Abstract
Cardiovascular disease (CVD) remains the primary cause of mortality both in New Zealand and worldwide. Due to the longstanding misconception that CVD predominantly afflicts men, much of the research designed to understand and mitigate CVD morbidity is tailored towards an aging male demographic, overlooking the substantial increase in CVD risk observed in women post-menopause. One proposed mechanism underlying this increased vulnerability is the decline in the bioavailability of nitric oxide (NO), a signalling molecule with well-established cardioprotective effects. An emerging target of NO is calcium/calmodulin-dependant protein kinase II (CaMKII), a cardiac regulatory protein involved in the modulation of calcium handling via the phosphorylation of key proteins, including the ryanodine type II receptor (RyR2). In the healthy heart, RyR2 channels are systematically arranged into orderly clusters to facilitate synchronous calcium release and contraction through the myocardium. However, in pathological conditions, hyperactivation of CaMKIIδ promotes pro-arrhythmic remodelling of the nanoscale organisation of RyR2 clusters, disrupting channel activity and triggering diastolic calcium leak. Previous work from our group has shown that baseline NO inhibits CaMKIIδ via S-nitrosylation of the C273 site, preventing autonomous activity and subsequent arrhythmogenesis. However, the interplay between NO and CaMKIIδ in regulating RyR2 clustering within cardiomyocytes remains unexplored.
This project aimed to characterise the effects of CaMKIIδ S-nitrosylation on the nanoscale organisation of RyR2 clusters and determine whether these effects were sexually dimorphic. Left ventricular myocardium was isolated from 12-week-old male and female transgenic knock-in mice insensitive to NO-mediated CaMKIIδ inhibition (C273S) alongside wildtype (WT) controls. Tissue was subsequently examined under super-resolution (dSTORM) imaging to quantify RyR2 cluster remodelling. No significant differences in individual cluster properties were observed with respect to either genotype or sex, with both cluster size and RyR2 packing density shown to be unchanged between groups. However, interestingly, C273S males exhibited increased cluster density relative to WT controls (P = 0.0154), an effect which was absent in the females. These findings indicate that loss of NO-mediated CaMKIIδ inhibition promotes inter-cluster remodelling of RyR2 under baseline conditions in a sexually dimorphic manner, occurring specifically in males but not females. This study provides mechanistic insight into NO’s influence on sexually dimorphic cardiac outcomes and establishes a foundation for investigating CaMKIIδ S-nitrosylation-driven RyR2 remodelling under acute and chronic stress.