Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA057

Poster Communications

Hypoxia regulates purine metabolism and adenosine-A2BR signalling of epicardium-derived cells formed after myocardial infarction

J. Hesse1, W. Groterath1, Z. Ding1, A. Marzoq1, J. Schrader1

1. Department of Molecular Cardiology, Heinrich Heine University Duesseldorf, Duesseldorf, Germany.

Epicardium-derived mesenchymal cells (EPDC) play a pivotal role in heart development by differentiating into coronary vascular precursors, fibroblasts and cardiomyocytes. In the adult heart, EPDC are reactivated by myocardial infarction (MI) and are considered as endogenous cell source involved in cardiac remodelling. EPDC proved to secrete paracrine factors, including VEGF and FGF2, which have beneficial effects on post-MI remodelling. We have recently shown that the paracrine activity of EPDC after MI, especially the secretion of major regulatory cytokines such as IL1β, IL6 and VEGF, is regulated via adenosine-A2BR and ATP-P2X7R signalling (Hesse et al., FASEB J 31:3040-3053, 2017). However, the individual factors modulating the purinergic signalling of EPDC are presently unknown. Since hypoxia has been reported to regulate tissue remodelling processes, we explored the effects of hypoxia on the purinergic metabolism and signalling of cultured adult EPDC, isolated from Wistar rat hearts 5 d after MI. MI was induced in mechanically ventilated rats with isoflurane (1.5% [vol/vol]) anaesthesia by ligation of the left coronary descending artery for 60 min and subsequent reperfusion. EPDC were digested from the heart surface with collagenase (1,200 U/ml) and cultured for up to 3 wk. Hypoxic conditions (1% O2) were established using a CO2 incubator with oxygen control. Values are means ± SD, compared by Student's t test. Immunofluorescence of EPDC after 24h of cultivation under hypoxic conditions confirmed nuclear accumulation of hypoxia-induced factor (HIF)1α (n=2). Quantitative Real-Time (qRT)-PCR revealed significantly increased expression of HIF1α target genes Car9 (CAIX, 2.7±0.6 fold, p<0.01), Vegfa (2.4±0.4 fold, p<0.01) and Nt5e (CD73, 1.2±0.1 fold, p<0.05) after 24 h of hypoxia (n=5). Functional analysis of ATP, NAD and AMP metabolism by the purinergic enzyme cascade on the EPDC cell surface after 24 h of hypoxia via HPLC showed a decline in ATP and AMP degradation (-1.4±0.3 fold and -1.3±0.1 fold), while NAD degradation was enhanced (1.3±0.3 fold) (4 min after substrate addition, n=3). Cultivation for 24h under hypoxic conditions significantly amplified A2BR (BAY 60-6583)-dependent induction of adenosine-generating CD73 (1.7±0.4 fold, p<0.05), A2BR (1.6±0.3 fold, p<0.05) and IL6 (3.7±1.3 fold, p<0.05) expression as measured by qRT-PCR (n=5). Interestingly, immunofluorescence after 24 h incubation showed that A2BR activation under normoxic conditions is sufficient to induce HIF1α stabilization in EPDC and this at least to the same extent as hypoxia (n=2). Our data demonstrate that hypoxia is a regulator of the purinergic metabolism and signalling in EPDC. Thus, local myocardial pO2 levels may crucially shape the potential pro-inflammatory and pro-fibrotic activities of EPDC in post-MI cardiac remodelling.

Where applicable, experiments conform with Society ethical requirements