Abstract
Cardiovascular disease (CVD) is the number one cause of death world-wide (Mathers & Loncar, 2006; McAloon et al., 2016). Mortality is higher in males when compared to females in all age groups because of earlier detectable prevalence (Mikkola et al., 2013). However, CVD is still the number one cause of death in females worldwide (Humphries et al., 2017) and increases significantly in the post-menopause age group (Mikkola et al., 2013). However, there are no clear indicators of the earliest age that females develop cardiac dysfunction. Thus, it has become imperative that female specific CVD risk factors are elucidated clearly to reduce the post-menopausal mortality risk (Mikkola et al., 2013; Appelman et al., 2015). Investigating underlying sex differences is therefore to be prioritised in the identification of new CVD drug targets (Förstermann & Kleinert, 1995; Casin & Kohr, 2020).
Calcium calmodulin kinase II (CaMKII) is a cardiac Ca2+ handling enzyme involved in the physiology of the cardiac cycle. However, pathological autonomous CaMKII hyperactivation has been established to be involved in cardiac dysfunction pathophysiology (Erickson & Anderson, 2008; Erickson, 2014). Recently S-nitrosylation has been identified as a post translational modification (PTM) of CaMKII residues C273 and C290 residues. S-nitrosylation of C273 inhibits autonomous activity, whereas S-nitrosylation of C290 induces autonomous activation (Erickson et al., 2011b; Gutierrez et al., 2013; Curran et al., 2014; Erickson et al., 2015).
Dysregulated autonomous CaMKII activity in CVD is attributable to PTMs persisting in disease states (Erickson & Anderson, 2008; Erickson et al., 2013). S-nitrosylation of CaMKII has been associated with arrhythmogenesis experimentally but requires investigation to determine the role in cardiac dysfunction (Curran et al., 2007; Curran et al., 2009; Gutierrez et al., 2013; Curran et al., 2014).
It has been postulated that the lack of estrogen dominated signalling in males may be imperative in dysregulating the homeostatic rate of C290 and C273 S-nitrosylation of CaMKII. This would implicate C290 S-Nitrosylation to be pathologically upregulated because of a lack of estrogen signalling. If this was to persist, then it may contribute to pathological CaMKII hyperactivity that causes males to present with cardiac dysfunction at an earlier age than females. Thus, this dual regulation of CaMKII provides a potential dynamic that could be targeted in the development of new CVD drugs.
To investigate this the C273S KO mouse strain was developed using CRISPR/Cas9 to knock out C273 functionally. I hypothesised that C273S mice would have cardiac dysfunction because of CaMKII hyperactivity. I also hypothesised that female C273S mice would have better cardiac function than male C273S mice.
Three experimental studies were conducted using male and female C273S mice and wildtype C57BL/6 mice. Experiments included: 1) lead II electrocardiography (ECG), 2) left ventricle short axis echocardiography and mitral valve long axis echocardiography, and 3) a Langendorff pilot study. Parametric unpaired t tests were used with statistical significance of p < 0.05. An additional secondary analysis was also completed by means of one-way ANOVA, followed by the false discovery rate post-hoc test, with the same statistical significance of p < 0.05.
The ECG study results suggested that the C273S mutation may predispose atrial fibrosis and first-degree atrio-ventricular block with no significant impact determined by biological sex. The echocardiography study results suggested that male C273S mice may have systolic and diastolic dysfunction with renin-angiotensin-aldosterone system (RAAS) hyperactivity. This was postulated to possibly include pathological ventricular hypertrophy and coronary artery disease. The results also suggested that female C273S mice may have preserved systolic function and high performing diastolic function. This was postulated to include physiological ventricular hypertrophy and a trend towards higher number of arrhythmias of a greater severity subtype.
I concluded that the C273S KO mutation may have caused CaMKII hyperactivity that resulted in cardiac dysfunction in male mice. The possibility of RAAS hyperactivity being involved only in male mice led to the interpretation that that NO and oxidative stress may have caused this phenotype of the C273S mutation because of biological sex. This supported my first and second hypothesis. I also concluded that estrogen signaling in the female mice may have prevented NO and oxidative induced CaMKII hyperactivity which preserved cardiac function. This supported my second hypothesis. Finally, I suggested the role of the C273 S-nitrosylation site to be involved in inhibiting autonomous activation molecularly and structurally by sterically hindering the oxidation sites of CaMKII, thus preventing subsequent pathological CaMKII hyperactivation.