Abstract 487: eNOS Potentiates the Differentiation of Adult Cardiac Side Population Cells in Co-Culture with Cardiac Myocytes
Side population (SP) cells from cardiac niches within the adult myocardium have the potential to differentiate into cardiomyocytes, thereby potentially contributing to the regeneration of the injured/diseased heart. The signaling mechanisms involved in their differentiation, however, remain poorly characterized. A growing body of evidence implicates paracrine signaling from either parenchymal cells or circulating progenitor cells. To dissect these signaling mechanisms, we established a co-culture assay of a sub-population of mouse adult cardiac SP cells plated in contact with cardiomyocytes. After MACS selection, 85–95% homogenous Sca-1 + cells by FACS analysis were labeled with an intracellular dye (CmdiI) and plated with freshly isolated mouse cardiomyocytes. Differentiation was monitored (at 14days) as the % cells immunolabeled for troponin I or alpha-actinin out of the total dye-labeled SP cells (>300 randomly chosen fields). To assess the potential contribution of nitric oxide and specific influence of eNOS on the incidence of differentiation, SP cells from C57Bl6 mice (WTspc) were treated with the NO-donor DETA-NO or co-cultured with cardiomyocytes from the same strain (WTcm) or from transgenic mice with cardiac-specific (alpha-MHC-driven) overexpression of NOS3 (TGcm). Reciprocally, cardiomyocytes from the same two strains were co-cultured with SP cells from TG hearts (TGspc). Treatment with DETA-NO dose-dependently increased troponin-I staining of WTspc (at 1μM: +81.0±6.7% vs solvent, n=3 prep; p<0.05). Moreover, co-culture of WTspc with TGcm increased their expression of differentiation markers with clear sarcomeric organization (+40.3±7.3%, n=3 prep; p<0.05) compared to co-culture with WTcm. This increase was abolished by NOS inhibition. Fusion events were unlikely, as few WTspc (that do not express detectable NOS3 protein in mono- or co-culture with WTcm) acquired staining for NOS3 upon co-culture with TGcm. We conclude that paracrine NO from cardiomyocytes promotes the differentiation of adult cardiac (Sca-1+) SP cells. Potentiation of myocardial eNOS expression/activity by specific drug treatment may promote myocardial regeneration from resident SP cells without the need for exogenous cell transplantation.