Abstract
Background: Delivery of c-kit positive cardiac stem cells (CSCs) is a promising approach to repairing the infarcted heart, but is severely limited by the poor survival of donor cells, >90% of which disappear within 7 days of transplantation. Carbon monoxide (CO), a byproduct of heme oxygenase 1 (HO-1), has potent anti-apoptotic, cytoprotective, and preconditioning actions. Cobalt protoporphyrin (CoPP) is a well-known HO-1 inducer and has been used to promote CO generation and protect against ischemia/reperfusion injury. Therefore, we determined whether preconditioning CSCs with CoPP promotes CSC survival. Methods and Results: C-kit positive, lineage negative CSCs were isolated from human hearts. Lactate dehydrogenase (LDH) release assays demonstrated that preconditioning CSCs with CoPP markedly enhanced cell survival after oxidative stress (1 mM H 2 O 2 ), concomitant with upregulation of HO-1 and other antiapoptotic proteins (e.g., Bcl2, Bcl2-A1, and Xiap). Conversely, knocking down HO-1 by shRNA inhibited the cytoprotective and antiapoptotic effects of CoPP. Both the annexin V/PI assay and the caspase-3 activity assay showed that pretreatment of CSCs with CoPP enhanced the cells’ resistance to apoptosis induced by oxidative stress. Preconditioning CPCs with CoPP also induced phosphorylation of NF-kappaB, Nrf-2, Erk1/2, and STAT1/3, which are known to modulate multiple cardioprotective genes. Further, preconditioning CSCs with CoPP led to a significant decrease in ROS generation and a global increase in release of cytokines such as EGF, FGFs, CSFs, and CXCL. Conditioned medium from cells pretreated with CoPP conferred naïve CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells play a key role in the antiapoptotic effects of CoPP. Conclusions: Preconditioning human cardiac stem cells with CoPP promotes cell survival and resistance to oxidative stress. The mechanism appears to involve activation of survival signaling pathways such as the NF-kappaB and ERK pathways, as well as paracrine effects via release of cytokines. These results provide a simple and effective strategy to enhance survival of CSCs after transplantation and, therefore, their efficacy in repairing infarcted myocardium.
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