Abstract 3670: Novel Stem Cell Paracrine Factor Protects Cardiomycytes Through Protein Kinase C Epsilon Selective Mechanism
Mesenchymal stem cells (MSC) overexpressing Akt release several paracrine factors to promote cardiac repair after acute myocardial infarction (AMI). Further gene expression and bioinformatics studies led us to the discovery of novel paracrine factors secreted from the Akt-transduced MSCs. In the current study, a novel protein, which we named Hypoxia Akt Stem Cell Paracrine Factor (HASP) was validated as dramatically upregulated in Akt-MSCs in response to hypoxia. To investigate the biologic function of HASF, we produced recombinant HASF protein using both bacterial and insect protein expression system. Addition of 10nM of recombinant HASF reduced apoptosis by ~50% which was induced by H2O2 in adult rat cardiomyocyte culture (n=3, p<0.001). Moreover, HASF significantly prevented mitochondrial permeability transition pore (mPTP) opening and preserved cell viability in cardiomyocytes subjected to ischemia/reperfusion (~27%, n=8, p<0.001). Intramyocardial injection of 1 μg of HASF dramatically reduced infarct size (~58%) and TUNEL positive nuclei (~69%, n=10, p <0.001) in an ischemia/reperfusion rat model. To address the mechanism underlying the protective effects of HASP treatment, we examined several cell-signaling pathways that play a pivotal role in determination of cell fate during apoptosis. Remarkably, we discovered that the addition of HASP in cardiomyocyte culture resulted in significant activation of the PKC/ERK pathway within 5–10 min. Moreover, specific inhibition of the PKC by bisindolymaleimide I hydrochloride (BIM) resulted in abolishment of the ERK phosphorylation and the anti-apoptotic effect of HASF treatment. Further experiments using isoform specific antibodies against phosphorylated-PKC α/β, δ, ϵ, 𝛉, ζ/λ and general pathway specific inhibitors suggested that the protective effects of HASF were exerted through PKC ϵ. The specific role of PKC ϵas downstream mediator of HASF was validated by use of the PKC ϵ inhibitor which abolished HASP mediated cardioprotective effects. Since PKC ϵ is a key molecule whose increased levels are associated with protective signaling processes during ischemic preconditioning, HASP may provide a potential novel therapeutic opportunity for myocardial ischemia/reperfusion injury.