Epicardium-derived mesenchymal stromal cells (EpiSC) play a pivotal role in heart development by differentiating into coronary vascular precursors, fibroblasts and cardiomyocytes. In the adult heart, EpiSC are reactivated by myocardial infarction (MI) and are considered as endogenous cell source involved in cardiac remodelling by paracrine factors, such as VEGF and IL6. We have recently shown that the secretion of these major regulatory cytokines by EpiSC is under control of adenosine signalling via the Gq/Gs-coupled A_2B receptor (A_2BR) (Hesse et al. 2017, FASEB J, 31: 3040-3053). A_2BR is a known target of hypoxia-inducible factor-1α (HIF-1α) and the epicardium/subepicardium has been identified as a cardiac hypoxic niche. Gq pathways triggered by endothelin-1 and angiotensin-II can induce HIF-1α even under normoxic conditions, however, whether this applies also to the A_2BR-coupled Gq pathway is unknown. Here we explored the crosstalk between adenosine-A_2BR and hypoxia-HIF-1α signalling in cultured adult EpiSC, isolated from rat hearts 5 days after ischemia/reperfusion injury.

A_2BR signalling was induced be the A_2BR-selective agonist BAY 60-6583 and hypoxic conditions (1% O₂) were established using a CO₂ incubator with oxygen control. We found by immunofluorescence that A_2BR signalling induced a strong nuclear accumulation of HIF-1α even under normoxic conditions (20% O₂). This effect was confirmed by using another specific A_2BR agonist, VCP 746, and was significantly attenuated by the PKC inhibitor GO 6983, indicating a pathway via the Gq-PKC axis. Quantitative Real-Time PCR analysis revealed that A_2BR activation enhanced HIF-1α mRNA levels, implying that the mechanism of A_2BR-mediated HIF-1α induction involves an increase in HIF-1α generation. Both hypoxia and A_2BR signalling increased gene expression of the angiogenic HIF-1α target VEGFA, individually or together cumulatively. Respirometric analysis showed that A_2BR activation alone was sufficient to trigger the HIF-1α-associated cell-protective metabolic switch towards less oxygen consumption. The normoxic A_2BR-mediated HIF-1α induction was not restricted to EpiSC but was also found in cardiac fibroblasts, indicating that this mechanism is operative in other A_2BR-expressing cell types as well.

Together, our data provide first evidence for a close, bidirectional crosstalk between adenosine-A_2BR and hypoxia-HIF-1α signalling that might be importantly involved in tissue protection and wound healing after MI.