Rationale Autologous stem cell therapy using human c-Kit+ cardiac progenitor cells (hCPCs) is a promising therapeutic approach for treatment of heart failure (HF). However, hCPCs derived from aged HF patients with genetic predispositions and/or comorbidities of chronic diseases exhibit poor proliferative and migratory capabilities, which impairs overall reparative potential for injured myocardium. Therefore, empowering functionally compromised hCPCs with pro-regenerative molecules ex vivo is crucial for improving the therapeutic outcome in HF patients. Objective To improve hCPC proliferation and migration responses that are critical for regeneration by targeting pro-regenerative P2Y2 nucleotide receptor (P2Y2R) activated by extracellular ATP and UTP molecules released following injury/stress. Methods and Results c-Kit+ hCPCs were isolated from cardiac tissue of HF patients undergoing left ventricular assist device (LVAD) implantation surgery. Correlations between P2 nucleotide receptor expression and hCPC growth kinetics revealed downregulation of select P2 receptors, including P2Y2R, in slow-growing hCPCs compared to fast-growers. hCPC proliferation and migration significantly improved by overexpressing or stimulating P2Y2R. Mechanistically, P2Y2R-induced proliferation and migration were dependent upon activation of yes-associated protein (YAP), the downstream effector of Hippo signaling pathway. Conclusions Proliferation and migration of functionally impaired hCPCs are enhanced by P2Y2R-mediated YAP activation, revealing a novel link between extracellular nucleotides released during injury/stress and Hippo signaling, a central regulator of cardiac regeneration. Functional correlations exist between hCPC phenotypic properties and P2 purinergic receptor expression. Lack of P2Y2R and other crucial purinergic stress detectors could compromise hCPC responsiveness to presence of extracellular stress signals. These findings set the stage for subsequent studies to assess purinergic signaling modulation as a potential strategy to improve therapeutic outcome for use of hCPCs in HF patients.

Heart failure (HF) is a leading cause of death in the US due to limited capability of adult mammalian heart to regenerate after myocardial infarction (MI). Autologous stem cell therapy holds promise for promoting regeneration of injured heart but stem cells derived from diseased organs exhibit poor proliferative, migratory and survival capabilities. Empowering cardiac-derived progenitor cells (CPC) with prosurvival genes has been attempted. However, molecular mechanisms by which stem cells detect stress signals to subsequently initiate appropriate regenerative responses are poorly understood. In this regard, purinergic receptors represent a major detector for extracellular nucleotides released during injury/stress and serve as an intracellular platform harboring numerous signaling pathways that regulate proinflammatory and regenerative responses required for the healing process. Despite the established roles of purinergic signaling in cardiovascular diseases, it has not been well-defined in CPCs. This study shows, for the first time, that the majority of P2 purinergic receptors are expressed and exhibit functional responses to ATP and UTP in human CPCs (hCPC) isolated from HF patients. The G protein-coupled P2Y 2 R is a pivotal stress detector that senses ATP and UTP accumulated in extracellular space after injury and mediates regenerative responses in various injury models, including MI model, and in stem cells from diverse origins. Interestingly, hCPCs with relatively slower growth kinetics and enhanced senescence show dramatic decreases in P2Y 2 receptor (P2Y 2 R) expression compared to fast-growing hCPCs, consistent with our hypothesis that overexpressing P2Y 2 R enables diseased hCPCs to better detect stress stimuli and react with the proper regenerative responses. Along this line, P2Y 2 R stimulation with UTP enhances hCPC proliferation and migration. Interestingly, preliminary results demonstrate that UTP treatment induces YAP activation and nuclear shuttling. Moreover, inhibition of YAP/TEAD interaction impairs UTP-induced proliferation and migration revealing a novel link between extracellular nucleotides released during cardiac ischemia and Hippo signaling that has been recently implicated in cardiac regeneration.