Abstract 395: Transient Inactivation of Retinoblastoma Induces De-Novo Cardiomyogenesis From Human Myocardial Progenitors but Not Pre-Existing Cardiomyocyte Replication

Abstract

Introduction: Activation of cardiac cell cycle re-entry is considered the primary therapeutic strategy for cardiomyocyte (CM) regeneration. However, the role of cardiac cell-cycle control in cardiomyogenesis remains elusive. Here, we combined RNA interference and stem cell modeling to investigate the role of Retinoblastoma (RB) in human cardiomyogenesis. Hypothesis: RB regulates proliferation and differentiation of cardiac progenitors (CPCs) but not CM replication. Methods: H9 human embryonic stem cells (hESCs) stably expressing tetracycline (tet)-inducible shRNAs against RB (hESCshRB) or hemagglutinin-tagged RB (hESCHA-RB) were tet-induced at selected time-points during or after CM differentiation. Results: Analysis of ser-608 illustrated stage-specific differences in the degree of RB inactivation during normal hESCs-cardiogenesis. Transient shRB knockdown in hESCshRB-derived embryoid bodies (EBs) during the CPC-stage (EB-days 5-8), significantly upregulated GATA4, ISL1, CTNNI, and cKit transcription (p<0.05), while increasing the yield of beating EBs by 2.4-fold (n=6/group, p<0.0001 vs. vehicle). Gene-expression arrays of 22 RB-related genes, illustrated that shRB-knockdown upregulated CCND1, CCND2, CCND3, and CDK4, CDK6 (p<0.05), followed by a 3.6-fold increase in E2F3 (p<0.05) expression. Moreover, expression of p107 and p130, p27, p57, ARF and CDKN3 were also significantly increased (p<0.05), whereas TP53 and MDM2 remained unchanged. Ectopic HA-RB in CPCs did not significantly affect cardiogenesis (n=18). Conversely, shRB knockdown in EB-day 60-derived CMs (n=15) did not stimulate cell cycle re-entry, as assessed by analysis of EdU incorporation and Aurora-B kinase (AurB). Remarkably, co-culture of hESCHA-RB-derived CMs with adult cardiac (CSCs) and/or mesenchymal (MSCs) stem cells (n=15/group), increased cell-cycle re-entry ~2.8-fold, assessed by ser-10 Histone H3 (p=0.0002) and AurB (p<0.0001). Conclusions: These findings suggest that RB regulates proliferation and differentiation of human CPCs in a cell-autonomous manner, via a CCND-CDK4/6-E2F3 mechanism. Conversely, CM replication may be enhanced via cell-cell interactions with MSCs and/or CSCs, but not cell-autonomously via RB inactivation.