Peptide array-derived optimized secretoneurin variants enhance CaMKII inhibition and reduce pathological cardiomyocyte Ca2+ handling

Background Disrupted Ca2+ regulation in cardiac myocytes can lead to ventricular arrhythmias and sudden cardiac death. Pathological cardiac myocyte Ca2+ regulation is closely linked to CaMKIIδ (Ca2+/calmodulin-dependent protein kinase II delta) overactivity, which makes CaMKIIδ inhibition a promising therapeutic strategy in patients with myocardial stress. Purpose Building on prior findings that secretoneurin (SN), a 33-amino acid peptide, inhibits CaMKIIδ, this study used multiplex-strategy to develop optimized SN candidates. Our objective is to identify highly potent SN variants for CaMKIIδ binding that also mitigate aberrant Ca2+ handling and thereby have potential to reduce arrhythmogenic risk. Methods We assessed the effect of the SN derivatives by bioinformatics and experimental models in vitro. Bioinformatic analysis suggested several amino acid changes to increase affinity to CaMKIIδ. A peptide array on cellulose membranes was used to screen suggested SN derivatives for CaMKII binding. Truncated SN sequences were synthesized to identify the minimal CaMKII binding region, while amino acid substitutions were introduced to identify which residues are critical for enhancing CaMKII affinity. Pull-down assays confirmed the binding of SN derivatives to endogenous CaMKII, and confocal fluorescence microscopy assessed the internalisation of FITC-labelled peptides into isolated rat cardiomyocytes. Functional effects on Ca2+ sparks and waves were evaluated using fluorescence imaging in cardiomyocytes exposed to isoproterenol (ISO) to simulate β-adrenergic stress. Results Different regions of SN were analysed for CaMKII binding, identifying the central region (aa 7-26) as the most effective. Following this, peptide array results highlighted the significance of specific residues and their positions in CaMKII binding. Substitution analysis generated over 50 peptides with enhanced CaMKII affinity compared to SN. Notably, some positions showed significant flexibility. For example, substitution of proline at position 11 increased CaMKII affinity, suggesting P11 may hinder binding due to steric hindrance. Promising mutations, including L17F, S19F, and V20F, also improved CaMKII binding. Truncation studies revealed a minimal 7-amino-acid binding site. Twelve SN derivatives were selected for further analysis, and eleven successfully internalized into rat cardiomyocytes. Notably, two derivatives showed superior reductions to the effect from native SN when assessing rat cardiomyocyte Ca2+ sparks during ISO stimulation. Conclusions We have developed optimized SN derivatives that enhance CaMKIIδ inhibition and improve Ca2+ handling in cardiomyocytes. Optimized SN derivatives hold the potential as promising therapeutic agents to reduce CaMKIIδ overactivity and thereby also influence risk of ventricular arrhythmia.