BPS2026 – Molecular effects of CaMKIIδ S-nitrosylation in cardiomyocytes

Calcium/calmodulin (CaM)-dependent protein kinase II delta (CaMKIIδ) is a central regulator of calcium signaling and electrophysiological function in cardiac myocytes. Persistent CaMKIIδ activation is implicated in heart failure (HF) and arrhythmia. Several distinct post-translational modifications (PTMs) affect kinase activity, either suppressing it or prolonging it even after CaM dissociates (autonomous activation). While autophosphorylation at T287 is well understood, the molecular properties of other PTMs, including CaM affinity, dissociation kinetics, and kinase conformation are less clear in native myocytes. S -nitrosylation at C273 (within the hinge region) suppresses CaM-dependent CaMKII activation which can be protective against arrhythmia. In contrast, S -nitrosylation at C290 (which is exposed after CaM binding) promotes autonomous activity, pathological Ca/Na dysregulation and arrhythmic triggers. In myocytes expressing WT, C273S, or C290A mutants of GFP-CaMKIIδ, we used FRET to measure the affinity of fluorescently tagged AF-568-CaM binding to CaMKIIδ upon elevation of Ca in saponin-permeabilized myocytes, with or without pretreating with S-nitrosylating agent GSNO. WT GFP-CaMKIIδ C has a Kd of ∼50 nM for CaM binding. In WT GFP-CaMKIIδ C expressing cells pre-incubated with GSNO, S- nitrosylation diminished CaM affinity (∼3-fold), and this affinity was similarly reduced in the C290A S -nitrosoresistant mutant, where GSNO could only target C273. Untreated C273S S -nitrosoresistant mutant had comparable affinity to the untreated WT GFP-CaMKIIδ C . Initial affinity measurements of GSNO-treated C273S S-nitrosoresistant mutant (only S-nitrosylatable at C290), showed increased CaM affinity compared to control. Likewise, GSNO-treated WT GFP-CaMKIIδ C , post Ca/CaM incubation, exhibited an increased affinity for CaM compared to control. Preliminary data suggest that S -nitrosylation at C273 inhibits myocyte CaMKII activation at least partly due to a reduced Ca/CaM affinity, while the autonomous activation by S -nitrosylation at C290 increases the Ca/CaM-CaMKII affinity.