Effects of A Gap Junction Inhibitor on Stem Cell Retention and Efficacy During Early Myocardial Ischemia

Bone marrow-derived mesenchymal stem cell (BM-MSC) replacement therapy is beneficial to the heart following ischemia but a significant loss of these cells within hours of administration could diminish their effect. Early coupling between BM-MSC and ischemic cardiomyocytes through gap junction (GJ) may play a detrimental role in stem cell survival and retention in the acute phase of cell therapy. We seeded 1×105 HL-1 atrial myocytes and place them in either normoxic or ischemic condition for four hours. Then, 2×104 lineage negative murine BM-MSC were seeded over the HL-1 monolayer and the cocultures were returned to incubation either in their previous conditions (normoxia, ischemia) or switched from ischemic to normoxic condition (ischemia–reperfusion) for an additional two hours after which they were lifted, labeled with fluorescent markers for dead and apoptotic cells, and subjected to flow cytometry analysis. Ischemia induced a greater proportion of dead BM-MSC over the two-hour coculture compared to the normoxic group. Ischemia–reperfusion resulted in significantly higher apoptotic but fewer dead BM-MSC. The presence of non-selective GJ inhibitor carbenoxolone (CBX) in the coculture significantly reduced the number of dead and apoptotic cells in ischemia and ischemia-reperfusion groups, respectively. We then created ischemia in mice by LAD ligation for 90 minutes after which the suture was removed to allow reperfusion. The animals were then given an intramyocardial injection of either 1×105 BM-MSC, 1×105 CBX-treated BM-MSC, CBX or vehicle. At 24 hours after the injury, their hemodynamic parameters, number of transplanted BM-MSC remained in the heart, and infarct size were assessed. Ischemia–reperfusion caused impaired cardiac function which can be attenuated by BM-MSC injection. CBX-treated BM-MSC further enhanced the cardiac function while the CBX alone did not. Flow cytometry analysis of whole heart digests demonstrated significantly increased number of BM-MSC in the hearts that had been given CBX-treated BM-MSCs as compared to those that received untreated BM-MSCs. However, no significant change in infarct size was observed in the heart at this specific time point. Our data suggest that early GJ communication may represent a novel paradigm whereby ischemic cardiomyocytes induce a detrimental effect on newly transplanted stem cells and thus reduce their efficacy.