Adipose, sex steroids and atrial arrhythmia vulnerability

© 2018 Dr. Gabriel Brian BernasochiBackground: Pericardial adipose deposition occurs in ageing and obesity, and independently contributes to the development of atrial fibrillation. The mechanisms underlying this association are not yet understood. Investigations to date have focused on physical conduction block posed by infiltrating adipose and the secretion of pro-inflammatory/pro-fibrotic paracrine factors into the atria. Though not yet investigated in the pericardial adipose, white adipose depots are established sites of oestrogen synthesis. Considering the reported actions of oestrogens on the heart, it is hypothesised that pericardial adipose may represent an important source of local oestrogen synthesis, exerting paracrine actions on the myocardium. Research questions: 1. Do myocardial and pericardial adipose tissues express aromatase, and do locally-derived oestrogens affect the vulnerability to atrial arrhythmia? (Chapter 2) 2. Does disruption of aromatase activity in aged and obese mice influence basal cardiac electrophysiology and the susceptibility to atrial arrhythmia? (Chapter 3) 3. Can liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) sensitive methodology be used to quantify androgens and oestrogens in human and mouse myocardium and pericardial adipose tissues? (Chapter 4) Methods: Aromatase expression in human and rodent myocardium and pericardial adipose was measured by Western immunoblotting. Arrhythmia vulnerability was assessed in isolated hearts from male C57BL/6 mice (‘young’, ‘aged’ or ‘aged’ + high fat diet). Hearts were perfused with a hypokalaemic solution (2 mmol [K+]) and subjected to programmed electrical stimulation to provoke arrhythmias. In addition, hearts were perfused with acute perfusion with 17β-oestradiol (or vehicle) and arrhythmic provocation repeated. Aromatase knockout and wild type mice (male and female) were fed a control or high fat diet for 40 weeks. Mice were subjected to electrocardiographic and echocardiographic assessment prior to isolated heart atrial arrhythmia provocation experiments. Human and mouse myocardium and adipose tissues were homogenised, derivatised with dansyl chloride and subjected to LC-MS/MS for sex steroid quantification. Mass spectrometric technique was developed using the aromatase knockout as a positive control for androgens and a negative control for oestrogens. Results: 1. Aromatase is expressed in human/rodent myocardium and pericardial adipose, conferring the capacity for local androgen to oestrogen synthesis. Pericardial adipose capacity to synthesise oestrogens increased by 30-50x in aged hearts, which were significantly more vulnerable to atrial arrhythmias. (Chapter 2) 2. The aromatase knockout model of oestrogen depletion and androgen excess revealed a sex-differential phenotype in the susceptibility to atrial arrhythmia. Left atrial action potential duration was prolonged and arrhythmia vulnerability greater in female aromatase knockout mice compared to all other groups. The combined influence of extensive pericardial adipose deposition and a highly androgenic/oestrogen-depleted environment was unique to the female aromatase knockout mice and may have been decisive in driving the exacerbated vulnerability to atrial arrhythmias. (Chapter 3) 3. LC-MS/MS methodologies were optimised for the detection and quantification of sex steroids in human/mouse myocardium and adipose. Successful quantification of testosterone and progesterone was achievable, but concentrations of oestrogens in tissues were below the technical limits of detection. (Chapter 4) Conclusions: This thesis identifies that pericardial adipose expresses aromatase and indicates a probable capacity for oestrogen synthesis, hence supporting the presence of a local cardiac androgen-oestrogen system. Pericardial adipose derived oestrogens (and androgens) are recognised as probable paracrine mediators capable of altering atrial arrhythmic vulnerability. In addition, the data support the clinically observed correlation between pericardial adipose accumulation and atrial fibrillation. Mass spectrometric methodology is capable of quantifying tissue testosterone and progesterone concentrations, but tissue oestrogens are below the limits of detection. Taken together, this thesis advances the mechanistic understanding of the link between pericardial adipose accumulation and greater atrial arrhythmia vulnerability