Identification of a small SN derivative that inhibits CaMKII activity

Background Ventricular arrhythmia, a major cause of sudden cardiac death, is driven by Ca2+ imbalance in cardiac myocytes, often linked to the overactivation of CaMKIIδ (Ca2+/calmodulin-dependent protein kinase II delta). As such, inhibiting CaMKIIδ represents a promising therapeutic strategy. Purpose Based on our previous finding that native secretoneurin (SN, 33 amino acids) is a weak CaMKIIδ inhibitor, we aimed to develop a more potent and shorter derivative of SN to effectively counter aberrant Ca2+ handling and arrhythmia risk. Methods Interaction of the novel SN derivative to CaMKIIδ was identified and analysed by structural homology modelling, pull-down, ELISA-based assays and surface plasmon resonance experiments. The inhibitory effect of the SN derivative on CaMKIIδ activity was investigated indirectly by measuring CaMKIIδ-dependent phosphorylation of Ser2814-RYR2 (ryanodine receptor) and Ser17-PLN (phospholamban). The internalisation into isolated rat cardiomyocytes was verified by confocal fluorescence microscopy of FITC-labelled peptides. Effects of the novel SN derivative on Ca2+ sparks and waves were examined by fluorescence in isolated rat cardiomyocytes treated with isoproterenol to mimic a β-adrenergic challenge. Results Various regions of SN were tested for CaMKIIδ binding, identifying the core region (amino acids 7-26), which is notably the most conserved across species, to have the strongest binding capacity. This region was subsequently optimized with two phenylalanine substitutions (L17F, V20F), resulting in the novel SN derivative; SN-db-short. Detailed mapping analyses show that SN-db-short bound both to the substrate-binding (S-site) region of CaMKIIδ, in addition to the ATP-binding region, with an 8-fold stronger binding compared to native SN. Consistent with only a partial calmodulin binding motif, SN-db-short exhibited no calmodulin binding, indicating a selective CaMKIIδ inhibition. In functional studies, SN-db-short inhibited CaMKIIδ-mediated phosphorylation of Ser2814-RYR2 and reduced the incidence of Ca2+ sparks and Ca2+ waves to a greater extent than native SN. SN-db-short also more markedly inhibited CaMKIIδ phosphorylation of Thr17-PLN, slowed Ca2+ reuptake, and reduced the magnitude of Ca2+ transients during isoproterenol stimulation. Conclusion SN-db-short effectively inhibits CaMKIIδ, and significantly counters aberrant Ca2+ handling in cardiomyocytes. Thus, this optimized peptide holds therapeutic potential for reducing the risk of ventricular arrhythmias.