PKG and PKC Are Down-Regulated during Cardiomyocyte Differentiation from Embryonic Stem Cells: Manipulation of These Pathways Enhances Cardiomyocyte Production

Figure 4

FACS analysis was used to quantify the percentage of cardiomyocytes differentiated from ES cells using a conventional protocol (control: DMSO carrier + no drugs [5, 6, 17]) and using PKG and/or PKC isotypic inhibitors. (a)–(e) Representative raw FACS data. The $x$ axis is the fluorescence level of cardiac troponin. The y axis is the cell count. M1 marks cardiac troponin (+) cells. (a) Inhibiting PKG alone (blue line) markedly pushed more cells towards the cardiomyocyte lineage when compared to control EBs differentiated down the cardiomyocte pathway (red line). (b)-(c) However, combining PKG and PKC inhibitors skewed ES cell differentiation further towards the cardiomyocyte lineage when compared to DT-3-treated EBs (a) and control EBs with the highest number of cardiac troponin-positive cells found in EBs treated with DT-3 and PKCbeta specific inhibitor (b). (d)-(e) PKC inhibitors alone also increased the number of cardiomyocytes generated from ES cells but not to the extent of dual inhibition of PKCbeta and PKG. (f) Proportions of cardiac troponin expressing cells from four experiments were analyzed using the two-sample test for binomial proportions. The proportion of CCE cells expressing cardiac troponin was significantly highest in PKG/PKCbeta-(purple bar) inhibited cells when compared to EBs differentiated without drugs (red bar; $P<.001$). Inhibiting both PKC$\beta$ and PKG resulted in the most significant increase in cardiomyocytes when compared to all other treatments ($P$ values are shown for each comparison). Two types of controls were used for FACS analyses: $(1)$ FACS machine parameters were set by analyzing cells from differentiated EBs not subjected to any antibodies and cells from differentiated EBs subjected to only secondary antibodies, and $(2)$ cells from treated EBs were compared to cells from untreated (or DMSO carrier) EBs subjected to primary (anticardiac troponin) and secondary antibodies. EBs differentiated without drugs (i.e., untreated) or without drugs but in DMSO carrier demonstrated virtually identical proportions of differentiated cardiomyocytes (data not shown).