Understanding the role of NOS1AP on ECG parameters and arrhythmogenesis: insights from mice and men

Of the parameters on a surface ECG, the QT interval, which represents ventricular repolarisation, has arguably been the most studied. In humans, observational data suggests both short and long QT intervals are associated with a higher risk of ventricular and atrial arrhythmias. However, these observational studies are subject to limitations such as confounding and reverse causality, so the causal relevance is unclear. Unexpectedly, the strongest common non-coding genetic variants associated with a longer QT interval across populations map within an enhancer of the Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP) gene, which are associated with higher myocardial NOS1AP transcript expression. However, the mechanism by which the NOS1AP protein affects the QT interval and the risk of arrhythmia remains unclear.
The aim of this thesis was to investigate the mechanistic effects of cardiac-limited NOS1AP over-expression on ECG parameters and arrhythmogenesis in a transgenic mouse model and subsequently investigate the causal relevance of ECG parameters more broadly in human arrhythmogenesis using large-scale population data.
A transgenic mouse overexpressing human NOS1AP in the myocardium (NOS1AP-Tg) was generated and phenotyped. NOS1AP-Tg showed a modest increase in NOS1AP protein (~2.5-fold) in all cardiac chambers. Subsequent phenotyping revealed a longer P-wave duration, PR interval and QRS interval, but a shorter measured QT interval on ECG. The mice had a higher propensity to induced ex-vivo ventricular arrhythmias and in-vivo atrial arrhythmias in the absence of echocardiographic differences in cardiac structure or function. Investigation of the cardiac electrical substrate resulting from NOS1AP overexpression revealed no significant difference in action potential durations (APD) between genotypes at physiological heart rates, although a shorter APD was seen at slower heart rates in NOS1AP-Tg hearts. There was a significant reduction in conduction velocity in the left ventricles of transgenic mice in the absence of an increase in cardiac fibrosis. The slower conduction velocity in NOS1AP-Tg was associated with lower connexin-43 protein content at the intercalated disc. No differences in calcium handling, NOS1 content or NOS activity were detected between genotypes. Exploratory analyses did not suggest gross differences in the rapid sodium current INa.
We subsequently investigated the causal relevance in humans of altered ECG parameters (observed in NOS1AP-Tg mice) and risks of development of atrial fibrillation (AF). To overcome limitations from existing observational data, we employed Mendelian randomisation techniques using weighted genetic scores for P-wave duration, PR interval and QT interval representing lifelong differences in cardiac electrical parameters. We showed novel evidence supporting causal relationships between lifelong differences in electrical parameters and risks of developing both AF and non-AF supraventricular tachycardias in large human population datasets. Unexpectedly, results supported a causal association between lifelong differences in ECG parameters representing longer atrial conduction times within the normal range, and a lower risk of AF.
Overall, the findings suggested that NOS1AP impairs cardiac electrical conductance and coupling by reducing connexin-43 protein stability and highlight the need for investigations of the impact of gene variation on NOS1AP protein and subcellular localisation in the human myocardium. Future investigation of the electrical substrate using an integrative combination of fundamental molecular knowledge, genetics and electrophysiology may yield novel insights and new therapeutic options.

Keywords: UK Biobank
NOS1AP
QT interval
transgenic mice
ECG
arrhythmias
Mendelian randomisation
Advisor: Simon, J
Hopewell, J
Casadei, B