Abstract 326: Nucleostemin Induced by Pim-1 Kinase Antagonizes Senescence of Cardiac Progenitor Cells

Abstract

Decline in stem cell functionality and regenerative capability is a crucial contributor to aging, upon which the c-kit+ cardiac progenitor cell (CPC) pool with enhanced regenerative potential decreases and CPCs with limited proliferative and differentiation potential progressively accumulate. With cardiac stem cells currently being used in the clinic, especially in the elderly population, there is a dire need to understand the phenotypic, molecular and functional characteristics of senescent stem cells to effectively manipulate them for therapy. Nucleostemin (NS), a nucleolar stress sensor protein associated with stem cell pluripotency and regeneration declines upon cardiac aging. We hypothesize that NS is a critical antagonizer of senescence in CPCs. NS expression decreases significantly (-59%, p<0.05), while the % of senescent cells increases in CPCs isolated from old mice (13 month, OCPC) relative to CPCs isolated from young mice (3 month, YCPC). OCPCs are morphologically different, characterized by flat, round cells (p<0.05) with increased bi- & poly-nucleation (p<0.05), slower proliferation (-80%, p<0.01) and altered cell cycle profiles, relative to YCPCs. Decreased NS level is causal for the phenotype observed in OCPCs, as silencing NS in YCPCs induces flattening of cells, increases bi- & poly-nucleation, decreases expression of stem cell marker c-Kit (-55%, p<0.05), upregulates cell cycle inhibitors p53 and p16 (4.2, 3.8 fold, p<0.01) and decreases proliferation (p<0.05). NS-mediated regulation of CPC senescence is p53 dependent, as silencing p53 rescues the phenotype induced by NS-silencing. Interestingly, NS is induced by Pim-1 kinase-mediated stabilization of transcription factor c-Myc, as NS protein expression decreases in CPCs upon knockdown of Pim-1 (p<0.05) or c-Myc (p<0.01); and in the absence of c-Myc, Pim-1 does not upregulate NS. Engineering OCPCs with NS is an effective strategy to antagonize senescence, as observed by decreased % senescent cells, increased proliferation and restored morphology. In conclusion, NS which is induced downstream of Pim-1 kinase, maintains multipotency and inhibits senescence in CPCs, consistent with cumulative evidence that Pim-1 induced cardiac regeneration is mediated in part by NS.