Investigation of HNO-Derived Modifications on Phospholamban

Abstract

Congestive heart failure frequently involves reduced cardiac contractility and slowed muscle relaxation. The reactive nitrogen species HNO (nitroxyl, azanone) has been shown to augment contractility and accelerate relaxation in both normal and failing hearts. In humans, greater than 70% of cytosolic Ca2+ is pumped into the sarco(endo)plasmic reticulum (SR) by SR Ca2+-ATPase (SERCA2a). Phospholamban (PLN) is a small transmembrane protein, which is associated with SERCA2a, and depending on its phosphorylation state, regulates the activity of the Ca2+ pump. Our previous work has shown that HNO modifies PLN and affects SERCA2a activity independent of the β-adrenergic pathway. We have applied our 15N-edited NMR method for sulfinamide detection and biochemical approaches to investigate HNO-derived modifications on PLN. These studies demonstrate that PLN cysteines 46 and 41 are the major sites of HNO reactivity. Furthermore, our results indicate that sulfinamide formation is the major HNO-derived modification in the presence of excess HNO donor, whereas disulfide formation is expected to dominate under physiologically relevant conditions.