Plk1 & Myocardial Regeneration Figure 2. Regulation of Plk2, Plk3 and Plk4 mRNA expression during cardiac myocyte development. Ventricular cardiac myocytes were isolated from hearts at various stages of development (embryonic day 18 [E18], 2, 5 and 10 days postnatal [P2, P5 and P10] and adult [250 g males]) and assessed for Plk2, Plk3 and Plk4 mRNA (black bars, A, B and C, respectively). Samples were normalised to GAPDH. Each bar chart represents data from at least 3 separate experiments, * p,0.05. doi:10.1371/journal.pone.0006752.g002 cardiac myocyte cell number when the cells were incubated in either serum free media (n = 3, WT p = 0.378; T210D p = 0.100) or media supplemented with 5% FCS (n = 3, WT p = 0.277, T210D p = 0.892) compared to EGFP control (Figure 5E). Cell counts were performed on permeabilised, fixed samples stained with cardiac specific markers so rule out increases in cell number by contaminating cardiac fibroblast cells. Over-expression of Plk1 does not increase the activity of Cdc2/cyclin B1 in P3 cardiac myocytes Whilst over-expression of Plk1 had no effect on binucleation in adult cardiac myocytes, it was surprising that Plk1 had no effect on P3 cardiac myocytes at all. P3 cardiac myocytes express the mitotic regulator Cdc2/cyclin B1[18] and retain a limited proliferative capacity. Plk1 promotes activation of Cdc2/cyclin B1[37,42] and inhibits the activity of the Cdc2/cyclin B1 inhibitors, Wee1 and Myt1[43,45,61]. Therefore, we evaluated the effect of Plk1 over expression on Cdc2/cyclin B1 activity in P3 rat cardiac myocytes to determine whether this might explain the lack of cell cycle progression. Interestingly, the activity of Plk1 increased in cells infected with both wild-type and constitutively active Plk1 adenovirus, whereas the activity of Cdc2/cyclin B1 in these cells was unaffected (Figure 6). This data shows that the activation of Cdc2/cyclin B in differentiating rat cardiac myocytes cannot be achieved solely by the over expression of Plk1 and highlights the complexity of cell cycle regulation in cardiac myocytes. This work shows that careful consideration is required when choosing which molecules should be selected for these studies. It is clear that the cell cycle must be manipulated in precisely the right way in order to elicit cell cycle progression. Cardiac myocytes do not express G2/M markers The current study aimed to determine whether Plk1 could be a potential therapeutic target for the initiation of cardiac repair in the mammalian heart. This hypothesis was based on previous studies that have shown that a significant population of adult cardiac myocytes is G2/M arrested [14,15,62,63]], and that over expression of the G2/M regulator Cdc2/cyclin B1 is sufficient to PLoS ONE | www.plosone.org 5 August 2009 | Volume 4 | Issue 8 | e6752