Activation of P53 Via Nutlin-3a Reveals Role for P53 In ROS Signaling During Cardiac Differentiation of hiPSCs.

ABSTRACT

Activation of the transcription factor P53 within cancer cells is a well-characterized pathway, whereas the effects of P53 activation during development remain largely unexplored. Previous research has indicated that increased levels of P53 protein during key murine developmental stages cause defects in multiple embryonic tissues, including the heart. These findings were confirmed in several different mouse models of congenital heart defects, but P53 activation in a human system of cardiovascular development is not available. Utilizing human induced pluripotent stem cells (hiPSCs), we characterized the normal levels of P53 during cardiac differentiation and showed that levels of P53 are high in hiPSCs and decrease upon cardiac lineage commitment. We also observed P53 localization changed from mainly cytoplasmic in iPS colonies to the nucleus in the Nkx2-5 + cardiac progenitor stage. Pharmacological-mediated increase of P53 protein levels with the Mdm2 inhibitor Nutlin-3a during early (mesoderm to cardiac mesoderm) stages of cardiogenesis resulted in a sizeable loss of cardiomyocytes due to increased apoptosis and cell cycle arrest. Interestingly, increasing P53 levels did not result in apoptosis at later (cardiac progenitor to beating cardiomyocytes) stages of the cardiac differentiation. These results illustrate the temporal sensitivity to increased P53 levels during cardiogenesis. We conducted RNA-Seq on these cells with or without Nutlin-3a to ascertain transcriptional differences due to increased P53 at the different stages during the differentiation. Our results from the RNA-Seq revealed up-regulation of Sestrins after Nutlin-3a treatment suggesting a new role for P53 in the metabolism of cardiac regeneration.