Figure 6. Exogenous Plk1 does not increase the activity of endogenous Cdc2/cyclin B1 in isolated P3 rat cardiac myo- cytes. Ventricular cardiac myocytes were isolated from Wistar rats 3 days after birth (P3). Adenovirus expressing EGFP, WT or T210D was used to infect cells for 48 hours. Cells were harvested and assessed for Plk1 and Cdc2/cyclin B1 kinase activity. Plk1 activity was assessed by its ability to phosphorylate casein (A, upper panel). Cdc2/cyclin B1 activity was assessed by phosphorylation of histone H3 (A, lower panel). B) Representative western blot of Plk1 over expression in P3 cardiac myocytes infected with EGFP, WT or T210D adenovirus. GAPDH was used to verify equal loading. doi:10.1371/journal.pone.0006752.g006 the adult stage. Adult cardiac myocytes are thought to be blocked in G2/M and it is possible that Plk3 could contribute to the arrested state of these cells. The decline in Plk1 mRNA and protein levels and activity observed during cardiac myocyte development is consistent with previous studies from our laboratory, and others, examining the expression of other such positive regulators of the cell cy- cle[8,11,12,18,21,23–25] and extends the work by Georgescu et Plk1 & Myocardial Regeneration al [60]. To investigate the role of Plk1 in nuclear and cellular division in rat cardiac myocytes, we over expressed wild type and constitutively active Plk1 in adult myocytes for 48 hours in the absence or presence of serum to determine whether Plk1 activity could induce cell division. Neither the wild type nor the constitutively active mutant induced any changes in cell cycle progression, in terms of the percentage of binucleated cells, or cell number. Surprisingly, we also found that catalytically active Plk1 was unable to increase the activity of endogenous Cdc2/cyclin B1 in P3 cardiac myocytes, suggesting that Plk1 is unable to complete with the other factors that regulate activity of the Cdc2/cyclin B1 complex. As well as dephosphorylation of Cdc2 at Thr14 and Tyr15 by Cdc25C, phosphorylation of Thr161 in the catalytic T- loop by Cdk activating kinase (CAK) is essential for Cdc2/cyclin B1 activity.[79] It is likely that some of these other factors limit the activation of endogenous Cdc2/cyclin B1; for example, CAK phosphorylates Cdc2 at Thr161 [80] and this modification is required for full activation of the Cdc2/cyclin B1 complex. If Plk1 can only enhance the activity of Cdc2/cyclin B1 that already is phosphorylated at Thr161, the amount of active Plk1 present would be irrelevant if CAK is the rate-limiting factor in Cdc2/ cyclin B1 activation. In addition to this, whilst Plk1 has been reported to inhibit the Cdk inhibitor, Wee1, it has been shown that an initial Cdk-mediated phosphorylation of Wee1 at Ser121 is necessary to produce a Plk1 docking site, and that this phosphorylation event is required for increased Plk1 binding[43]. If the Cdk activity in P3 cardiac myocytes is not sufficient to prime Wee1 for Plk1 binding, over expression of Plk1 alone will not be sufficient for Wee1 inhibition and, therefore, an increase in Cdc2/ cyclin B1 activity. Cdc2/cyclin B1 is involved in some of the early stages of mitosis, such as the breakdown of the nuclear envelope and chromosome condensation[81]. Because ectopic Cdc2/cyclin B1 can elicit an increase in the number of adult cardiac myocytes, whilst Plk1 can not, it is possible that in the heart these cells are in an early/mid- G2, rather than in late-G2/M arrest. As such, the introduction of Plk1 into adult myocytes could be too far downstream of this arrest, and therefore unable to bypass the other events that are necessary to occur before mitosis can take place. Based upon the observations reported here, we sought to characterize the G2/M arrest in more detail. Previous reports have suggested that a G2/M arrested population of cardiac myocytes exists in the rat adult heart.[14,15] To examine this further, we prepared heart sections and assessed them for any evidence of mitotic arrest by the phosphorylation of histone H3 on serine 10 or serine 28. Cells staining positive for cardiac myocyte markers and phosphorylation of either serine residue declined with age, with virtually no adult cardiac myocytes staining positive. The expression of the cell cycle marker, Ki67, was also assessed during rat heart development and was shown to be absent from the adult heart. This data suggests that adult cardiac myocytes are not in a state of ‘G2/M’ arrest as previously reported[15], but that the arrest could be earlier, such as late S/G2. This raises questions about the involvement of mechanisms of endoreduplication, rather than mitosis, being responsible for cell cycle arrest in the adult cardiac myocyte. Cardiac myocytes are known to increase in terms of ploidy following myocardial infarction[82]. It is possible that the increase in ploidy seen in the myocardium surrounding an infarct[82] is an attempt at cell division that is inhibited by the absence of Cdk or Plk1 activity at G2, leading to rounds of DNA synthesis in the absence of cell division. This may partially explain the presence of the previously reported population of G2/M nuclei;[15] these nuclei may in fact be the product of replication origin re-licensing and endoreduplication rather than an aborted mitosis. PLoS ONE | www.plosone.org 9 August 2009 | Volume 4 | Issue 8 | e6752