P2X4 receptor limits adverse cardiac remodeling following ischemia–reperfusion through regulation of autophagy in cardiac fibroblasts

Pathological ventricular remodeling is an adverse tissue response following acute myocardial infarction (AMI), characterized by an exaggerated fibrosis leading to tissue disorganization and chronic heart failure. Cardiac fibroblasts have a prominent role in the healing process, but also in the fibrosis development, through their differentiation into myofibroblasts, which produce the extracellular matrix, in response to ischemia–reperfusion. Recently, the ATP-gated P2X4 ionotropic receptor has been identified as a key regulator of macro-autophagy, activated under metabolic challenges. In this study, we investigated the participation of the P2X4 receptor in the metabolic response of cardiac fibroblasts to ischemia–reperfusion and the myofibroblast switch. To this end, we submitted a primary culture of cardiac fibroblasts to an ischemia–reperfusion simulation and assessed autophagic flux, fibrotic differentiation markers and the correlation to P2X4 expression and activity. Then, using pharmacological inhibition and siRNA-mediated knockdown receptor under-expression, we investigated its role in the response of cardiac fibroblasts to this metabolic challenge. Finally, we validated our findings in an in vivo murine model of coronary ligation in wild-type and P2rx4 −/− mice. Our results reveal that human cardiac fibroblasts subjected to simulated ischemia present an increased autophagic activity associated with a concomitant overexpression of P2X4 and its activity at the plasma membrane of cells. Pharmacological antagonism of P2X4, or its silencing, impaired autophagy, as evidenced by the accumulation of autophagy markers, and induced the expression of fibrotic markers. In an in vivo model of AMI through transient coronary ligation, P2rx4 −/− mice presented the accumulation of autophagic markers both in healthy and ischemic zones, an extended tissue inflammation and fibrosis compared to wild type mice. This study suggests, for the first time, that the P2X4 receptor may contribute to steering cardiac fibroblasts toward a healing-associated profile under ischemia–reperfusion conditions, possibly by modulating autophagic activity and limiting extensive pro-fibrotic differentiation.