Abstract
Ischemia/Reperfusion (I/R) injuries are involved in many myocardial pathologic processes. Cardiac fibroblasts (CF) respond to this stress by excessive proliferation and secretion of soluble factors, such as ATP, leading to purinergic receptors activation. We already showed that P2Y11 receptor (P2Y11R) played an immunomodulatory role in human dendritic cells (DC). Our aim was to investigate the role of human CF P2Y11R stressed by I/R on immune response modulation and cardiomyocytes (CM) survival. Adult ventricular human CF were subjected to simulated I/R (5 h at 1%O2 and 24 h at 21%O2). CF secretome (CFS) was analyzed by bioluminescence, ELISA and Mutiplex assays. DC's maturation markers were analyzed by flow cytometry after 48 h incubation in CFS, and cytokine expression was assessed by qPCR and ELISA. Human CM were subjected to 5 h/1 h I/R and CFS was added at the onset of reperfusion. CM viability after I/R was assessed by MTT, AnnexinV/Pi, and LDH release. I/R induced CF proliferation (+8.7%, P < 0.001), ATP secretion (+31.7%, P < 0.001), and IL-6 secretion (+27.1%, P < 0.05). Stimulation of CF P2Y11R with NF546 reduced I/R-induced proliferation (−10% vs I/R, P < 0.01), and decreased ATP and IL-6 secretion (respectively: −28%, P < 0.05 and −31%, P < 0.01 vs I/R). CFS increased CD83, CD25 and CD86 expression (respectively +30%, +26% and +23%), suggesting a switch of DC from immature to mature phenotype. CFS pre-conditioned with NF546 decreased CD83, CD25 and CD86 expression (respectively −16%, −8 and −7% vs I/R). P2Y11R inhibition with NF340 abolished this effect. CFS protected CM from I/R injury (+28.7%, P < 0.001). NF340 inhibited CFS protection on CM (−22% vs NF546 condition). Our data demonstrated a paracrine link between CF, CM and DC after I/R suggesting a key role of CF in the cellular reponses after reperfusion. P2Y11R seems to modulate myocardial I/R injuries, especially acting on CF secretome. Additional experiments are needed to understand P2Y11R mechanisms of action.
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