Mesenchymal Stem Cell Conditioned Tyrode is a Potent Activator of Akt in Cardiomyocytes

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Transplantation of bone marrow-derived mesenchymal stem cells (MSCs) in clinical trails has been reported to decrease infarct size and benefit ventricular ejection fraction of the heart. Differentiation of the MSCs into cardiac myocytes has been postulated, but stronger evidence points toward a paracrine mechanism. We tested the hypothesis that MSC conditioned tyrode (conT) results in improved cardiomyocyte survival through activation of the anti-apoptotic Akt protein kinase pathway. HEPES/ Bicarbonate buffered tyrode (pH 7.4) was placed on MSCs for 16 hrs at 37°C for conditioning. Isolated mouse ventricular cardiomyocytes (VMs) were treated with conT. Immunoblotting of VM lysates was used to examine the activation Akt, a downstream effector of the receptor-mediated PI3-Kinase pathway in conjunction with confocal imaging of intracellular Ca2+ (FLUO 4-AM). Superfusion of VMs with conT resulted in a progressive decrease of the Ca2+ transient duration (31±3.4 %) and an increase in Ca2+ transient amplitude (84±1.5 %; n=218 ) that reached steady-state approx. 3 hrs post-treatment. ConT increased the activation of Akt as indicated by phosphorylation of Akt (p-Akt) on Ser473 at 15 min and remained elevated relative to non-treated VMs at 3 hrs. A faster migrating p-Akt immunoreactive band was also identified. This putative Akt-cleavage product was not seen with insulin (10 μM) stimulation of p-Akt and not prevented with inhibitors of caspase activity (Z-DEVD-FMK (40 μM), Boc-D-FMK (50 μM)). This activation was not myocyte specific and conT treatment produced similar results in fibroblasts. The results demonstrate a paracrine mechanism of MSCs on improved cardiomyocyte survival through activation the PI3K/Akt pathway that also triggers remodeling of EC coupling. Stimulation of Akt in fibroblasts presents an additional indirect means by which cardiac repair could be modulated following injury.