Serine 663-dependent phosphorylation of SERCA2 in ischemic heart disease: Regulation of calcium homeostasis, cell death and myocardial infarct size

Abstract

Ischemic heart disease is one of the leading cause of death and disability worldwide. Calcium handling in the post-ischemic myocardium is a prime target to treat patients with acute myocardial infarction. Although SERCA2 is a promising target for the treatment of Ca2+ dyshomeostasis and contractile dysfunction following ischemia-reperfusion, the mechanisms through which SERCA2 is regulated remains still unclear. Post-translational modifications of SERCA2 have been recently shown to modulate its activity, however specific phosphorylation sites of SERCA2 have not yet been identified. We hypothesized that a direct phosphorylation of SERCA2 controls SERCA2 activity, Ca2+ homeostasis and subsequent cell death in heart. We combined in silico analysis with in vitro and genetic manipulations to determine potential SERCA2 phosphorylation sites and their effect on Ca2+ homeostasis and cell death in heart. Importantly, we developed an in vivo AAV-myocardial rescue strategy to assess the effect of the SERCA2 phosphorylation event on infarct size and we investigated the potential application of our finding in patient hearts. Our results show for the first time that SERCA2 is a potential substrate for the enzyme GSK3β, whose interaction is improved during an ischemic stress, associated with an increase of the phosphorylation level of SERCA2 at S663 in human hearts. Using a CRISPR/Cas9 approaches, we demonstrated that when SERCA2 cannot be phosphorylated at serine 663, the activity of SERCA2 is significantly increased. As a consequence, we provided evidences that when SERCA2 is resistant to serine-663 phosphorylation, cells are protected cells against reperfusion injury, through a cellular detoxification of calcium into reticulum lumen. As in vivo proof of concept, using AAV-myocardial strategy, phosphoresistant S663 mutant showed an improved excitation-contraction coupling and developed significantly smaller infarct as compared to WT, whereas infarct size was drastically increased in phosphomimetic S663 rescued mice. We conclude that the increased phosphorylation of SERCA2 at serine 663 is a contemporary event of cardiovascular diseases in human hearts, and that avoiding the phosphorylation of SERCA2 at S663 offers a novel therapeutic strategy against acute myocardial infarction, through detoxification of cellular Ca2+ excess into reticulum lumen.