OBJECTIVETo investigate the role of homeobox gene Nkx2-5 in cardiac myogenesis.

METHODSTwo P19 cell lines, namely cells transfected with exogenous expression of Nkx2-5 gene and non-transfected cells, were cultured in suspension for 4 days to induce cell aggregation, and the cell aggregates were transferred to the Petri dish for further adherent culture. On days 4, 8, 12 and 16 of adherent culture, the expressions of α-sarcomeric actin (α-SA) and cardiac troponin T (cTnT) protein were detected by immunocytochemistry, and the mRNA expressions of GATA-4, α-myosin heavy chain (α-MHC) and atrial natriuretic factor (ANF) genes by RT-PCR.

RESULTSIn the transfected cells, α-SA and cTnT protein expressions were detected on days 8, 12 and 16 of adhere culture, and their expressions increased gradually with time. α-SA and cTnT expression was significantly higher on day 16 than on day 8 of culture (P<0.01). RT-PCR analysis of the transfected cell showed the presence of GATA-4 expression on day 4 of adherent culture, and the expression increased on days 8 and 12 but decreased on day 16. ANF and α-MHC expressions were found on days 8, 12, and 16, increasing gradually over time and showing significant differences from those on day 4 (P<0.05 or P<0.01). The expression of α-MHC was significantly higher on days 12 and 16 than on day 8 (P<0.05 or P<0.01), and ANF expression was significantly higher on day 16 than on days 8 and 12 (P<0.01). The non-transfected cells were negative for the expressions of all these genes.

CONCLUSIONExogenous expression of Nkx2-5 gene can induce P19 cells to express cardiac markers in vitro.