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  • br Experimental section br Acknowledgments br Introduction A

    2023-03-17


    Experimental section
    Acknowledgments
    Introduction Adipose is a major endocrine organ which releases a range of bioactive agents [1]. Selected adipose depots have been established as sites of sex steroid metabolism [1]. Subcutaneous and visceral adipose express high levels of aromatase, capable of converting androgens to estrogens [1]. No studies to date have investigated aromatase occurrence or expression regulation in pericardial adipose. Aging and obesity are both associated with a marked increase in pericardial adipose deposition, and represent significant cardiac risk factors [2]. With ongoing rises in obesity rate and life expectancy, there is considerable interest in identifying the underlying cellular mechanisms by which pericardial adipose accumulation may exert pathogenic influence. Clinically, a correlation between the extent of pericardial adipose (constituting both epicardial and paracardial) and atrial fibrillation (AF) risk has now been established [2]. AF is the most common sustained arrhythmia, and is particularly prevalent in the aged population [3]. Cather ablation procedures have been successful in treating paroxysmal AF, though high rates for repeat procedures highlight the necessity for new adjunct therapies or stand-alone preventative measures. Investigations relating to adipose accumulation and AF have focused on the identification of pro-inflammatory and pro-fibrotic mediators produced by epicardial adipose [2]. These mediators have been proposed to exert paracrine actions on atrial myocardium, culminating in the fibrosis, scarring and conduction heterogeneity which contribute to the atrial structural substrate for AF [2]. Our contention is that, beyond adipokines, other adipocyte/myocyte-related metabolites may constitute important paracrine mediators. A possible role for regulated pericardial adipose aromatase modulation of local tissue steroid levels has not been previously considered – in physiological or pathophysiological settings. Similarly, the potential for pericardial adipose influence on AF induction risk through modulation of local ddhC synthetic capacity has not yet been explored. Observational studies of cardiovascular disease have supported the conventional view of estrogen ‘protection’, yet clinical trial outcomes (including the Women's Health Initiative, WHI) have not demonstrated definitive benefit of estrogen supplementation and in some cases provided qualified evidence of increased risk [4]. The WHI reported that estrogen-only therapy in women elevated AF incidence through unknown mechanisms [5], indicating that augmented estrogenic influence may exacerbate cardiac vulnerability. Experimentally, we and others have shown that in the heart, estrogens influence electromechanical properties, myocyte viability signaling and disease progression [6]. We have also previously identified aromatase expression in rodent ventricular tissues, and demonstrated that aromatase transgenic and knockout animals exhibit distinct cardiac phenotypes with differential arrhythmogenicity [7], [8].
    Methods A detailed methodology is available in the online supplement. Briefly, rodents were anesthetized with an intra-peritoneal injection of sodium pentobarbitone (70mg/kg) and sodium heparin (200IU/kg) prior to heart excision. Experiments were conducted and animals handled in the manner specified by the NHMRC/CSIRO/ACC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2013) and the EU Directive 2010/63/EU for animal experiments, with approval and oversight of the project by the University of Melbourne Animal Ethics Committee. Patients undergoing elective coronary surgery and/or valve surgery were recruited and consented for study participation which involved collection of atrial appendage and pericardial fat biopsy from resected tissue. Human placental tissues were collected from women providing written and informed consent undergoing surgical termination of pregnancy. The protocols were approved by the Monash University Human Research Ethics Committee, the Melbourne Health Human Research and Ethics Committee and the Southern Health Research & Ethics Committee. All tissues were homogenized, prepared for SDS-PAGE immunoblotting and aromatase expression detected with the use of a mouse anti-aromatase primary antibody (Serotec MCA2077S, Bio-Rad, Hercules, CA). Mouse hearts were isolated and retrogradely perfused with oxygenated (95% O2–5% CO2) bicarbonate buffer (37.0°C, pH7.4) in the non-recirculating Langendorff mode (80mmHg). The left atrium and left ventricle were instrumented with bipolar electrogram electrodes and hearts arrhythmias provoked through perfusion with a hypokalemic solution (2mmol [K+]) and programmed electrical stimulation. A subset of hearts was exposed to acute perfusion with either 17β-estradiol (10nM) or ethanol vehicle under hypokalemic conditions. Results are presented as mean±SEM, with statistical differences considered significant at p<0.05. All statistical calculations were performed using SPSS v.21.0 (IBM, Armonk, NY) and Prism 6 (GraphPad, La Jolla, CA).