Plk1 & Myocardial Regeneration Figure 4. Effect of adenovirus-delivered Plk1 on HeLa cell number and Plk1 activity. Plk1 enhanced cell division in HeLa cells at 48 hours post infection (A and B). The activity of adenovirus-delivered Plk1 was assessed in P10 cardiac myocytes (C). doi:10.1371/journal.pone.0006752.g004 increase cell number in vitro[18]. Although the data presented here shows that Plk1 is unable to do this, it does highlight that a more definitive understanding of the arrest in cardiac myocytes is necessary in order to successfully identify potential targets for a gene therapy approach. In order to further characterise the arrest in cardiac myocytes, and a therefore provide a smaller number of potential targets, we decided to more precisely define the point within the G2/M arrest that these cells are arrested in. Four micrometre sections were taken from E18, P3, P10 and adult Wistar rat hearts (Figure 6) and assessed for the expression of Ki67 and phosphorylation of histone H3 at serine 10 (Ser10) and at serine 28 (Ser28). The proliferation marker, Ki67, is present in all phases of the cell cycle, increasing from G1 to maximal intensity at the metaphase-anaphase transition, but is not found in telophase nor in quiescent, G0, cells[64]. Histone H3 becomes phosphorylated on Ser10 during late G2 and is important for chromosome condensation[65–67]. Ser28 is also phosphorylated during mitosis, and like Ser10 phosphorylation, contributes to chromosome condensation [68]. However, unlike Ser10, Ser28 is not phos- phorylated until the onset of mitosis[68] and therefore serves as a more faithful mitotic marker. The expression of Ki67 and the phosphorylation of histone H3 at Ser10 and Ser28 declined with age (Figure 7). Very few (Ki67, 0.4960.25%; Ser10, 0.3560.27%; Ser28, 0.2960.15%; n = 3, mean6SEM) troponin-I-positive adult cardiac myocytes demon- strated any staining or mitotic figures. Previous studies have investigated the isolated nuclei of adult cardiac myocytes using FACS analysis using dual staining for bromodeoxyuridine (BrdU) and propidium iodide (PI) [14,15]. This type of analysis determines cell cycle stage by assessing the DNA content of each event (cell or nucleus) as it passes through the laser. Any event displaying a double DNA content, such as a post-S phase, mitotic or binucleated cell, will be classified as a G2/M cell. The lack of any Ser10 or Ser28 staining or mitotic structures suggests that the G2/M population originally observed in adult rat cardiac myocytes most likely represents a population of late S-phase/ early G2-arrested cells, rather cells in late G2/M. This might explain why Plk1 is unable to induce cell cycle progression while Cdc2/cyclin B1 can; Cdc2/cyclin B1 reaches its peak activity before the G2/M transition while Plk1 peaks at metaphase[69]. Discussion In this study, we have determined the expression of the mammalian Plk family of serine/threonine protein kinases in the developing rat cardiac myocyte in an effort to better understand why adult cardiac myocytes are unable to proliferate and repair the damaged myocardium, and to potentially identify novel therapeutic targets that could be manipulated to encourage myocardial repair. Developing cardiac myocytes undergo binucleation during neonatal growth,[70–73] whilst adult cells undergo a G1/S transition during the process of hypertrophy.[2,74]. Both Plk2 and Plk4 are PLoS ONE | www.plosone.org 7 August 2009 | Volume 4 | Issue 8 | e6752