Pathological ventricular remodelling and purinergic signaling: Emerging roles of the ATP-gated P2X4 receptor

Abstract

Pathological ventricular remodelling is an abnormal healing process following myocardial infarction (MI), which mechanisms remain debated. Several hypotheses have been proposed to explain the links between inflammatory response secondary to MI and the evolution towards pathological remodelling, including the involvement of ATP and its cellular receptors: the purinergic receptors. Our working hypothesis is that there is a maintenance/resurgence of a tissue inflammatory response in MI patients with pathological remodelling that would hinder the healing process and may be associated with a specific purinergic profile. The objective of the project is to study the impact of ischemia-reperfusion on cardiac fibroblast's’ profiles and to identify an associated purinergic signature. First, we studied the effect of ischaemia (5 h) and reperfusion (24, 48 h) on human primary cardiac fibroblasts (HCF) phenotype by studying the expression of markers associated with the activated state: periostin and the differentiated state: smooth muscle alpha-actin (αSMA) by Western blot. Simultaneously, the expression level of P2 purinergic receptors (P2R) was quantified by RT-qPCR and WB over time. In a second step, we chose to investigate the involvement of P2X4 in the activation and differentiation of cardiac fibroblasts. Therefore, we blocked its activity during reperfusion with its specific antagonist 5-BDBD (5 μM). In addition, wound healing studies were conducted with and without P2X4's antagonist to investigate its involvement in the migratory capacity of cardiac fibroblasts. Ischaemia-reperfusion (IR) appears to induce an initial increase in periostin expression at 24 h followed by a decrease at 48 h as compared to normoxia. This may be related to a more important secretion of periostin at 48 h, as suggested by its increase in supernatants. Concerning αSMA, IR induces a decrease in its expression in a sustained manner over time. Regarding P2X4, there is a decrease in its mRNA and protein levels. The use of its antagonist in normoxia and IR decreases αSMA expression but does not affect periostin expression. Similarly, its blockade decreases the basal migratory capacity of fibroblasts. IR seems to prevent fibroblast differentiation by maintaining them into an activated state. P2X4 could potentially be involved by decreasing fibroblast differentiation, thus impacting αSMA expression. Yet, other purinergic receptors whose expression levels vary following MI might also be involved.