Adenovirus-infection studies Recombinant adenoviruses expressing wild-type (WT) or constitutively active Plk1 (T210D) or EGFP alone were generated using the AdEasy System (QBiogene, Carlsbad, USA). For adult cardiomyocyte infection studies, isolated adult myocytes were plated in serum-free modified M199 medium (M199 containing 2 mM creatine, 5 mM taurine and 2 mM carnitine) for 1 hr on MatrigelTM-coated six-well dishes. Adherent adult cardiomyocytes were washed and then infected at a MOI (multiplicity of infection) of 50 plaque forming units of each virus per cell in modified M199 medium. Infected cultures were maintained in modified M199 medium for 24 h before analysis. Neonatal cells were transfected 24 hours after isolation. Cells were washed with PBS and infected in either serum-free M199 media or M199 supplemented with 5% FCS. Cells were incubated for 48 hours prior to analysis. Western Blotting Protein expression levels were determined by SDS-PAGE analysis as previously described [10,13]. In vitro kinase assays Cdc2/cyclin B1 immunocomplexes were immunoprecipitated from 250 mg of protein extracted from infected cultures using anti- cyclin B1 antibody and kinase activity measured as described previously [18]. For Plk1 kinase assays, protein lysate was prepared from infected cell cultures in TBSN lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EGTA, 25 mM b-glycerophos- phate, 25 mM NaF, 0.5 mM Na3VO4, 10 mM p-nitro phenyl- phosphate (pNPP), 0.1% Nonidet P-40, 72 mg/ml AEBSF, 10 mg/ ml leupeptin, 5 mg/ml aprotinin). Samples were precleared with 30 ml Protein-G Agarose beads at 0.1 g/ml (Sigma) at 4uC on a rotating wheel for 1 hour. Plk1 was immunoprecipitated with 1.5 ml monoclonal mouse anti-Plk1 antibody cocktail (Zymed) and pulled down with 30 ml Protein G-Agarose. Immunoprecipitated Plk1 was washed twice in TBSN buffer and twice in TDSN (50 mM Tris pH 7.5, 10 mM MgCl2, 5 mM dithiothreitol (DTT), 2 mM EGTA, 0.5 mM sodium vanadate, 20 mM p-nitrophenyl phosphate) and incubated with 4 mg casein, 10 mCi AT32P and 10 mM ATP in 25 ml TDSN buffer at 30uC for 30 minutes. The reaction was terminated by boiling the samples in 1X Laemmli buffer with 0.2 M dithiothreitol (DTT) for 5 minutes. Samples were loaded on a 10% polyacrylamide gel and transferred onto nitrocellulose membrane. Membranes were assessed with a phosphorimager cassette (Molecular Dynamics) for the desired time. Immunocytochemistry Neonatal cardiomyocytes were cultured in 5% myocyte media on gelatin-coated glass coverslips for 24 h before infection with recombinant adenoviruses. Adult cardiomyocytes were plated on to MatrigelTM-coated chamber slides for 1 hr before infection with recombinant adenoviruses. Cell monolayers were fixed in 1% (v/v) formaldehyde in PBS for 10 min at room temperature (21uC), permeabilised with 0.5% (v/v) Triton X-100 in PBS for 15 min and blocked in 10% (v/v) normal goat serum/2% (w/v) BSA in PBS for 30 min. Cardiac myocytes were identified by staining with cardiac myocyte markers: anti-tropomyosin (sarcomeric) mono- clonal antibody (clone CH1) was detected with anti-mouse IgG AlexaFluorH 568 or anti-troponin I was detected with anti-rabbit IgG AlexaFluorH 488 conjugate. These markers were used to distinguish cardiomyocytes from non-myocytes. Images were collected using an Axioskop2 microscope in conjunction with AxioVision software (Zeiss). Plk1 & Myocardial Regeneration Statistical analysis All results are presented as means6standard error of the mean (SEM). Data were analyzed by one-way ANOVA followed by the appropriate t-test, with P,0.05 considered to be statistically significant. Results Expression and activity of Plk1 declines during rat cardiac myocyte development Based upon previous studies showing the downregulation of positive regulators of cell cycle molecules during cardiac myocyte development, we hypothesized that the expression of Plk1 would be downregulated as these cells progressed towards full differen- tiation. Quantitative PCR analysis revealed a progressive decline in Plk1 mRNA levels during development (Figure 1A) and this was also seen at the protein level (Figure 1B). No Plk1 protein was detected in adult cardiac myocytes. Interestingly, the activity of Plk1 does not change significantly at P10, compared to P3 levels, despite a decline in Plk1 expression (Figure 1B–E). These results suggest that there is a change in the regulation of Plk1 leading to increased activity at P10 compared to levels in P3 cardiac myocytes that is independent of protein synthesis. mRNA and protein levels of Plk2, Plk3 and Plk4 during rat cardiac myocyte development Quantitative PCR revealed differential patterns of regulation for Plk2, Plk3 and Plk4 mRNA during the postnatal developmental period; all are significantly downregulated in the adult. Plk2 showed very little change from E18 levels throughout postnatal development (Figure 2A) whereas Plk3 showed a small, but significant, increase in mRNA levels during the early postnatal growth phase (P2 and P5) (Figure 2B). Plk4 showed a small, significant decrease at P2 and is downregulated by ,90% in the adult compared to E18 levels (Figure 2C). At the protein level, Plk2, Plk3 and Plk4 are all expressed in the adult (Figures 3A to F), unlike Plk1, which is not be detected in adult cardiac myocytes (Figure 1B). Plk2 protein levels showed a considerable degree of variability between samples (n = 5); protein levels do not appear to change significantly during development. Plk3 protein expression also remains unchanged during early postnatal development, but does appear to be downregulated in the adult heart by ,40% compared to embryonic day 18 (E18). Plk4 protein levels decrease as development proceeds, with a significant downregulation at P2 and in the adult (Figure 3E and F). Based upon the data showed here, Plk1 is the most interesting candidate for re-initiation of cell cycle in adult cardiac myocytes. No protein expression or activity is detectable in the adult heart, whereas Plk2–4 are all expressed, albeit at lower levels, compared to E18. Adenoviral expression of active Plk1 in both HeLa cells and cardiac myocytes Petronczki et al (2007)[48] have shown that inhibition of Plk1 in HeLa cells released from a metaphase block results in binuclea- tion. The majority of adult cardiac myocytes are binucleated and do not express Plk1. We hypothesized that reintroduction of Plk1 into isolated adult rat cardiac myocytes might drive the cells through a successful round of nuclear and/or cell division, thereby increasing cell number. Accordingly, we generated adenoviruses carrying Plk1 in conjunction with an internal ribosomal entry site (IRES) and EGFP marker. To confirm the ability of the Plk1 construct to induce cell cycle progression in proliferating cells, HeLa cells were infected with adenovirus containing either EGFP, PLoS ONE | www.plosone.org 3 August 2009 | Volume 4 | Issue 8 | e6752