Over Expression of Plk1 Does Not Induce Cell Division in Rat Cardiac Myocytes In Vitro Carmen H. Coxon1,3, Katrina A. Bicknell1, Fleur L. Moseley1¤, Gavin Brooks1,2* 1 School of Pharmacy, University of Reading, Reading, United Kingdom, 2 School of Biological Sciences, University of Reading, Reading, United Kingdom, 3 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom Abstract Background: Mammalian cardiac myocytes withdraw from the cell cycle during post-natal development, resulting in a non- proliferating, fully differentiated adult phenotype that is unable to repair damage to the myocardium, such as occurs following a myocardial infarction. We and others previously have shown that forced expression of certain cell cycle molecules in adult cardiac myocytes can promote cell cycle progression and division in these cells. The mitotic serine/ threonine kinase, Polo-like kinase-1 (Plk1), is known to phosphorylate and activate a number of mitotic targets, including Cdc2/Cyclin B1, and to promote cell division. Principal Findings: The mammalian Plk family are all differentially regulated during the development of rat cardiac myocytes, with Plk1 showing the most dramatic decrease in both mRNA, protein and activity in the adult. We determined the potential of Plk1 to induce cell cycle progression and division in cultured rat cardiac myocytes. A persistent and progressive loss of Plk1 expression was observed during myocyte development that correlated with the withdrawal of adult rat cardiac myocytes from the cell cycle. Interestingly, when Plk1 was over-expressed in cardiac myocytes by adenovirus infection, it was not able to promote cell cycle progression, as determined by cell number and percent binucleation. Conclusions: We conclude that, in contrast to Cdc2/Cyclin B1 over-expression, the forced expression of Plk1 in adult cardiac myocytes is not sufficient to induce cell division and myocardial repair. Citation: Coxon CH, Bicknell KA, Moseley FL, Brooks G (2009) Over Expression of Plk1 Does Not Induce Cell Division in Rat Cardiac Myocytes In Vitro. PLoS ONE 4(8): e6752. doi:10.1371/journal.pone.0006752 Editor: Anja-Katrin Bielinsky, University of Minnesota, United States of America Received May 8, 2009; Accepted July 17, 2009; Published August 25, 2009 Copyright: 2009 Coxon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a PhD Studentship from Heart Research UK, ref 2463/02/06, awarded to G.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Fleur Moseley currently works for Innovex Medical Communications, but was working at the University of Reading as a postodoctoral researcher at the time of the study. * E-mail: g.brooks@rdg.ac.uk ¤ Current address: Innovex Medical Communications, Bracknell, United Kingdom Introduction Mammalian cardiac myocytes lose the ability to divide shortly after birth, [1–4], differentiating into cell cycle arrested binucleated cells. Subsequent growth of the myocardium is achieved through an increase in myocyte cell size, known as hypertrophy. Following myocardial injury, cardiac myocytes are lost from the damaged area and replaced by scar tissue. Any loss in cardiac function is compensated by a physiological hypertrophic response and cardiac remodeling. Although this process initially helps to maintain cardiac output, the remodeling process continues and eventually leads to the development of pathological hypertrophy and heart failure [5–7]. The change in growth potential of cardiac myocytes during cardiac development and hypertrophy is reflected in the expressions and activities of certain cell cycle molecules.[8–15] Thus, positive regulators of the cell cycle machinery, such as the Cdk/cyclin complexes and the pro-proliferative E2F transcription factors (E2F1-3) are down regulated, whilst negative regulators, including retinoblas- toma protein (pRb) and the cyclin-dependent kinase inhibitors (CDKIs), p21 and p27, are upregulated. As a consequence, the majority of adult mammalian cardiac myocytes are arrested in G0/G1, with the remaining cells being present at the G2/M transition[15]. Current strategies aimed at encouraging repair of the heart focus on the use of stem cells or gene therapy to induce cell division and differentiation at the site of damage to reduce the size of the infarct and improve patient outcome.[16,17] In support of the gene therapy approach, work from our laboratory[12,13,18] and others (see below) have shown that manipulation of the cardiac myocyte cell cycle machinery could be a suitable therapeutic approach for promoting myocardial repair. Alterations in the expressions of certain cell cycle molecules can increase cardiac myocyte proliferation in vitro,[18–25] inhibit the induction of hypertrophy in vitro[12] or increase cardiac myocyte num- ber[14,24] and promote myocardial repair[25,26] in vivo. These studies have highlighted a number of potential targets for the reinitiation of cardiac myocyte cell division, with the aim of increasing the number of cardiac myocytes and reducing infarct size to improve patient outcome. The Polo-like kinases (Plks) are a family of serine/threonine kinases that exhibit sequence identity in both their N-terminal catalytic domain and their C-terminal region, which is termed the polo-box domain (PBD). This C-terminal region contains phospho- peptide binding motifs[27,28], called ‘polo-boxes’, that are essential for Plk localization and regulation.[29–32] This family of kinases PLoS ONE | www.plosone.org 1 August 2009 | Volume 4 | Issue 8 | e6752