Abstract 17974: Sulphonylation of SERCA Cysteine-674 Contributes to Cardiac Dysfunction in Endotoxemic Mice
Background: Functional inhibition of the sarcoplasmic reticulum (SR) calcium (Ca2+) pump (SERCA) contributes to cardiac dysfunction and cardiac Ca2+ dysregulation in sepsis and endotoxemia. Here we aimed to identify the molecular mechanisms responsible for SERCA inhibition in endotoxemic mice.
Methods: Male C57BL/6 mice (15-25 weeks old) were administered lipopolysaccharide (LPS, 25 μg/g, ip) and euthanized 12 h later. Cellular Ca2+ transients were recorded (using fura-2 AM) in isolated, externally paced cardiomyocytes.
Results: SERCA function was depressed in LPS-challenged mice to 90 ± 4% of baseline (p<0.01, n>30 cells from >4 mice for this and following, unless specified), as evidenced by prolongation of the time constant of Ca2+ decay (τCa), in association with a decrease in Ca2+ transient amplitude (ΔCai, to 84 ± 4% of baseline) and SR Ca2+ load (CaSR, measured using caffeine, to 86 ± 5%). Among other Ca2+ transporters, LPS did not alter the function of the sarcolemmal Na+/Ca2+ exchange or the ryanodine receptor leak flux. LPS did not alter protein expression of SERCA or phospholamban (PLB), nor affect PLB phosphorylation (at Ser16 and Thr17, assessed by immunoblotting). LPS caused sulphonylation of SERCA cysteine-674, as measured immunohistochemically (to 156 ± 18% of baseline, n>5 hearts each group) and supported by decreased SERCA cysteine-674 labeling with biotinylated iodoacetamide (to 61 ± 6%, n >5 hearts each group). We next aimed to identify whether cGMP synthesized by the enzyme soluble guanylyl cyclase (sGC, which is proposed to play a protective role in sepsis) prevents SERCA sulphonylation in LPS-challenged mice. After LPS challenge, mice deficient in sGC main isoform (sGCα1-/-) showed more pronounced levels of SERCA cysteine-674 sulphonylation (to 227 ± 30% of baseline, n>5 hearts each group), as well as a more severe decrease in SERCA function (to 80 ± 3%), ΔCai (to 57 ± 3%) and CaSR (to 62 ± 4% of baseline), confirming the protective effect of cGMP.
Conclusions: Cardiac Ca2+ dysregulation in endotoxemic mice is mediated, in part, by sulphonylation of SERCA cysteine-674. In contrast, cGMP produced by sGC plays a protective role by opposing SERCA sulphonylation.
- © 2013 by American Heart Association, Inc.