The cardiac response to ischemic injury is a complex process involving the concerted action of various cell types. Among them are cardiac fibroblasts (CF) that undergo dynamic cellular state changes after injury and mediate cardiac repair by forming a collagenous scar. Secretion of the pleiotropic cytokine IL-6 and generation of collagen by CF are under adenosine control, signalling via the Gq/Gs-coupled A2B receptor (A_2BR). Antifibrotic effects of Gs-triggered elevation of intracellular cAMP are mediated via the actions of exchange protein directly activated by cAMP 1 (EPAC1), which has been shown to be a component of the A_2BR signalling pathway. A_2BR activation was reported to prevent ventricular remodelling after myocardial infarction (MI).  Extracellular adenosine is generated by the purinergic ectoenzyme cascade involving the rate-limiting enzyme CD73, which converts AMP to adenosine. Levels of extracellular adenosine are profoundly increased under conditions of inflammatory hypoxia due to upregulation of the adenosine-generating enzymes together with downregulation of adenosine degradation and its cellular uptake. Here we explored the impact of dynamic cell state changes of CF in shaping adenosine metabolism and signalling, using cultured mouse CF in vitro as well as single-cell RNA sequencing (scRNAseq) data from mouse hearts 5 days after MI.

CF were isolated from mouse hearts by Langendorff-based perfusion with collagenase. In cultured CF, cell state changes were induced by extended time in culture (up to 16 days), stimulation with isobutylmethylxanthine (IBMX) to elevate intracellular cAMP, and induction of hypoxia (1% O₂) using a CO₂ incubator with oxygen control. We found by qRT-PCR, that hypoxia increased CD73 expression, while it remained stable during prolonged culture. In contrast, A_2BR expression was significantly downregulated after prolonged culture, while A_2BR expression was not affected by hypoxia. However, we found the expression of another prevalent adenosine receptor in CF, A_2AR, was reduced by hypoxia, thereby changing the A_2AR-A_2BR expression balance. This change in A_2AR-A_2BR balance was also observed in CF after IBMX treatment. To assess adenosine metabolism and signalling in the different CF stages induced by MI, we interrogated a scRNAseq data set recently published by us on 11 transcriptionally distinct CF populations observed in infarcted mouse hearts 5 days post MI (Hesse and Owenier et al. 2021, eLife, 10: e65921). Comparison of resident, non-activated CF populations and infarct-activated CF populations revealed that A_2BR expression was significantly increased in activated CF, while the expression of CD73 and the ATP-degrading enzyme CD39 remained unaltered. Surprisingly, expression of the enzymes CD38 and ENPP1, a pyrophosphatase that generates AMP from extracellular NAD, was increased in activated CF, suggesting that they can use NAD to provide AMP as substrate for CD73.