ATP Release and Signaling via P2Y Receptors Regulate Cardiac Fibroblast Phenotype and Activity

Cardiac remodeling is an essential process that facilitates heart development and wound healing responses post-injury. Cardiac fibroblasts (CFs) are the primary cell type responsible for maintaining myocardial structure and the cardiac extracellular matrix (ECM). CFs contribute to both basal ECM synthesis and its enhanced generation in response to cardiac injury: unregulated, these actions can lead to cardiac fibrosis and decreased cardiac performance. Soluble factors and mechanical cues can stimulate the transformation of CFs into myofibroblasts that increase ECM synthesis, proliferation and migration and that express numerous pro-fibrogenic genes. The work to be described here investigates a novel pro-fibrotic signaling pathway initiated by the release of cellular adenosine triphosphate (ATP) from CFs via connexin (Cx) gap junction hemi-channels following mechanical perturbations (Chapter 3). Importantly, released ATP functions as an autocrine/paracrine signaling molecule, which activates P2Y₂ nucleotide receptors and drives myofibroblast transformation, as indicated by increased collagen synthesis, CF migration and proliferation and the up-regulation of pro-fibrotic genes including [alpha]- smooth muscle actin ([alpha]-SMA) and plasminogen activator inhibitor (PAI)-1 (Chapters 3 & 4). Collagen synthesis and PAI-1 expression are sensitive to MAPK/ERK inhibition, while [alpha]-SMA expression and CF contractile responses are sensitive to RhoA/ROCK inhibition; both pathways are activated upon P2Y₂ stimulation by ATP. However, attenuating this signaling are ectonucleoside triphosphate diphosphohydrolases (ENTPDs), extracellular enzymes that hydrolyze ATP and play an essential role in regulating pro-fibrotic nucleotide signaling. ENTPD inhibition with siRNA or pharmacological agents increased basal extracellular ATP concentration and was sufficient to drive CF collagen synthesis and myofibroblast transformation (Chapter 5). Furthermore, ENTPD activity not only hydrolyzes pro- fibrotic ATP but also facilitates the generation of adenosine, a bioactive molecule that activates A2B adenosine receptors with anti-fibrotic effects. Together, these findings identify an autocrine/paracrine mechanism of CF regulation initiated by cellular ATP release and integrating pro-fibrotic ATP-P2Y and anti-fibrotic adenosine-P1 signaling, which are mediated by nucleotidase activity. This regulatory system contributes to both basal CF phenotype and response to acute cellular stress. These findings not only have direct implications in the understanding of cardiac fibrosis, but they may represent a general mechanism underlying the regulation of cellular homeostasis and response to injur