[3,4,51]. In addition, in very recent years, convincing evidence has shown that the normal heart is not a post mitotic organ since it contains a pool of progenitor cells and a population of immature, dividing myocytes that allow for a turnover of cardiomyocytes involving the generation of new cardiomyocytes in substitution of the damaged ones [52]. A new view on anthracycline cardiotox- icity was recently introduced with the demonstration that in comparison to differentiated cardiomyocytes, dividing cardiomy- ocytes are more sensitive to anthracyclines and that low doses of doxorubicin causes senescence-like changes in these cells [3,4,53,54].These effects may inhibit the regenerative capacity of the heart and, through this mechanism, impair the self-repairing potential of the heart, ultimately leading to ventricular dysfunction and late clinical events. Neonatal cardiomyocytes share some characteristics with the population of replicating cells that are present in the adult heart, and that are reminiscent of a fetal/ neonatal phenotype [52]. Thus, this experimental model may be considered a convenient indicator of what might happen to these cardioregenerative cells when the heart is exposed to anthracy- clines with or without. pre-treatment with a cardioprotective agent. The work presented here demonstrates for the first time that the PPARd activator L-165041 is a highly effective cardioprotective agent, and suggests that further testing including animal models of anthra- cycline cardiac injury should be carried out to assess the potential therapeutic role of this compound. Materials and Methods All materials, unless otherwise indicated, were supplied by Sigma-Aldrich (Poole,UK). Cell Culture and Treatment Neonatal rat ventricular myocytes (CloneticsH Rat Cardiac Myocytes, Lonza, Switzerland) were purchased and cultured with rCMC Medium Bulletkit (Clonetics, Lonza, Switzerland) as described [55]. H9c2 rat heart-derived embryonic myocytes (American Type Culture Collection) were cultured as previously described [9]. Cells were always used at less than 70% of confluence. Experimental design: Cells were pre-incubated for 1 hour with or without the ERK1/2 pathway inhibitor PD98059 (50 mmol/L) (Calbiochem, Merk, Germany), the JNK inhibitor SP600125 (20 mmol/L), the p38 MAPK inhibitor SB203580 (3 mmol/L), with the Akt pathway inhibitor Akt1/2 kinase inhibitor (30 mmol/ L), and then were incubated for 2 hours with or without L-165041 (10 mmol/L) (Calbiochem, Merk, Germany). They were then treated with or without various doses of doxorubicin for 3 hours [4] and analyzed at the time indicated for each experiment. In order to evaluate MAPK and Akt phosphorylation, cells were pre- treated for 20, 40, or120 minutes with or without L-165041, then they were treated for 20, 40, or 120 minutes with or without doxorubicin. Since both L-165041 and the MAPK inhibitors were dissolved in 0.1% dimethyl sulphoxide (DMSO), an equivalent amount of vehicle was added to both the control and to the drug- treated samples when the experiments were performed with these inhibitors. Semi-quantitative Reverse Transcription PCR RNA isolation and RT-PCR were performed using the previously described procedure [56]. The primers used for PCR were TRF2 forward (GGACAGCGAGACACTAAAGGC), re- verse (CTGGATGACAATGTCTG CTTC) GenBankTM acces- sion number NM031055. PPARd forward (AGGCCTCA Activated PPARd Protects from Senescence TGAACGTGCCACAG), reverse (AGCGGACTAATGGGCT- CACTGG) GenBankTM accession number NM013141. GAPDH forward (GAGGGGCCATCCACAGTCTT), reverse (TTCATTGA CCTCAACTACAT), GenBankTM accession number XM221254. The quantity of mRNA was normalized for Glyceraldehyde-3- Phosphate Dehydrogenase (GAPDH). The PCR products were quantified by a gel documentation system with analysis software (Syngene, Cambridge, UK). Quantitative Real Time PCR Analysis Total RNA was extracted from 6-wells of cultured cells from neonatal rat ventricular myocytes using RNeasy mini Kit according to the manufacturer’s protocol (Qiagen, Germany). Twenty ml of total RNA were reverse transcribed using the High- Capacity cDNA Archive Kit (Applied Biosystems by Life Technologies Foster City, CA, U.S.A.) and pre-amplified using the PreAmp Master Mix KitPrimers, and probes for PPARa, PPARc, PPARd and GAPDH were obtained from a pre- developed assay-on-demand (Applied Biosystems by Life Tech- nologies Foster City, CA, U.S.A.). Five ml of the resulting pre-amplified, cDNA dilution (1:2) were used for quantitative PCR amplification which was performed in quadruple on the Prism 7900HT Instrument (Applied Biosystems, Foster City, CA, U.S.A) with the fluorescent TaqMan method. The PPAR mRNA quantities were normalized to the control gene GAPDH and were expressed in relation to a calibrator sample. The levels of transcripts PPAR and GAPDH were determined using the standard curve. The standard curve was obtained with serial dilutions (106-10 molecules) of a cDNA calibrator. Total Cell Lysates Cells were lysed in lysis buffer [20 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP40, 2.5 mM Na2P2O7, and 1 mM b-glycerophosphate]. The follow- ing inhibitors were added immediately before adding the buffer to the cells: 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF, and protease inhibitor mixture (Roche, Switzerland). Subcellular Fractionation Cytoplasmic and nuclear fractions were obtained as previously described [57]. The nuclear fractions were supplemented with 50U endonuclease benzonase to obtain both the soluble and the chromatin-bound insoluble pool of nuclear proteins. Co-immunoprecipitation To examine the interaction between PPARd and Bcl6, the nuclear protein fraction was co-immunoprecipitated with the anti- PPARd antibody as previously described [17] and then analyzed by western blot using the anti-Bcl6 antibody. Western Blot Immunoblotting was performed using the previously described procedure [9]. After various treatments, cells were processed to determine the levels of TRF2 (clone 4S794.15, Imgenex, USA), PPARd (ab23673, Abcam USA) phosphorylated p38 (T180/ Y182 site, R&D Systems) Bcl6 (D-8) GAPDH (0411), p38 (H- 147), phosphorylated JNK (G-7) (pJNK) and JNK (FL), phosphorylated Akt (pAkt) (ser473)-R, Akt (H-136), phosphory- lated ERK1/2 (pERK1/2) (E-4), ERK1/2 (K-23) (Santa Cruz Biotechnology, Santa Cruz, USA). After incubation in horserad- ish peroxidase secondary antibody, blots were visualized with ECL substrate (Millipore, Merk, Germany) and films were PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e46126