Obesity is a common comorbidity for pulmonary arterial hypertension (PAH). Additionally, oestrogen and its metabolites are risk factors for the development of PAH. Visceral adipose tissue (VAT) is a major site of oestrogen production; however, the influence of obesity-induced changes in oestrogen synthesis and metabolism on the development of PAH is unclear. To address this we investigated the effects of inhibiting oestrogen synthesis and metabolism on the development of pulmonary hypertension in male and female obese mice.We depleted endogenous oestrogen in leptin-deficient (ob/ob) mice with the oestrogen inhibitor anastrozole (ANA) and determined the effects on the development of pulmonary hypertension, plasma oestradiol and urinary 16α-hydroxyestrone (16αOHE1). Oestrogen metabolism through cytochrome P450 1B1 (CYP1B1) was inhibited with 2,2′,4,6′-tetramethoxystilbene (TMS).ob/ob mice spontaneously develop pulmonary hypertension, pulmonary vascular remodelling and increased reactive oxygen species production in the lung; these effects were attenuated by ANA. Oestradiol levels were decreased in obese male mice; however, VAT CYP1B1 and 16αOHE1 levels were increased. TMS also attenuated pulmonary hypertension in male ob/ob mice. Intra-thoracic fat from ob/ob mice and VAT conditioned media produce 16αOHE1 and can contribute to oxidative stress, effects that are attenuated by both ANA and TMS.Obesity can induce pulmonary hypertension and changes in oestrogen metabolism, resulting in increased production of 16αOHE1 from VAT that contributes to oxidative stress. Oestrogen inhibitors are now in clinical trials for PAH. This study has translational consequences as it suggests that oestrogen inhibitors may be especially beneficial in treating obese individuals with PAH.

The pulmonary veins have an external sleeve of cardiomyocytes that are a widely recognised source of ectopic electrical activity that can lead to atrial fibrillation. Although the mechanisms behind this activity are currently unknown, changes in intracellular calcium (Ca) signalling are purported to play a role. Therefore, the intracellular Ca concentration was monitored in the pulmonary vein using fluo-4 and epifluorescence microscopy. Electrical field stimulation evoked a synchronous rise in Ca in neighbouring cardiomyocytes; asynchronous spontaneous Ca transients between electrical stimuli were also present. Immediately following termination of electrical field stimulation at 3 Hz or greater, the frequency of the spontaneous Ca transients was increased from 0.45 ± 0.06 Hz under basal conditions to between 0.59 ± 0.05 and 0.65 ± 0.06 Hz (P < 0.001). Increasing the extracellular Ca concentration enhanced this effect, with the frequency of spontaneous Ca transients increasing from 0.45 ± 0.05 Hz to between 0.75 ± 0.06 and 0.94 ± 0.09 Hz after electrical stimulation at 3 to 9 Hz (P < 0.001), and this was accompanied by a significant increase in the velocity of Ca transients that manifested as waves. Moreover, in the presence of high extracellular Ca, the spontaneous Ca transients occurred more synchronously in the initial few seconds following electrical stimulation. The ryanodine receptors, which are the source of spontaneous Ca transients in pulmonary vein cardiomyocytes, were found to be arranged in a striated pattern in the cell interior, as well as along the periphery of cell. Furthermore, labelling the sarcolemma with di-4-ANEPPS showed that over 90% of pulmonary vein cardiomyocytes possessed T-tubules. These findings demonstrate that the frequency of spontaneous Ca transients in the rat pulmonary vein are increased following higher rates of electrical stimulation and increasing the extracellular Ca concentration.

The pulmonary veins (PVs) are widely recognised as an important source of ectopic electrical activity that can lead to atrial fibrillation. While the ectopic activity most likely originates in the cardiomyocytes that form an external sleeve around the PVs, the underlying mechanism remains to be elucidated. Recently, changes in the intracellular Ca2+ concentration have been proposed to play a role. Studies were carried out on intact PVs isolated from the rat, using fluo‐4 to monitor intracellular Ca2+ within individual cardiomyocytes. Under basal unstimulated conditions, spontaneous Ca2+ transients were observed that were asynchronous in neighbouring cells. The frequency of spontaneous Ca2+ transients increased from 0.45 ± 0.06 Hz to 0.59 ± 0.05 Hz (P<0.001) immediately following electrical field stimulation at 5 Hz. Increasing the external Ca2+ concentration to 4.5 mM enhanced this effect, with the frequency of spontaneous Ca2+ transients increasing from 0.45 ± 0.05 Hz under basal conditions to 0.91 ± 0.07 Hz after electrical stimulation (P<0.001). Thus, although electrical field stimulation significantly increased the frequency of the spontaneous Ca2+ transients, their asynchronous nature would mean that they are unlikely to be capable of triggering an action potential. Other interventions that could lead to entrainment of the Ca2+ transients are currently being investigated.