Abstract 1120: Akt Modified MSCs Secrete a Novel Stem Cell Paracrine Factor that Protects Injured Heart, Activates Cyclin Dependent Kinase and Phosphorylates/Activates Akt Signaling Pathway in Adult Rat Cardiomyocytes
Previously, we have shown that intracardiac injection of mesenchymal stem cell (MSC) overexpressing Akt (Akt-MSCs) could dramatically reduced infarct size and restored cardiac function in a paracrine manner. Microarray analysis of Akt-MSCs led to the identification of a novel transcript encoding a secreted protein. Cloning of the cDNA encoding this candidate gene into pET15b allowed the purification as a 6×His tagged recombinant protein. Interestingly, a 30 minute pre-incubation of the H9C2 myocytes with this protein (10nM) reduced H2O2 induced apoptosis by 50% as measured by annexin V/PI staining on flow cytometry (n = 3, p<0.001). Moreover, this protein significantly prevent H2O2 induced DNA fragmentation and inhibited Caspase 9 and Caspase 3/7 activities in adult rat cardiomyocytes by ~36% and ~42%, respectively (n=3, p<0.01). Furthermore, this protein dramatically inhibited the mitochondrial release of Cytochrome C and maintained mitochondrial Bcl-2 protein level. Intramyocardial injection of 1 μg of this novel protein dramatically reduced infarct size (−58%, n=10, p<0.001) and TUNEL positive nuclei (−69%, n=10, p<0.001) in a 30 min ischemia/24h reperfusion rat model. Fibrosis evidenced by collagen deposition was greatly reduced (−61%, n=8, p<0.001) 4 weeks after ischemia/reperfusion injury. Signaling studies demonstrated that this novel protein phosphorylated rat adult cardiomyocytes AktThr308 but not AktSer473 in a time-depended manner peaking at 30 min. Consequently, downstream Akt substrates, GSK3ßSer9 and BadSer128, are also phosphorylated at 30 min or at a later time point. In contrast to the traditional concept that PDK1 phosphorylates both AktThr308 and AktSer473 , our mass-spec and in vitro enzymatic analysis of rat cardiomyocytes stimulated with this factor suggests cyclin-dependent kinase 7 as being responsible for phosphorylation of Akt specifically at Thr 308 position. Further studies would be of great interest to interrogate whether this novel factor could activate other cyclin dependent kinase(s) and render the cardiomyocytes re-entering cell cycle. In conclusion, we have identified a novel paracrine factor from Akt-MSCs that mediates cardioprotective effects through activation of novel pathways involving Akt.