158 - HNO Modifies Cysteines 41 and 46 in the Transmembrane Domain of Phospholamban Increasing SERCA2a Activity

Abstract

Heart failure (HF) is characterized by perturbed myocyte Ca2+ handling. Altered SR Ca2+-ATPase (SERCA2a) activity sizably contributes to impair Ca2+ cycling in the heart, affecting its contraction/relaxation. Strategies to rescue or improve SERCA2a expression/activity are being clinically tested in HF patients. Donors of nitroxyl (HNO) augment cardiac muscle performance in failing hearts, by modulating phospholamban (PLN) and SERCA2a interaction. They do so independently from β-adrenergic activation. In fact, HNO increases SERCA2a activity by promoting redox-dependent PLN oligomerization, thus reducing the amount of monomeric/inhibitory PLN. Yet, the precise sites (cysteines that are modified by HNO in PLN) and the chemical nature of these redox modifications (disulfide bond vs. sulfinamide formation) remain elusive. To fill this gap, we have applied our 15N-edited NMR method for sulfinamide detection and biochemical approaches to investigate HNO-derived modifications on PLN. We found that cysteines 46 and 41 are the sites of HNO action in PLN, and that sulfinamide formation is the major HNO-derived modification when HNO is in excess with respect to thiols. However, when HNO/thiol approach the 1:1 ratio (thus reflecting more physiological conditions), intramolecular disulfide bonds are expected to be the dominant HNO-induced species. To investigate the intramolecular disulfide bond formation further, we have started to employ fluorescence labeling methodologies. Present findings provide more mechanistic insight into the salutary actions of HNO donors that are currently tested for safety and efficacy in patients with acute decompensated HF.