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Intracellular accumulation of fatty acid metabolites in non-adipose tissues has been proposed to play an important role in the pathogenesis of diabetes mellitus. Moreover, lipotoxicity may contribute to cell death and cardiac dysfunction. Evidence is emerging that saturated fatty acids induce cell death through apoptosis and this effect is specific for palmitate and stearate. On the other hand, n-3 polyunsaturated fatty acids (PUFAs) have been implicated in the protection against cardiovascular diseases, cardiac ischemic damage and myocardial dysfunction. In the present study we show- that supplementation with the n-3 PUFA- eicosapentaenoic acid (EPA)- to culture medium of H9c2 rat cardiomyoblasts protects cells against palmitate-induced apoptosis. Experiments were performed by using H9c2 rat ventricular cardiomyoblasts. Apoptosis was detected by trypan blue exclusion, MTT or caspase activity assays. Gene expression was investigated by Real Time PCR. Signal transduction pathways were investigated by western blotting. Our results indicate that palmitate induces apoptosis in a dose dependent manner. In particular, an approximately 4-fold increase in caspase activity was observed after 24 h of treatment with 500 M palmitate. The incubation with 60 M EPA inhibited palmitate-induced apoptosis of H9c2 cells. Preliminary results indicate that the anti-apoptotic effect of EPA could be mediated by the modulation of the transcriptional peroxisome proliferator–activated receptor  coactivator-1 considered a master regulator of myocardial energy metabolism in diverse pathophysiological conditions, and may involve the rapamycin-insensitive companion of mammalian target of rapamycin (mTOR) (Rictor), a key member of mTOR complex-2 that promotes cellular survival.

The responses of AMP-Activated Protein Kinase (AMPK) and Ornithine decarboxylase (ODC) to isoproterenol have been examined in H9c2 cardiomyoblasts, a cell model of cardiac hypertrophy. AMPK represents the link between cell growth and energy availability whereas ODC, the key enzyme in polyamine biosynthesis, is essential for all growth processes including the development of cardiac hypertrophy. Isoproterenol rapidly induced ODC activity in H9c2 cardiomyoblasts by promoting the synthesis of the enzyme protein. The increase in enzyme activity became significant between 15 and 30 minutes after the treatment. At the same time, isoproterenol stimulated the phosphorylation of AMPKα catalytic subunits (Thr172), which could be observed as soon as 15 minutes. Downregulation of both α1 and α2 isoforms of the AMPK catalytic subunit by siRNA to knockdown AMPK enzymatic activity, led to superinduction of ODC in isoproterenol-treated cardiomyoblasts. Downregulation of AMPKα increased ODC activity even in cells treated with other adrenergic agonists and in untreated cells, but was without effect when ODC was induced by hypoxia, suggesting that ODC modulation by AMPK is not a general event, but is stimulus-dependent. In conclusion, isoproterenol quickly activates in H9c2 cardiomyoblasts two events that seem to contrast one another. The first one, an increase in ODC activity, is linked to cell growth, whereas the second, AMPK activation, is a homeostatic mechanism that negatively modulates the first. The modulation of ODC activity by AMPK can represent a self-regulating mechanism that may contribute to control the development of heart hypertrophy.

Purpose: Polyamines are naturally occurring, positively charged polycations able to control several cellular processes in different cell types, by interacting with negatively charged compounds and structures within the living cell. Several pieces of evidence from the literature point at a role of polyamines in promoting chondrocyte differentiation. Hence, we investigated the effects of exogenously added spermine or spermidine in chondrocyte maturation recapitulated in 3D micromass cultures, to tease out the effects on gene and protein expression of key chondrogenesis regulatory transcription factors, markers and effectors, as well as their posttranscriptional regulation. Methods: Micromasses were seeded in control medium or in the presence of either 5 µM spermine or spermidine. We evaluated the effects on molecular markers of chondrocyte differentiation at the level of gene (real time PCR) and protein (western blot and immunohistochemistry) expression as well as the effects on extracellular matrix deposition and remodeling, and caspase activation and mineralization. The subcellular localization of RUNX-2 in stimulated samples was investigated with confocal microscopy. Results: Both spermine and spermidine were able to increase the rate and the extent of chondrogenesis, with enhanced collagen 2 deposition and remodeling with downstream generation of collagen 2 bioactive peptides. These were able to promote nuclear localization of RUNX-2, the pivotal transcription factor in chondrocyte hypertrophy and osteoblast generation. Indeed, samples stimulated with polyamines showed an enhanced mineralization, along with increased caspase activity, indicating increased chondrocyte terminal differentiation. Conclusions: Polyamine pathway can represent a potential target to control and correct chondrocyte inappropriate maturation in osteoarthritis.

Introduction. Intracellular accumulation of fatty acid metabolites in nonadipose tissues has been proposed to play an important role in the pathogenesis of diabetes mellitus. Moreover, lipotoxicity may contribute to cell death and cardiac dysfunction. Evidence is emerging that saturated fatty acids induce cell death through apoptosis and this effect is specific for palmitate and stearate. n-3 polyunsaturated fatty acids (PUFAs) have been implicated in the protection against cardiovascular diseases, cardiac ischemic damage and myocardial dysfunction. In the present study we showed that supplementation with the n-3 PUFAs eicosapentaenoic acid (EPA), to culture medium of H9c2 rat cardiomyoblasts protects cell against palmitate-induced apoptosis. Methods: Experiments were performed by using H9c2 rat ventricular cardiomyoblasts. Apoptosis was detected by trypan blue exclusion, MTT or caspase activity assays. Gene expression was investigated by Real Time PCR. Signal transduction pathways was investigated by western blotting. Results: Our results indicate that palmitate induces apoptosis in a dose dependent manner. In particular at 500 M dose it increased caspase activity by about 4 fold after 24 h of treatment. The incubation with 60 M EPA inhibited palmitate-induced apoptosis of H9c2. Preliminary results indicate that the anti-apoptotic effect of EPA could be mediated by the modulation of the transcriptional peroxisome proliferator–activated receptor  coactivator-1 considered a master regulator of myocardial energy metabolism in diverse pathophysiological conditions, as well as activation of SIRT1. Interestlingy, the protective effect of EPA on cell viability seems also involve the rapamycin-insensitive companion of mammalian target of rapamycin (mTOR) (Rictor) a key member of mTOR complex-2 that promotes cellular survival.

Introduction. Intracellular accumulation of fatty acid metabolites in nonadipose tissues has been proposed to play an important role in the pathogenesis of diabetes mellitus. Moreover, lipotoxicity may contribute to cell death and cardiac dysfunction. Evidence is emerging that saturated fatty acids induce cell death through apoptosis and this effect is specific for palmitate and stearate. n-3 polyunsaturated fatty acids (PUFAs) have been implicated in the protection against cardiovascular diseases, cardiac ischemic damage and myocardial dysfunction. In the present study we showed that supplementation with the n-3 PUFAs eicosapentaenoic acid (EPA), to culture medium of H9c2 rat cardiomyoblasts protects cell against palmitate-induced apoptosis. Methods: Experiments were performed by using H9c2 rat ventricular cardiomyoblasts. Apoptosis was detected by trypan blue exclusion, MTT or caspase activity assays. Gene expression was investigated by Real Time PCR. Signal transduction pathways was investigated by western blotting. Results: Our results indicate that palmitate induces apoptosis in a dose dependent manner. In particular at 500 M dose it increased caspase activity by about 4 fold after 24 h of treatment. The incubation with 60 M EPA inhibited palmitate-induced apoptosis of H9c2. Preliminary results indicate that the anti-apoptotic effect of EPA could be mediated by the modulation of the transcriptional peroxisome proliferator–activated receptor  coactivator-1 considered a master regulator of myocardial energy metabolism in diverse pathophysiological conditions, and may involve the rapamycin-insensitive companion of mammalian target of rapamycin (mTOR) (Rictor) a key member of mTOR complex-2 that promotes cellular survival

Adipose derived stem cells (ADSCs) are mesenchymal stem cells that can be easly isolated from lipoaspirates. They show multilineage differentiative capacities, including the cardiac phenotype. Therefore they represent a promise for regenerative medicine (Zuk 2001), for the treatment of those diseases affecting organs with extremely low potential of repair and regeneration, such as the heart. The knowledge of the molecular mechanisms that control development and differentiation - such as epigenetic DNA promoter methylation at CpG sites and histone modifications inducing chromatin remodelling and changes in DNA accessibility (Bernstein 2007) - will help to understand their potential in this regard. Aim of this study was to evaluate the epigenetic status of cardiac specific transcription factors,such as GATA-4, MEF-2C and Nkx2.5 (Brand 2003) in undifferentiated human ADSCs. The analysis of DNA promoter methylation, using Methylation Specific PCR (MSP), revealed the unmethylated status of the promoters of the above cardiac master genes. This finding suggested that ADSCs are not prevented to differentiate towards the cardiac phenotype by DNA methylation, as otherwise reported for myogenic genes (Sørensen 2010). Chromatin immunoprecipitation (ChIP) showed association of a well known transcriptional repressive mark [trimethylation of Lys27 in H3 (H3K27me3)] with GATA-4 and Nkx2.5 promoters. On the other hand, an enrichment in acetylation of histone 3 (acH3), hallmark of transcriptional potential, was detected for MEF-2C promoter. These results are in agreement with those of the gene expression analysis, showing the presence of only MEF-2C transcript in total mRNA extracted from undifferentiated human ADSCs of three different donors. In conclusion our results suggest that the epigenetic signature of ADSCs represents a level for intervention to address their differentiation towards the cardiac lineage.

Heart failure, a major cause of morbidity and mortality in western countries, is the final outcome of a variety of cardiovascular pathologies, such as myocardial infarction and sustained overload of the heart. Myocardial hypertrophy in response to pathophysiological signals is a predictor of progressive cardiac disease that can result in heart failure. Pioneer studies from this and other laboratories have established a link between the aliphatic polyamines and cardiac hypertrophy, as it emerged that heart polyamine biosynthesis was rapidly stimulated in a number of animal models of cardiac hypertrohy. Recent studies from our laboratory have focused on the involvement of polyamines in various aspects of the remodeling occurring in cardiac diseases, such as growth, death and differentiation of cardiac cells. Polyamine metabolism and effects have been investigated even in relation to some extra- and intracellular signals and biochemical mechanisms involved in these pathophysiological responses. These lines of research have employed both cellular and animal models, comprising cultures of embryo, neonatal and adult cardiomyocytes, cardiomyoblast cell lines and stem cells, as well as wild-type and transgenic animals. In the present paper we have reviewed these studies.

Desmin constitutes the intermediate filaments (IFs) cytoskeleton in cardiac myocytes. Novel roles of the IFs, including the formation of toxic aggregates similar to those found in neurodegenerative diseases, have been recently linked to heart failure (HF). Post-translational modifications (PTMs) control desmin assembly and immunoreactivity, in vitro. Therefore PTMs likely play a role in desmin aggregation and could explain the controversy on desmin immuno-based quantitation in human HF. In the present study we explore the occurrence of desmin PTMs in canine and human HF and control specimens. We found a significant increase of three desmin forms, compatible with the mono- and bi-phosphorylated forms and a protein fragment (all 2-fold, p0.05), in a canine pacing model of dyssynchronous HF (DHF) compared to non-paced shams, using difference gel electrophoresis (DIGE). Importantly, these alterations were reverted by treatment (bi-ventricular or bi-V pacing for resynchronization). Two novel phosphorylation sites (Ser-27 and Ser-31) were identified by tandem mass spectrometry (MS2). Phosphorylated desmin species were also increased in the heart of class III NYHA patients when compared with healthy donors and phosphorylation of Ser-27 was observed, for the first time, in humans by MS2 and confirmed by multiple reaction monitoring. Finally, desmin-positive oligomers ( three bands, ~50, ~200 and ~600 kDa) were increased in DHF hearts compared to controls by blue-native gel electrophoresis (30%, p0.02). This is the first characterization of desmin phosphorylation in the heart, in vivo with identification of novel sites in both animal models and humans. We show that the levels of PTM-forms of desmin are modified in canine and human HF. Finally, these alterations correlate with the formation of oligomers and are reversed by bi-V pacing in dogs, suggesting that PTMs may regulate desmin-induced toxicity in HF.

Introduction - A good inverse correlation between progression-free survival and tumor levels of ornitine decarboxylase (ODC) was found in patients with anaplastic gliomas treated with the specific irreversible inhibitor of ODC difluoromethylornithine (DFMO) [Levin et al. 2007]. This observation fits well with a number of evidences that polyamine-mediated changes in gene expression may contribute to tumour development/progression. Moreover, it has recently shown that polyamines can exert both positive and negative influence on the function of chromatin modifying histone acetyltransferase (HAT) and deacetylase (HDAC) enzymes, suggesting that polyamines upregulate some genes while downregulating others via epigenetic modifications of the structure of chromatin [Hobbs et al. 2003, Wei et al. 2007]. Material and Methods - Here we investigated, by western blotting, how DFMO treatment (0.5 - 2 mM up to 24 h) affects (a) the acetylation status of histones H3 and H4 and (b) the concurrent level of expression of O6-methylguanine-DNA methyltransferase (MGMT) – a tumor suppressor gene whose transcription is known to be regulated via epigenetic modifications – in glioblastoma cells grown in vitro. Results and Discussion – Inhibiting ODC resulted in a dose- and time-dependent increase in the acetylation level of both H3 and H4 histones, with respect to untreated control. A concurrent upregulation of MGMT expression was also scored. These findings suggests that polyamines might contribute to tumour development/progression promoting chromatin condensation, thus transcriptional silencing, at the level of oncosuppressor gene sequences. In order to strengthen these results we are evaluating specific histone marks on MGMT promoter using chromatin immunoprecipitation.