Targeting of phospholamban by peroxynitrite decreases beta-adrenergic stimulation in cardiomyocytes.
OBJECTIVE
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.


RESULTS
Isolated myocytes from wild-type (WT, CF-1) and PLB knockout (PLB(-/-)) mice were stimulated at 1 Hz, and myocyte shortening and Ca(2+) transients were simultaneously recorded. PLB phosphorylation was measured via western blot. Myocytes were superfused with isoproterenol, a beta-adrenergic agonist, and SIN-1, a peroxynitrite donor. SIN-1 superfusion dramatically decreased isoproterenol-stimulated Ca(2+) 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 beta-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 beta-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 beta-adrenergic dysfunction observed in many cardiomyopathies.
Kohr
Mark J
MJ
Department of Physiology and Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, USA.
Wang
Honglan
H
Wheeler
Debra G
DG
Velayutham
Murugesan
M
Zweier
Jay L
JL
Ziolo
Mark T
MT
D000818:Animals; D002135:Calcium-Binding Proteins / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology*; D030421:Peroxynitrous Acid / Q000502:physiology*; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors / Q000502:physiology; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000502:physiology; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000502:physiology*; D012694:Serine / Q000378:metabolism; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D007545:Isoproterenol; D008981:Molsidomine; D030421:Peroxynitrous Acid; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D011943:Receptors, Adrenergic, beta; D012694:Serine; D000099211:Phospholamban; C002385:linsidomine