Targeting of phospholamban by peroxynitrite decreases β-adrenergic stimulation in cardiomyocytes

Aims: Peroxynitrite production increases during the pathogenesis of numerous cardiac disorders (e.g. heart failure). However, limited studies have investigated the mechanism through which peroxynitrite exerts anti-adrenergic effects. Thus, the purpose of this study is to investigate the contribution of phospholamban (PLB), a critical excitation-contraction coupling protein, to the peroxynitrite-induced dysfunction. Methods and results: Isolated myocytes from wild-type (WT, CF-1) and PLB knockout (PLB^-/-) mice were stimulated at 1 Hz, and myocyte shortening and Ca²⁺ transients were simultaneously recorded. PLB phosphorylation was measured via western blot. Myocytes were superfused with isoproterenol, a β-adrenergic agonist, and SIN-1, a peroxynitrite donor. SIN-1 superfusion dramatically decreased isoproterenol-stimulated Ca²⁺ transients and myocyte shortening in WT myocytes. These effects were inhibited upon addition of the peroxynitrite decomposition catalyst, FeTPPS. Surprisingly, SIN-1 had no functional effect on β-adrenergic-stimulated PLB^-/- myocytes. Western blot analyses revealed that SIN-1 significantly decreased isoproterenol-stimulated PLB _Ser16 phosphorylation. Experiments with the protein phosphatase inhibitor, okadaic acid, alleviated the SIN-1-induced functional effects and the decrease in PLB phosphorylation. Conclusions: The peroxynitrite donor SIN-1 decreases β-adrenergic stimulation by reducing PLB_Ser16 phosphorylation via protein phosphatase activation. This peroxynitrite-induced decrease in PLB phosphorylation may be a key mechanism in the β-adrenergic dysfunction observed in many cardiomyopathies. Published on behalf of the European Society of Cardiology. All rights reserved.