, arrhythmias and stimulation, cardiovascular imaging, vascular medicine and hypertension, epidemiology and risk factors, and large multicenter studies. Archives of Cardiovascular Diseases also publishes abstracts of papers presented at the annual sessions of the Journées Européennes de la Société Française de Cardiologie and the guidelines edited by the French Society of Cardiology.
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