S100A1’s single cysteine is an indispensable redox-switch for the protection against diastolic calcium leakage in cardiomyocytes

The EF-hand calcium (Ca 2+ ) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CM). Oxidative posttranslational modification (ox-PTM) of S100A1’s conserved, single cysteine residue (C85) via reactive nitrogen species (i.e. S-nitrosylation or glutathionylation) was proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecule’s affinity towards Ca 2+ . In light of the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox-switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wildtype (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca 2+ transient amplitudes under basal conditions and β-adrenergic receptor (βAR) stimulation. However, in contrast ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca 2+ leak and ryanodine receptor (RyR2) hypernitrosylation during β-AR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca 2+ -dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1’s molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox-switch that is indispensable for S100A1’s antiarrhythmic but not its inotropic potency in CM. We therefore propose a model where C85’s ox-PTM determines S100A1’s ability to beneficially control diastolic but not systolic RyR2 activity.