Plk1 & Myocardial Regeneration Figure 1. Plk1 expression and activity declines during rat cardiac myocyte development. Ventricular cardiac myocytes were isolated from Wistar rat 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 Plk1 mRNA (A, normalised to GAPDH) and protein (B, normalised to GAPDH) expression and activity (C). D) representative western blot of Plk1 expression. E) Representative kinase assay and corresponding protein expression (GAPDH was used as a loading control). Data is from three separate experiments, * p,0.05. doi:10.1371/journal.pone.0006752.g001 wild-type or constitutively active (T210D) Plk1 at a multiplicity of infection (MOI) of 50 (50 virus particles per cell). Forty eight hours post infection, we found that over-expression of wild-type (WT) Plk1 increased the number of HeLa cells by 2365.6% (n = 3, p = 0.02), compared to the EGFP control (Figure 4A and B). Constitutively active Plk1 (T210D) also increased HeLa cell numbers by 2061.5% above the EGFP control. This data shows that adenovirus-delivered Plk1 was able to induce cell division in proliferative cells. We then assessed the activity of adenovirus- delivered Plk1 by in vitro kinase assay in non-proliferating P10 cardiac myocytes: Plk1 kinase activity was increased in both WT and T210D Plk1 expressing cells (Figure 4C). Over-expression of catalytically active Plk1 has no effect on cardiac myocyte binucleation We next determined the effect of over expressing catalytically active WT and T210D Plk1 on cardiac myocyte cell binucleation. Ventricular cardiac myocytes were isolated from P3, P7 and adult Wistar rat hearts and subjected to adenovirus infection as above; P7 cardiac myocytes were chosen due to their improved viability following isolation compared to P10 cells (data not shown). The effect of EGFP, WT or constitutively active (T210D) Plk1 expression on cardiac myocyte binucleation was assessed by calculating the number of nuclei per cardiac myocyte. For uninfected postnatal cells, the number of binucleated cells increased from ,20% at P3 to ,40% at P7. Adult cardiac myocytes showed extensive (,90%) binucleation (Figure 5A). Surprisingly, we found adenovirus-delivered Plk1 did not have a significant effect on the percentage of binucleated cardiac myocytes at any stage of development (Figures 5B, C and D). This was somewhat surprising, especially at P3 where there is still a capacity for cell division. To determine whether there had been an effect on cell number but not on binucleation, we performed cell counts at P3 and found that there was no significant increase in PLoS ONE | www.plosone.org 4 August 2009 | Volume 4 | Issue 8 | e6752