Phosphorylation of SERCA2 at the heart of cardioprotection in human-derived cardiomyocytes

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.