S100A1 has emerged as a key factor in the control of cardiomyocyte (CM) contractile performance in vivo and in vitro and S100A1 gene therapy presents a promising approach for the treatment of heart failure. Improved sarcoplasmic reticulum (SR) function with enhanced Ca2+ resequestration appears critical for its cAMP-independent inotropic effects but raises concerns about potential diastolic SR Ca2+ leakage that might trigger fatal arrhythmias. Thus, the major goal of this study is to determine the impact of S100A1 on the development of diastolic SR Ca2+ leak, triggered Ca2+ waves and after-contractions in response to Ca2+- and cAMP-dependent stressors using cardiac cellular and multi-cellular models as an in vitro test platform.Methods and Results
Employing confocal Ca2+ imaging, adenoviral-mediated S100A1 overexpression in adult rat CMs revealed both decreased SR Ca2+-spark frequency and prevented β-adrenergic receptor (β-AR)-triggered Ca2+ waves despite augmented SR Ca2+ load compared to controls. Equal efficacy was observed in electrically stimulated failing CMs, where S100A1 overexpression protected against β-AR triggered diastolic Ca2+ waves (occurring in 25% of CMs compared to 83% in controls). Analysis of the apparent protective effect of S100A1 in Engineered rat Heart Tissue (EHT) preparations revealed a profound anti-arrhythmic potency against Ca2+-triggered after-contractions as measured in a shift of EC50 from 1mM to 1.5mM extracellular Ca2+, while inotropy was preserved under isometric load ( + 40% in twitch tension, TT). After chronic endothelin-1 treatment, hypertrophied EHTs exhibited severe contractile failure (-50% in TT compared to control) and pro-arrhythmogenic SR Ca2+ leakage, which could be rescued by adenoviral-mediated S100A1 overexpression. Mechanistically, ryanodine receptor (RyR2) immunoprecipitation confirmed a direct interaction with S100A1 but unveiled unchanged compartmentalized protein kinase A (PKA) and Ca2+/ calmodulin dependent protein kinase II (CamKII) activity as analyzed by RyR2 Ser2808 and Ser2814 phosphorylation.Conclusion
Our data provide first proof-of-concept for a potential anti-arrhythmic efficacy of S100A1 in CMs using EHT as a test platform. S100A1 appears to convey a rather unique molecular profile combining cAMP-independent inotropy with protection against Ca2+- and β-AR-triggered pro-arrhythmogenic events. Our data point towards the suppression of the SR Ca2+-leak by a direct interaction of S100A1 with the RyR2. Anyhow, further research is clearly needed and ongoing studies focus on the precise molecular interaction of S100A1 at the RyR2.