Abstract

Cell-based therapy using mesenchymal stem cells (MSC) has emerged as a promising approach to regenerating failing hearts. However, studies need to be done to optimise and enhance the beneficial effects of mesenchymal stem cell therapy. Here we show that genetic silencing of Plasma Membrane Calcium ATP-ase 4 (PMCA4) in MSC produces a protective effect when co-cultured with cardiomyocytes in response to hypoxia.

Using the lentivirus system we have generated MSC with stable  integration of PMCA4 shRNA (MSC-PMCA4KD). PMCA4 expression was reduced by more than 75% in these cells (p<0.01). To test the protective effect of MSC-PMCA4KD we co-cultured these cells with neonatal rat cardiomyocytes and cultured them in hypoxic conditions (95% N2, 5% CO2, 1% O2) for 24 hours. We found that cardiomyocytes co-cultured with MSC-PMCA4KD displayed higher survival and less apoptosis, as indicated by a significantly lower level of caspase 3/7 activity (n=3, p<0.05) compared to cells co-cultured with control MSC. We also found that MSC-PMCA4KD expressed and secreted higher levels of sFRP2 (secreted frizzled related protein 2) compared to control, as indicated by qPCR, Western blot and ELISA analyses (n=3, p<0.05). sFRP2 is a potent inhibitor of the Wnt signalling pathway and may be responsible for the protective effect of MSC-PMCA4KD.

In conclusion, our data show that PMCA4 genetic silencing increases the protective effect of MSC against hypoxic stimulus. Thus, PMCA4 may be a target to enhance the therapeutic effects of MSC.