Abstract 15778: HNO Enhances SERCA2a Activity and Cardiomyocyte Function by Altering Key Cysteine Residues 41 and 46 in Phospholamban
Rationale. Nitroxyl (HNO) exerts positive cardiac inotropy/lusitropy partially via the acceleration of sarcoplasmic reticulum (SR) Ca2+ re-uptake/release. However, the role of phospholamban (PLN) in the HNO-induced acceleration of SR-Ca2+ uptake and myocyte functional enhancement is unknown.
Hypothesis. HNO modifies critical cysteine residues in PLN thereby uncoupling PLN from SERCA2a and enhancing its Ca2+ uptake activity.
Methods and Results. Ventricular myocytes and SR vesicles were isolated from control (WT) or PLN knockout (PLN-/-) mice to measure sarcomere shortening, whole Ca2+ transient and ATP-dependent Ca2+ uptake. Compared to WT, PLN-/- heart cells displayed enhanced basal contraction and a blunted response to isoproterenol. When exposed to HNO, these cells exhibited blunted nitroxyl-induced inotropy (n=20, 37.7±7%, p=0.0002 vs. 179±25% in WT) and Ca2+ transient amplitude (n=20, 8.2±4.5 vs. 35.5±8% in WT, p<0.005), along with diminished basal and caffeine-induced SR Ca2+ release. HNO accelerated SR Ca2+ uptake in WT SR vesicles (n=6, 0.3 to 0.7 sec-1, p<0.001), but had no effect in PLN-/- (n=7, 0.38 to 0.45 sec-1, p=NS). These effects occurred independent of β-agonist induced modification of PLN. Spectroscopic studies in microsomes show that in the presence of PLN, HNO decreased the rotational mobility of SERCA2a (from 60 ± 5 μsec to 90 ± 8 µsec), thereby increasing preferred oligomeric contacts between SERCA2a units. This led to an increased affinity for Ca2+ and catalytic efficiency of SERCA2a. Overall, our data show that HNO likely functions to uncouple PLN from SERCA2a through the modification of PLN cysteine residues 41 and 46, as the effect could be abolished when these residues were mutated to alanine.
Conclusions. HNO functionally uncouples PLN from SERCA2a via a cysteine-centered redox change, independent of PLN phosphorylation. This supports the use of HNO donors as therapeutic agents in cardiac diseased conditions such as heart failure where PKA-based modulation of SR targets is impaired.
- © 2011 by American Heart Association, Inc.