and differentiation [20]. In the light of its effects on metabolism and inflammation, PPARd activation has been seen as a promising approach for the treatment of atherosclerosis [21]. A number of studies using transgenic approaches and pharmaco- logical interventions have shown that PPARd also plays a crucial role in cardiomyocyte growth and survival, thus suggesting that PPARd activation may be a therapeutic target in heart diseases [22]. Moreover, two studies demonstrated that PPARd and Bcl6 may play a role in the regulation of cellular senescence [23,24]. In this study we demonstrate that L-165041, a PPARd agonist, is a cardioprotective agent that prevents senescence and apoptosis induced by low and high doses of doxorubicin, respectively. We prove that Bcl6 and Bcl6:PPARd interference plays a central role in the regulation of senescence in cardiac muscle cells, and that the protective effects of the PPARd agonist involve Mitogen-activated protein kinases (MAPKs) and Akt activation. Results Pre-treatment with the PPARd Agonist Prevents the Prosenescent Effects of Doxorubicin 0.1 mM in Neonatal Rat Ventricular Myocytes and H9c2 Previous studies have shown that brief exposure to low (0.1 mM) or high (1 mM) doses of doxorubicin induces either senescence or apoptosis, respectively, in neonatal rat ventricular myocytes. We examined the effects of pre-treatment with the PPARd agonist L- 165041 on neonatal cardiomyocytes exposed to a low, prosenes- cent dose of doxorubicin (Figs. 1 and 2). Since previous studies demonstrated that down regulation of TRF2 (a nuclear protein that governs chromosomal stability) is at the core of the pathways that lead to doxorubicin-induced premature senescence [4,25], we first examined the expression levels of TRF2. TRF2 maintains the telomere t-loop ‘‘end- capping’’ structure, thus preventing chromosome end-to-end fusion and chromosomal abnormalities. We found that doxorubi- cin down-regulates TRF2 (Fig. 1A), causes cell cycle alterations by increasing both the S phase and the hyperploid (.4N DNA) cell population (Fig. 1B), and also blocks cell proliferation (Fig. 1C). Pre-treatment with L-165041 prevented TRF2 downregulation, partially restored the cell cycle, and partially rescued the blocking of cell proliferation, (Figs. 1A, 1B, 1C). Doxorubicin 0.1 mM also induced a senescence-like phenotype characterized by enzymatic SA-b-gal activity expression at pH 6.0 (Figs. 2A and 2C) as well as by an increase in size and a change in shape of the cells which became flatter (Figs. 2B and 2C). These changes were accompanied by increases in both the length and density of the cytoplasmic actin fibers, as evaluated by phalloidin staining, and by the early loss of cytoplasmic membrane integrity, as documented by Annexin/Propidium double staining (Fig. 2C). In fact, 24 hours after a brief incubation with doxorubicin, the majority of annexin positive cells were also propidium positive. This double positivity is predictive of late death for mitotic catastrophe in cells treated with low doses of doxorubicin and is in contrast with the typical pattern of early-stage apoptosis that is present in cells treated with pro-apoptotic doses and which is characterized by annexin positivity and propidium negativity (Fig. 2E). Pre-treatment with the PPARd agonist L-165041 lowered the increase in SA-b-gal activity and significantly attenuated all the cell morphology and structural changes induced by the exposure to low (Figs. 2A, 2B, 2C) and high doses of doxorubicin (Fig. 2E). We also evaluated the effects of doxorubicin 0.1 mM on p16INK4A, a cyclin-dependent kinase inhibitor thought to be a Activated PPARd Protects from Senescence senescence–associated marker. Western blot analysis documented that doxorubicin induces changes in p16INK4A expression levelsand that L-165041 inhibits the increase of doxorubicin- induced p16INK4A (Fig. 2D). Even though L-165041 is thought to be a specific ligand for the delta isoform which is the most highly expressed in the heart, we were interested in evaluating whether the obtained results could be in part attributed to the other isoforms. To this aim, we performed a quantitative Real Time PCR analysis (RTPCR) which demonstrated that PPARd are much more highly expressed in neonatal cardiomyocytes than PPARa and PPARc. The cells were treated for two hours with L-165041 and analyzed at 4 and 22 hours after the treatment. At 22 hours, L-165041 decreased the transcription ratios of PPARa and PPARc and did not significantly increase the transcription ratio of PPARd (Fig. 3). After having carried out studies on neonatal cardiomyocytes, we performed experiments on H9c2 cells and obtained similar results (data not shown). H9c2 cells abundantly express the PPARd subtype, where PPARa is mildly expressed and PPARc is undetectable. Therefore, these cells represent a suitable model to investigate the role of PPARd activation without the potential interference of other PPAR subtypes [26]. In the following paragraphs we report data collected from the experiments on H9c2. MAPK-mediated Signal Transduction Pathways Play a Key Role in the Cytoprotective Effects of the PPARd Agonist L-165041 in H9c2 Cells In order to analyze which signaling pathways influence the protective effects exerted by L-165041, we blocked p38, JNK, Akt, ERK1/2 signaling by using the specific inhibitors SB203580, SP600125, Akt1/2 kinase inhibitor, and PD98059, respectively. Cells were assayed for SA-b-gal activity. Pre-incubation with the ERK inhibitor did not influence the protective effects of L-165041. In contrast, the effects of L-165041 on doxorubicin-induced SA-b- gal activity were attenuated by p38, JNK and Akt inhibition (Fig. 4). These results show the importance of p38, JNK and Akt signaling pathways in the cytoprotective effects of the PPARd agonist L-165041 against the pro-senescent effects of doxorubicin 0.1 mM in H9c2 cells. These findings prompted us to investigate the effects of pre- treatment with L-165041 on doxorubicin-induced MAPK activa- tion. To this aim, we first examined the effects of doxorubicin 0.1 mM given alone for 120 minutes. Figure 5 shows that doxorubicin induced an early increase in pp38, pJNK and pAkt levels, while an increase in pERK levels was observed 120 min after exposure to doxorubicin. We then examined the effects of L- 165041 given alone. We found that L-165041 increased pp38, pJNK, pERK, and pAkt levels. Finally, we examined the effects of the sequential treatment with L-165041for 2 hours followed by the treatment with doxorubicin for 2 hours. It is interesting to note that the doxorubicin-induced changes in MAPK and Akt activation were influenced by pre-treatment with the PPARd agonist L-165041. In fact, pre-treatment with L-165041 prevented the doxorubicin-induced increases in pJNK, pAkt and pERK levels and led to higher doxorubicin-induced pp38 levels as compared to the levels that were reached by doxorubicin alone. No changes in total MAPK and Akt protein levels were found (data not shown). PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e46126