SIRT3-mediated inhibition of FOS through histone H3 deacetylation prevents cardiac fibrosis and inflammation.

Sirtuin 3 (SIRT3) is a deacetylase that modulates proteins that control metabolism and protects against oxidative stress. Modulation of SIRT3 activity has been proposed as a promising therapeutic target for ameliorating metabolic diseases and associated cardiac disturbances. In this study, we investigated the role of SIRT3 in inflammation and fibrosis in the heart using male mice with constitutive and systemic deletion of SIRT3 and human cardiac AC16 cells. SIRT3 knockout mice showed cardiac fibrosis and inflammation that was characterized by augmented transcriptional activity of AP-1. Consistent with this, SIRT3 overexpression in human and neonatal rat cardiomyocytes partially prevented the inflammatory and profibrotic response induced by TNF-α. Notably, these effects were associated with a decrease in the mRNA and protein levels of FOS and the DNA-binding activity of AP-1. Finally, we demonstrated that SIRT3 inhibits FOS transcription through specific histone H3 lysine K27 deacetylation at its promoter. These findings highlight an important function of SIRT3 in mediating the often intricate profibrotic and proinflammatory responses of cardiac cells through the modulation of the FOS/AP-1 pathway. Since fibrosis and inflammation are crucial in the progression of cardiac hypertrophy, heart failure, and diabetic cardiomyopathy, our results point to SIRT3 as a potential target for treating these diseases.

Targeted cytoplasmic irradiation and autophagy.

The effect of ionizing irradiation on cytoplasmic organelles is often underestimated because the general dogma considers direct DNA damage in the nuclei to be the primary cause of radiation induced toxicity. Using a precision microbeam irradiator, we examined the changes in mitochondrial dynamics and functions triggered by targeted cytoplasmic irradiation with α-particles. Mitochondrial dysfunction induced by targeted cytoplasmic irradiation led to activation of autophagy, which degraded dysfunctional mitochondria in order to maintain cellular energy homeostasis. The activation of autophagy was cytoplasmic irradiation-specific and was not detected in nuclear irradiated cells. This autophagic process was oxyradical-dependent and required the activity of the mitochondrial fission protein dynamin related protein 1 (DRP1). The resultant mitochondrial fission induced phosphorylation of AMP activated protein kinase (AMPK) which leads to further activation of the extracellular signal-related kinase (ERK) 1/2 with concomitant inhibition of the mammalian target of rapamycin (mTOR) to initiate autophagy. Inhibition of autophagy resulted in delayed DNA damage repair and decreased cell viability, which supports the cytoprotective function of autophagy. Our results reveal a novel mechanism in which dysfunctional mitochondria are degraded by autophagy in an attempt to protect cells from toxic effects of targeted cytoplasmic radiation.

17ß-Estradiol-induced interaction of estrogen receptor α and human atrial essential myosin light chain modulates cardiac contractile function.

Chronic increased workload of the human heart causes ventricular hypertrophy, re-expression of the atrial essential myosin light chain (hALC-1), and improved contractile function. Although hALC-1 is an important positive inotropic regulator of the human heart, little is known about its regulation. Therefore, we investigated the role of the sex hormone 17ß-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function. We showed that E2 attenuated hALC-1 expression in human atrial tissues of both sexes and in human ventricular AC16 cells. E2 induced the nuclear translocation of estrogen receptor alpha (ERα) and hALC-1 in AC16 cells, where they cooperatively regulate the transcriptional activity of hALC-1 gene promoter. E2-activated ERα required the estrogen response element (ERE) motif within the hALC-1 gene promoter to reduce its transcriptional activity (vehicle: 15.55 ± 4.80 vs. E2: 6.51 ± 3.69; ~2 fold). This inhibitory effect was potentiated in the presence of hALC-1 (vehicle: 11.13 ± 3.66 vs. E2: 2.18 ± 1.10; ~5 fold), and thus, hALC-1 acts as a co-repressor of ERα-mediated transcription. Yeast two-hybrid screening of a human heart cDNA library revealed that ERα interacts physically with hALC-1 in the presence of E2. This interaction was confirmed by Co-Immunoprecipitation and immunofluorescence in human atrium. As a further novel effect, we showed that chronic E2-treatment of adult mouse cardiomyocytes overexpressing hALC-1 resulted in reduced cell-shortening amplitude and twitching kinetics of these cells independent of Ca2+ activation levels. Together, our data showed that the expression of hALC-1 gene is, at least partly, regulated by E2/ERα, while hALC-1 acts as a co-repressor. The inotropic effect of hALC-1 overexpression in cardiomyocytes can be significantly repressed by E2.

miR-146a targets Fos expression in human cardiac cells.

miR-146a is a microRNA whose transcript levels are induced in the heart upon activation of NF-κB, a transcription factor induced by pro-inflammatory molecules (such as TNF-α) that is strongly related to the pathogenesis of cardiac disorders. The main goal of this study consisted of studying new roles of miR-146a in cardiac pathological processes caused by the pro-inflammatory cytokine TNF-α. Our results demonstrate that miR-146a transcript levels were sharply increased in cardiac ventricular tissue of transgenic mice with specific overexpression of TNF-α in the heart, and also in a cardiomyocyte cell line of human origin (AC16) exposed to TNF-α. Among all the in silico predicted miR-146a target genes, Fos mRNA and protein levels notably decreased after TNF-α treatment or miR-146a overexpression. These changes correlated with a diminution in the DNA-binding activity of AP-1, the Fos-containing transcription factor complex. Interestingly, AP-1 inhibition was accompanied by a reduction in matrix metalloproteinase (MMP)-9 mRNA levels in human cardiac cells. The specific regulation of this MMP by miR-146a was further confirmed at the secretion and enzymatic activity levels, as well as after anti-miR-mediated miR-146a inhibition. The results reported here demonstrate that Fos is a direct target of miR-146a activity and that downregulation of the Fos-AP-1 pathway by miR-146a has the capacity to inhibit MMP-9 activity. Given that MMP-9 is an AP-1 target gene involved in cardiac remodeling, myocardial dysfunction and progression of heart failure, these findings suggest that miR-146a might be a new and promising therapeutic tool for treating cardiac disorders associated with enhanced inflammation in the heart.

Tetramethylpyrazine (TMP) protects against sodium arsenite-induced nephrotoxicity by suppressing ROS production, mitochondrial dysfunction, pro-inflammatory signaling pathways and programed cell death.

Although kidney is a target organ of arsenic cytotoxicity, the underlying mechanisms of arsenic-induced nephrotoxicity remain poorly understood. As tetramethylpyrazine (TMP) has recently been found to be a renal protectant in multiple kidney injuries, we hypothesize that TMP could suppress arsenic nephrotoxicity. In this study, human renal proximal tubular epithelial cell line HK-2 was used to elucidate the precise mechanisms of arsenic nephrotoxicity as well as the protective mechanism of TMP in these cells. Sodium arsenite exposure dramatically increased cellular reactive oxygen species (ROS) production, decreased levels of cellular glutathione (GSH), decreased cytochrome c oxidase activity and mitochondrial membrane potential, which indicated mitochondrial dysfunction. On the other hand, sodium arsenite activated pro-inflammatory signals, including ß-catenin, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor alpha and cyclooxygenase-2 (COX-2). Small molecule inhibitors of NF-κB and p38 MAPK blocked arsenic-induced COX-2 expression, suggesting arsenic-induced COX-2 up-regulation was NF-κB- and p38 MAPK-dependent. Finally, sodium arsenite induced autophagy in HK-2 cells at early phase (6 h) and the subsequent apoptosis at 24 h. Treatment by TMP or by the antioxidant N-acetylcysteine decreased arsenic-induced ROS production, enhanced GSH levels, prevented mitochondria dysfunction and suppressed the activation of pro-inflammatory signals and the development of autophagy and apoptosis. Our results suggested that TMP may be used as a new potential therapeutic agent to prevent arsenic-induced nephrotoxicity by suppressing these pathological processes.

Mitochondria regulate DNA damage and genomic instability induced by high LET radiation.

High linear energy transfer (LET) radiation including α particles and heavy ions is the major type of radiation find in space and is considered a potential health risk for astronauts. Even though the chance that these high LET particles traversing through the cytoplasm of cells is higher than that through the nuclei, the contribution of targeted cytoplasmic irradiation, to the induction of genomic instability and other chromosomal damages induced by high LET radiation is not known. In the present study, we investigated whether mitochondria are the potential cytoplasmic target of high LET radiation in mediating cellular damage using a mitochondrial DNA (mtDNA) depleted (ρ0) human small airway epithelial (SAE) cell model and a precision charged particle microbeam with a beam width of merely one micron. Targeted cytoplasmic irradiation by high LET α particles induced DNA oxidative damage and double strand breaks in wild type ρ+ SAE cells. Furthermore, there was a significant increase in autophagy, micronuclei, which is an indication of genomic instability, together with the activation of nuclear factor kappa-B (NF-κB) and mitochondrial inducible nitric oxide synthase (iNOS) signaling pathways in ρ+ SAE cells. In contrast, ρ0 SAE cells exhibited a significantly lower response to these same endpoints examined after cytoplasmic irradiation with high LET α particles. The results indicate that mitochondria are essential in mediating cytoplasmic radiation induced genotoxic damage in mammalian cells. Furthermore, the findings may shed some light in the design of countermeasures for space radiation.

[PPARα attenuates palmitate-induced endoplasmic reticulum stress in human cardiac cells by enhancing AMPK activity]. / PPARα atenúa el estrés del retículo endoplasmático inducido por palmitato en células cardíacas humanas por medio de la inducción de la actividad AMPK.

INTRODUCTION: Endoplasmic reticulum (ER) stress has been linked to several cardiovascular diseases, such as atherosclerosis, heart failure and cardiac hypertrophy. ER stress impairs insulin signalling, thus contributing to the development of insulin resistance and diabetes. Since several studies have reported that PPARα may inhibit ER stress, the main aim of this study consisted in investigating whether activation of this nuclear receptor is able to prevent lipid-induced ER stress in cardiac cells, as well as studying the mechanisms involved. METHODS: A cardiomyocyte cell line of human origin, AC16, was treated with palmitate in the presence or absence of several AMPK and PPARα pharmacological agonists and antagonists. For the in vivo studies, wild-type male mice were fed a standard diet, or a high-fat diet (HFD), for two months. At the end of the experiments, several ER stress markers were assessed in cardiac cells or in the mice hearts, using real-time RT-PCR and Western-blot analyses. RESULTS: The results demonstrate that both palmitate and the HFD induced ER stress in cardiac cells, since they upregulated the expression (ATF3, BiP/GRP78 and CHOP), splicing (sXBP1), and phosphorylation (IRE-1α and eIF2α) of several ER stress markers. Interestingly, treatment with the PPARα agonist Wy-14,643 prevented an increase in the majority of these ER stress markers in human cardiac cells by means of AMPK activation. CONCLUSION: These data indicate that PPARα activation by Wy-14,643 might be useful to prevent the harmful effects of ER stress and associated cardiovascular diseases in obese patients, and even during diabetic cardiomyopathy, by enhancing AMPK activity.

PPARß/δ attenuates palmitate-induced endoplasmic reticulum stress and induces autophagic markers in human cardiac cells.

BACKGROUND: Chronic endoplasmic reticulum (ER) stress contributes to the apoptotic cell death in the myocardium, thereby playing a critical role in the development of cardiomyopathy. ER stress has been reported to be induced after high-fat diet feeding in mice and also after saturated fatty acid treatment in vitro. Therefore, since several studies have shown that peroxisome proliferator-activated receptor (PPAR)ß/δ inhibits ER stress, the main goal of this study consisted in investigating whether activation of this nuclear receptor was able to prevent lipid-induced ER stress in cardiac cells. METHODS AND RESULTS: Wild-type and transgenic mice with reduced PPARß/δ expression were fed a standard diet or a high-fat diet for two months. For in vitro studies, a cardiomyocyte cell line of human origin, AC16, was treated with palmitate and the PPARß/δ agonist GW501516. Our results demonstrate that palmitate induced ER stress in AC16 cells, a fact which was prevented after PPARß/δ activation with GW501516. Interestingly, the effect of GW501516 on ER stress occurred in an AMPK-independent manner. The most striking result of this study is that GW501516 treatment also upregulated the protein levels of beclin 1 and LC3II, two well-known markers of autophagy. In accordance with this, feeding on a high-fat diet or suppression of PPARß/δ in knockout mice induced ER stress in the heart. Moreover, PPARß/δ knockout mice also displayed a reduction in autophagic markers. CONCLUSION: Our data indicate that PPARß/δ activation might be useful to prevent the harmful effects of ER stress induced by saturated fatty acids in the heart by inducing autophagy.

Sex differences in exercise-induced physiological myocardial hypertrophy are modulated by oestrogen receptor beta.

AIMS: Oestrogen receptor alpha (ERα) and beta (ERß) are involved in the regulation of pathological myocardial hypertrophy (MH). We hypothesize that both ER are also involved in physiological MH. Therefore, we investigated the role of ER in exercise-induced physiological MH in loss-of-function models and studied potential mechanisms of action. METHODS AND RESULTS: We performed 1 and 8 weeks of voluntary cage wheel running (VCR) with male and female C57BL/6J wild-type (WT), ERα- and ERß-deleted mice. In line with other studies, female WT mice ran more than males (P ≤ 0.001). After 8 weeks of VCR, both sexes showed an increase in left ventricular mass (females: P ≤ 0.01 and males: P ≤ 0.05) with more pronounced MH in females (P < 0.05). As previously shown, female ERα-deleted mice run less than female WT mice (P ≤ 0.001). ERß-deleted mice showed similar running performance as WT mice (females vs. male: P ≤ 0.001), but did not develop MH. Only female WT mice showed an increase in phosphorylation of serine/threonine kinase (AKT), ERK1/2, p38-mitogen-activated protein kinase (MAPK), and ribosomal protein s6, as well as an increase in the expression of key regulators of mitochondrial function and mitochondrial respiratory chain proteins (complexes I, III, and V) after VCR. However, ERß deletion abolished all observed sex differences. Mitochondrial remodelling occurred in female WT-VCR mice, but not in female ERß-deleted mice. CONCLUSION: The sex-specific response of the heart to exercise is modulated by ERß. The greater increase in physiological MH in females is mediated by induction of AKT signalling, MAPK pathways, protein synthesis, and mitochondrial adaptation via ERß.

Cytoplasmic irradiation results in mitochondrial dysfunction and DRP1-dependent mitochondrial fission.

Direct DNA damage is often considered the primary cause of cancer in patients exposed to ionizing radiation or environmental carcinogens. Although mitochondria are known to play an important role in radiation-induced cellular response, the mechanisms by which cytoplasmic stimuli modulate mitochondrial dynamics and functions are largely unknown. In the present study, we examined changes in mitochondrial dynamics and functions triggered by α particle damage to the mitochondria in human small airway epithelial cells, using a precision microbeam irradiator with a beam width of 1 µm. Targeted cytoplasmic irradiation using this device resulted in mitochondrial fragmentation and a reduction of cytochrome c oxidase and succinate dehydrogenase activity, when compared with nonirradiated controls, suggesting a reduction in respiratory chain function. In addition, mitochondrial fragmentation or fission was associated with increased expression of the dynamin-like protein DRP1, which promotes mitochondrial fission. DRP1 inhibition by the drug mdivi-1 prevented radiation-induced mitochondrial fission, but respiratory chain function in mitochondria inhibited by radiation persisted for 12 hours. Irradiated cells also showed an increase in mitochondria-derived superoxide that could be quenched by dimethyl sulfoxide. Taken together, our results provide a mechanistic explanation for the extranuclear, nontargeted effects of ionizing radiation.

Resveratrol induces nuclear factor-κB activity in human cardiac cells.

BACKGROUND: Resveratrol is a grape polyphenol that prevents cardiac hypertrophy and protects the heart from ischemic injury, metabolic dysregulation, and inflammatory processes in several murine models. METHODS AND RESULTS: The aim of this study was to investigate the effects of resveratrol on the inflammatory processes in human cardiac AC16 cells in order to gain a better understanding of its cardioprotective mechanisms in the human heart. Resveratrol induced the DNA-binding activity of the pro-inflammatory transcription factor NF-κB in AC16 cells, and exacerbated the increase caused by tumor necrosis factor-α (TNF-α). In accordance with this, resveratrol increased the expression of the pro-inflammatory genes ICAM-1 (intercellular adhesion molecule-1) and TNF-α. In contrast, resveratrol decreased the expression of pro-inflammatory genes IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1). Likewise, resveratrol also induced inflammation in rat neonatal cardiomyocytes, and in the heart of mice fed a standard chow diet supplemented with resveratrol (1g/kg diet) for four months. Western-blot analyses revealed that NF-κB p65 subunit levels were upregulated in an IκB-dependent manner in the nuclei of resveratrol-treated human cardiac cells. Finally, resveratrol activated the signal transducer and activator of transcription 3 (STAT3) signaling and induced the expression of its anti-apoptotic downstream effector Bcl-xL, both involved in the cardioprotective survival activating factor enhancement (SAFE) pathway. CONCLUSIONS: Resveratrol enhanced NF-κB activity in human and murine cardiac cells, in a process that coincided with the activation of STAT3 and anti-apoptotic downstream effectors. Therefore, activation of the SAFE pathway by resveratrol might be involved in the cardioprotective effects of this compound.

17ß-Estradiol-induced interaction of ERα with NPPA regulates gene expression in cardiomyocytes.

AIMS: 17ß-Oestradiol (E2) and its receptors (ERα and ERß) are important regulators of physiological and pathological processes in the cardiovascular system. ER act in concert with other regulatory factors mediating oestrogenic effects. However, the underlying mechanisms modulating ER transcriptional activity are not fully elucidated. To gain better understanding of E2-induced ERα action in the human heart, we aimed to identify and functionally analyse interaction partners of ERα. METHODS AND RESULTS: Using yeast two-hybrid assays with a human heart cDNA library, we identified atrial natriuretic peptide precursor A (NPPA), a well-known cardiac hypertrophy marker, as a novel ERα interaction partner interacting in an E2-dependent manner. Mutation analyses and immunofluorescence data indicated that the LXXLL motif within NPPA is necessary for its E2-induced interaction with ERα, its action as a co-repressor of ERα, and its translocation into the nucleus of human and rat cardiomyocytes. Expression analysis and chromatin immunoprecipitation assays in a human left ventricular cardiomyocyte cell line, AC16, showed that NPPA interacts with E2/ERα, suppressing the transcriptional activity of ERα on E2-target genes, such as NPPA, connexin43, αactinin-2, nuclear factor of activated T-cells, and collagens I and III. CONCLUSION: We characterize for the first time an E2-regulated interaction of NPPA with ERα in cardiomyocytes, that may be crucial in physiological and/or pathological cardiac processes, thereby representing a potential therapeutic target.

Cardiomyocyte specific deficiency of serine palmitoyltransferase subunit 2 reduces ceramide but leads to cardiac dysfunction.

The role of serine palmitoyltransferase (SPT) and de novo ceramide biosynthesis in cardiac ceramide and sphingomyelin metabolism is unclear. To determine whether the de novo synthetic pathways, rather than ceramide uptake from circulating lipoproteins, is important for heart ceramide levels, we created cardiomyocyte-specific deficiency of Sptlc2, a subunit of SPT. Heart-specific Sptlc2-deficient (hSptlc2 KO) mice had a >35% reduction in ceramide, which was limited to C18:0 and very long chain ceramides. Sphingomyelinase expression, and levels of sphingomyelin and diacylglycerol were unchanged. But surprisingly phospholipids and acyl CoAs contained increased saturated long chain fatty acids. hSptlc2 KO mice had decreased fractional shortening and thinning of the cardiac wall. While the genes regulating glucose and fatty acid metabolism were not changed, expression of cardiac failure markers and the genes involved in the formation of extracellular matrices were up-regulated in hSptlc2 KO hearts. In addition, ER-stress markers were up-regulated leading to increased apoptosis. These results suggest that Sptlc2-mediated de novo ceramide synthesis is an essential source of C18:0 and very long chain, but not of shorter chain, ceramides in the heart. Changes in heart lipids other than ceramide levels lead to cardiac toxicity.

Mitochondria-derived reactive intermediate species mediate asbestos-induced genotoxicity and oxidative stress-responsive signaling pathways.

BACKGROUND: The incidence of asbestos-induced human cancers is increasing worldwide, and considerable evidence suggests that reactive oxygen species (ROS) are important mediators of these diseases. Our previous studies suggested that mitochondria might be involved in the initiation of oxidative stress in asbestos-exposed mammalian cells. OBJECTIVE: We investigated whether mitochondria are a potential cytoplasmic target of asbestos using a mitochondrial DNA-depleted (ρ(0)) human small airway epithelial (SAE) cell model: ρ(0) SAE cells lack the capacity to produce mitochondrial ROS. METHODS: We examined nuclear DNA damage, micronuclei (MN), intracellular ROS production, and the expression of inflammation-related nuclear genes in both parental and ρ(0) SAE cells in response to asbestos treatment. RESULTS: Asbestos induced a dose-dependent increase in nuclear DNA oxidative damage and MN in SAE cells. Furthermore, there was a significant increase in intracellular oxidant production and activation of genes involved in nuclear factor κB and proinflammatory signaling pathways in SAE cells. In contrast, the effects of asbestos were minimal in ρ(0) SAE cells. CONCLUSIONS: Mitochondria are a major cytoplasmic target of asbestos. Asbestos may initiate mitochondria-associated ROS, which mediate asbestos-induced nuclear mutagenic events and inflammatory signaling pathways in exposed cells. These data provide new insights into the molecular mechanisms of asbestos-induced genotoxicity.

Defects of mtDNA replication impaired mitochondrial biogenesis during Trypanosoma cruzi infection in human cardiomyocytes and chagasic patients: the role of Nrf1/2 and antioxidant response.

BACKGROUND: Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)-regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. METHODS AND RESULTS: We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi-infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)-regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. CONCLUSIONS: The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi-infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for replication and gene expression in Chagas disease.

The interplay between NF-kappaB and E2F1 coordinately regulates inflammation and metabolism in human cardiac cells.

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARß/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARß/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription.

PPARß/δ activation blocks lipid-induced inflammatory pathways in mouse heart and human cardiac cells.

Owing to its high fat content, the classical Western diet has a range of adverse effects on the heart, including enhanced inflammation, hypertrophy, and contractile dysfunction. Proinflammatory factors secreted by cardiac cells, which are under the transcriptional control of nuclear factor-κB (NF-κB), may contribute to heart failure and dilated cardiomyopathy. The underlying mechanisms are complex, since they are linked to systemic metabolic abnormalities and changes in cardiomyocyte phenotype. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate metabolism and are capable of limiting myocardial inflammation and hypertrophy via inhibition of NF-κB. Since PPARß/δ is the most prevalent PPAR isoform in the heart, we analyzed the effects of the PPARß/δ agonist GW501516 on inflammatory parameters. A high-fat diet induced the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6, and enhanced the activity of NF-κB in the heart of mice. GW501516 abrogated this enhanced proinflammatory profile. Similar results were obtained when human cardiac AC16 cells exposed to palmitate were coincubated with GW501516. PPARß/δ activation by GW501516 enhanced the physical interaction between PPARß/δ and p65, which suggests that this mechanism may also interfere NF-κB transactivation capacity in the heart. GW501516-induced PPARß/δ activation can attenuate the inflammatory response induced in human cardiac AC16 cells exposed to the saturated fatty acid palmitate and in mice fed a high-fat diet. This is relevant, especially taking into account that PPARß/δ has been postulated as a potential target in the treatment of obesity and the insulin resistance state.

Neutral lipid storage disease with subclinical myopathy due to a retrotransposal insertion in the PNPLA2 gene.

An 18-year-old girl referred to a rheumatologist with malar flush and Gottran papules was found to have a markedly elevated serum CK. She was a good student and an avid ballet dancer. A muscle biopsy showed massive triglyceride storage, which was also found in peripheral blood granulocytes (Jordan anomaly) and cultured skin fibroblasts. Assessment using computerized dynamometry and cycle ergometry showed normal strength and muscle energetics, but proton spectroscopy revealed severe triglyceride accumulation in both skeletal and cardiac muscle. Sequencing of PNPLA2, the gene responsible for neutral lipid storage disease with myopathy (NLSDM), revealed a retrotransposal insertion of about 1.8kb in exon 3 that abrogates transcription of PNPLA2. The sequences of CGI-58, the gene responsible for Chanarin-Dorfman syndrome (CDS), another multisystem triglyceride storage disease, and of two genes encoding lipid droplets-associated proteins, perilipin A and adipophilin, were normal. This case shows that NLSDM can be a transposon-associated disease and that massive lipid storage in muscle can present as asymptomatic hyperCKemia.

The p65 subunit of NF-kappaB binds to PGC-1alpha, linking inflammation and metabolic disturbances in cardiac cells.

AIMS: Nuclear factor-kappaB (NF-kappaB) is a transcription factor induced by a wide range of stimuli, including hyperglycaemia and pro-inflammatory cytokines. It is associated with cardiac hypertrophy and heart failure. It was previously reported that the NF-kappaB-mediated inhibition of proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) might explain the shift in glucose metabolism during cardiac pathological processes induced by pro-inflammatory stimuli, although the specific mechanisms remain to be elucidated. We addressed the specific mechanisms by which exposure to tumour necrosis factor-alpha (TNF-alpha) results in PGC-1alpha down-regulation in cardiac cells and, as a consequence, in the metabolic dysregulation that underlies heart dysfunction and failure. METHODS AND RESULTS: By using coimmunoprecipitation studies, we report for the first time that the p65 subunit of NF-kappaB is constitutively bound to PGC-1alpha in human cardiac cells and also in mouse heart, and that NF-kappaB activation by TNF-alpha exposure increases this binding. Overexpression and gene silencing analyses demonstrated that the main factor limiting the degree of this association is p65, because only the modulation of this protein modified the physical interaction. Our data show that the increased physical interaction between p65 and PGC-1alpha after NF-kappaB activation is responsible for the reduction in PGC-1alpha expression and subsequent dysregulation of glucose oxidation. CONCLUSION: On the basis of these data, we propose that p65 directly represses PGC-1alpha activity in cardiac cells, thereby leading to a reduction in pyruvate dehydrogenase kinase 4 (PDK4) expression and the subsequent increase in glucose oxidation observed during the proinflammatory state.