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Despite major leaps in regenerative medicine, the regeneration of cardiomyocytes after ischemic conditions remains to elucidate. It is crucial to understand hypoxia induced cellular mechanisms to provide advanced treatment options, including the use of stem cell paracrine factors for myocardial regeneration.    Materials and Methods  In this study, the regenerative potential of hypoxic human cardiomyocytes (group Hyp-CMC)  in vitro  was evaluated when co-cultured with human bone-marrow derived MSC (group Hyp-CMC-MSC) or stimulated with the secretome of MSC (group Hyp-CMC-S MSC ). The secretome of normoxic MSC and CMC, and the hypoxic CMC was analyzed with a cytokine panel. Gene expression changes of  HIF-1 α, proliferation marker  Ki-67  and cytokinesis marker  RhoA  over different reoxygenation time periods of 4, 8, 24, 48, and 72 h were analyzed in comparison to normoxic CMC and MSC. Further, the proinflammatory cytokine IL-18 protein expression change, metabolic activity and proliferation was assessed in all experimental setups.    Results and Conclusion   HIF-1 α was persistently overexpressed in Hyp-CMC-S MSC  as compared to Hyp-CMC (except at 72 h). Hyp-CMC-MSC showed a weaker  HIF-1 α expression than Hyp-CMC-S MSC  in most tested time points, except after 8 h. The  Ki-67  expression showed the strongest upregulation in Hyp-CMC after 24 and 48 h incubation, then returned to baseline level, while a temporary increase in  Ki-67  expression in Hyp-CMC-MSC at 4 and 8 h and at 48 h in Hyp-CMC-S MSC  could be observed.  RhoA  was increased in normoxic MSCs and in Hyp-CMC-S MSC  over time, but not in Hyp-CMC-MSC. A temporary increase in IL-18 protein expression was detected in Hyp-CMC-SMSC and Hyp-CMC. Our study demonstrates timely dynamic changes in expression of different ischemia and regeneration-related genes of CMCs, depending from the culture condition, with stronger expression of  HIF-1 α,  RhoA  and IL-18 if the hypoxic CMC were subjected to the secretome of MSCs.

Comparative Effect of MSC Secretome to MSC Co-culture on Cardiomyocyte Gene Expression Under Hypoxic Conditions in vitro