M. Otero

Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism

In osteoarthritis (OA), adult articular chondrocytes undergo phenotypic modulation in response to alterations in the environment owing to mechanical injury and inflammation. These processes not only stimulate the production of enzymes that degrade the cartilage matrix but also inhibit repair. With the use of in vitro and in vivo models, new genes, not known previously to act in cartilage, have been identified and their roles in chondrocyte differentiation during development and in dysregulated chondrocyte function in OA have been examined. These new genes include growth arrest and DNA damage (GADD)45β and the epithelial-specific ETS (ESE)-1 transcription factor, induced by bone morphogenetic protein (BMP)-2 and inflammatory cytokines, respectively. Both genes are induced by NF-κB, suppress COL2A1 and upregulate matrix meatalloproteinase-13 (MMP-13) expression. These genes have also been examined in mouse models of OA, in which discoidin domain receptor 2 is associated with MMP-13-mediated remodelling, in order to understand their roles in physiological cartilage homoeostasis and joint disease.

Lack of effect of the ghrelin gene-derived peptide obestatin on cardiomyocyte viability and metabolism

Obestatin is a recently discovered peptide encoded by the ghrelin gene that opposes ghrelin effects on food intake and gastrointestinal function. The biological activity of obestatin depends on amidation at its carboxyl terminus and on its postulated binding to the orphan G protein-coupled receptor 39 (GPR39). We have previously demonstrated that ghrelin is synthesized by cardiomyocytes and has direct effects on its viability. Our aim was to know if obestatin, derived from the same gene as ghrelin, also affects cardiomyocyte physiology. By RT-PCR and immunocytochemistry we have demonstrated that murine cardiomyocytes cultured in vitro and human atrial tissue express GPR39 receptor. Competitive binding studies with radioiodine 125I-labeled obestatin recognized specific binding sites for this peptide in the murine cardiomyocyte cell line HL-1. However, obestatin did not modify the cell cycle or viability of these cells, and it was not able to prevent the cytosine arabinoside-induced apoptosis of HL-1 cardiomyocytes, as assessed by Hoechst dye vital staining, flow cytometry analysis and determination of lactate dehydrogenase in the culture media. Finally, treatment with obestatin did not affect fatty acid or glucose uptake by HL-1 cardiomyocytes. In conclusion, obestatin is not a relevant metabolic or viability modifier for cardiomyocytes.

Nesfatin-1 in human and murine cardiomyocytes: synthesis, secretion, and mobilization of GLUT-4.

Nesfatin-1, a satiety-inducing peptide identified in hypothalamic regions that regulate energy balance, is an integral regulator of energy homeostasis and a putative glucose-dependent insulin coadjuvant. We investigated its production by human cardiomyocytes and its effects on glucose uptake, in the main cardiac glucose transporter GLUT-4 and in intracellular signaling. Quantitative RT-PCR, Western blots, confocal immunofluorescence microscopy, and ELISA of human and murine cardiomyocytes and/or cardiac tissue showed that cardiomyocytes can synthesize and secrete nesfatin-1. Confocal microscopy of cultured cardiomyocytes after GLUT-4 labeling showed that nesfatin-1 mobilizes this glucose transporter to cell peripherals. The rate of 2-deoxy-D-[(3)H]glucose incorporation demonstrated that nesfatin-1 induces glucose uptake by HL-1 cells and cultured cardiomyocytes. Nesfatin-1 induced dose- and time-dependent increases in the phosphorylation of ERK1/2, AKT, and AS160. In murine and human cardiac tissue, nesfatin-1 levels varied with diet and coronary health. In conclusion, human and murine cardiomyocytes can synthesize and secrete nesfatin-1, which is able to induce glucose uptake and the mobilization of the glucose transporter GLUT-4 in these cells. Nesfatin-1 cardiac levels are regulated by diet and coronary health.

Increased expression of fatty-acid and calcium metabolism genes in failing human heart.

Background Heart failure (HF) involves alterations in metabolism, but little is known about cardiomyopathy-(CM)-specific or diabetes-independent alterations in gene expression of proteins involved in fatty-acid (FA) uptake and oxidation or in calcium-(Ca2+)-handling in the human heart. Methods RT-qPCR was used to quantify mRNA expression and immunoblotting to confirm protein expression in left-ventricular myocardium from patients with HF (n = 36) without diabetes mellitus of ischaemic (ICM, n = 16) or dilated (DCM, n = 20) cardiomyopathy aetiology, and non-diseased donors (CTL, n = 6). Results Significant increases in mRNA of genes regulating FA uptake (CD36) and intracellular transport (Heart-FA-Binding Protein (HFABP)) were observed in HF patients vs CTL. Significance was maintained in DCM and confirmed at protein level, but not in ICM. mRNA was higher in DCM than ICM for peroxisome-proliferator-activated-receptor-alpha (PPARA), PPAR-gamma coactivator-1-alpha (PGC1A) and CD36, and confirmed at the protein level for PPARA and CD36. Transcript and protein expression of Ca2+-handling genes (Two-Pore-Channel 1 (TPCN1), Two-Pore-Channel 2 (TPCN2), and Inositol 1,4,5-triphosphate Receptor type-1 (IP3R1)) increased in HF patients relative to CTL. Increases remained significant for TPCN2 in all groups but for TPCN1 only in DCM. There were correlations between FA metabolism and Ca2+-handling genes expression. In ICM there were six correlations, all distinct from those found in CTL. In DCM there were also six (all also different from those found in CTL): three were common to and three distinct from ICM. Conclusion DCM-specific increases were found in expression of several genes that regulate FA metabolism, which might help in the design of aetiology-specific metabolic therapies in HF. Ca2+-handling genes TPCN1 and TPCN2 also showed increased expression in HF, while HF- and CM-specific positive correlations were found among several FA and Ca2+-handling genes.

Differential Gene Expression of Cardiac Ion Channels in Human Dilated Cardiomyopathy

Background Dilated cardiomyopathy (DCM) is characterized by idiopathic dilation and systolic contractile dysfunction of the cardiac chambers. The present work aimed to study the alterations in gene expression of ion channels involved in cardiomyocyte function. Methods and Results Microarray profiling using the Affymetrix Human Gene® 1.0 ST array was performed using 17 RNA samples, 12 from DCM patients undergoing cardiac transplantation and 5 control donors (CNT). The analysis focused on 7 cardiac ion channel genes, since this category has not been previously studied in human DCM. SCN2B was upregulated, while KCNJ5, KCNJ8, CLIC2, CLCN3, CACNB2, and CACNA1C were downregulated. The RT-qPCR (21 DCM and 8 CNT samples) validated the gene expression of SCN2B (p < 0.0001), KCNJ5 (p < 0.05), KCNJ8 (p < 0.05), CLIC2 (p < 0.05), and CACNB2 (p < 0.05). Furthermore, we performed an IPA analysis and we found a functional relationship between the different ion channels studied in this work. Conclusion This study shows a differential expression of ion channel genes involved in cardiac contraction in DCM that might partly underlie the changes in left ventricular function observed in these patients. These results could be the basis for new genetic therapeutic approaches.

Heart mitochondrial proteome study elucidates changes in cardiac energy metabolism and antioxidant PRDX3 in human dilated cardiomyopathy.

Background Dilated cardiomyopathy (DCM) is a public health problem with no available curative treatment, and mitochondrial dysfunction plays a critical role in its development. The present study is the first to analyze the mitochondrial proteome in cardiac tissue of patients with DCM to identify potential molecular targets for its therapeutic intervention. Methods and Results 16 left ventricular (LV) samples obtained from explanted human hearts with DCM (n = 8) and control donors (n = 8) were extracted to perform a proteomic approach to investigate the variations in mitochondrial protein expression. The proteome of the samples was analyzed by quantitative differential electrophoresis and Mass Spectrometry. These changes were validated by classical techniques and by novel and precise selected reaction monitoring analysis and RNA sequencing approach increasing the total heart samples up to 25. We found significant alterations in energy metabolism, especially in molecules involved in substrate utilization (ODPA, ETFD, DLDH), energy production (ATPA), other metabolic pathways (AL4A1) and protein synthesis (EFTU), obtaining considerable and specific relationships between the alterations detected in these processes. Importantly, we observed that the antioxidant PRDX3 overexpression is associated with impaired ventricular function. PRDX3 is significantly related to LV end systolic and diastolic diameter (r = 0.73, p value<0.01; r = 0.71, p value<0.01), fractional shortening, and ejection fraction (r = −0.61, p value<0.05; and r = −0.62, p value<0.05, respectively). Conclusion This work could be a pivotal study to gain more knowledge on the cellular mechanisms related to the pathophysiology of this disease and may lead to the development of etiology-specific heart failure therapies. We suggest new molecular targets for therapeutic interventions, something that up to now has been lacking.

Aliskiren affects fatty-acid uptake and lipid-related genes in rodent and human cardiomyocytes.

PURPOSE We investigated whether the direct renin inhibitor aliskiren can affect metabolism in cardiomyocytes from rat, mouse and human sources. METHODS AND RESULTS At 10-50 μmol/L, aliskiren significantly increased medium-chain-fatty-acid uptake in primary-cultured neonatal-rat and HL-1 adult-mouse-derived cardiomyocytes (BODIPY-induced fluorescence intensity). The fatty-acid transporter CD-36 was correspondingly translocated to, but the glucose transporter Glut-4 away from, the sarcoplasmic reticulum/plasma membrane, in primary-cultured neonatal-rat (CD-36, Glut-4) and adult-human (CD-36) cardiomyocytes (confocal immunocytochemistry). Immunoblotting showed that aliskiren induced phosphorylation of ERK1/2 in cardiomyocytes from all three sources; responses were dose- and time-dependent, unaffected by renin treatment, and did not cause alterations in expression of (P)R or Igf2/M6P receptors. Microarray analysis of the complete genome of aliskiren-treated neonatal-rat cardiomyocytes, with RT-qPCR and immunoblot confirmation assays in rat and human primary cardiomyocytes, showed that aliskiren up-regulated mRNA and increased protein expression of several enzymes important in lipid and glucose metabolism and in cholesterol biosynthesis. Cardiomyocyte cell-cycle and viability were unaffected by aliskiren. CONCLUSIONS Aliskiren can induce changes in fatty-acid and glucose uptake and expression of key enzymes of lipid and cholesterol metabolism, which are not associated with increased expression of (P)R or Igf2/M6P receptors, in cultured cardiomyocytes.

217 MECHANISMS OF ACTION OF ESE1, A NOVEL TRANSCRIPTIONAL REGULATOR OF CARTILAGE REMODELING, IN MMP-13 REGULATION

Purpose: Matrix metalloproteinase (MMP)-13 plays a major role in cartilage degradation. MMP-13 expression and activity are up-regulated in osteoarthritis (OA) cartilage as compared to non-OA cartilage. We previously reported that ELF3/ESE1 levels are higher in OA cartilage and that ESE-1 transactivates the MMP13 promoter via a proximal highly conserved ETS binding site. The aim of this study was to better define the mechanism/s and signalling pathways that modulate the ESE-1-driven MMP13 promoter activation in chondrocytes. Methods: We investigated the contribution of ESE-1 to MMP-13 expression by siRNA knock down (KD) transfection experiments in human primary chondrocytes and real time PCR analysis of MMP-13 expression induced by IL-1β. The response of the MMP13 promoter to ESE-1 was analyzed in luciferase reporter assays, after co-transfection of immortalized chondrocytes with human MMP13 promoter constructs and expression vectors encoding ESE-1, p38, JNK, ERK-1, MKK-6, MKK-7, MEK-1 and MKP-1. ESE-1 binding to the MMP13 promoter was analyzed by chromatin immunoprecipitation (ChIP) assays. IL-1β-induced ESE-1 nuclear translocation was addressed by Western blotting analysis of cytoplasmic and nuclear fractions of human immortalized chondrocytes. Results: Real time PCR analysis revealed a significant reduction of IL1β-induced MMP-13 mRNA levels associated with ESE-1 KD in human primary chondrocytes. Luciferase reporter assays showed that MEK1/ERK1 overexpression enhanced ESE1-driven activities of the -1528/+27 and 267/+27 MMP13 promoter constructs, whereas MKK6/p38 or MKK7/JNK overexpression did not affect ESE-1 transactivation of MMP-13. Overexpression of MKP-1 reduced both the ESE-1 activation of MMP13 and also the MEK1/ERK1 enhancement of ESE-1-driven MMP13 activation. Accordingly, pre-treatment with the MEK1/2 inhibitor, U0126, decreased the MMP13 promoter activity induced by ESE-1 overexpression. Finally, Western blotting analysis revealed that IL-1β stimulation induced ESE-1 nuclear translocation, which correlated with increased ESE-1 binding to the endogenous MMP13 promoter, as addressed by ChIP assays. Conclusions: The role of MMP-13 as a central factor for OA progression has been highlighted by recent studies in Mmp13 knockout mice. We previously reported that ESE-1 is a key factor in controlling MMP13 transcription. ESE-1 belongs to the ETS family of transcription factors, which are classic MAPK effectors in different tissues and cell types. Here, we show that ESE-1 participates in the IL-1β-induced MMP-13 expression and that IL-1β induces ESE-1 nuclear translocation and binding to the MMP13 promoter. In addition, we show that the ESE1-driven transactivation of MMP13 is enhanced by MEK1/ERK1. In chondrocytes, ERK1/2 phosphorylation is increased in vitro in response to IL-1β and in vivo in OA cartilage, and MEK/ERK activation has been reported to participate in the cytokine-induced MMP-13 expression. Therefore, ESE-1 could be one of the effectors of the MEK/ERK pathway in controlling MMP-13 expression in chondrocytes, and the relative contributions of the MEK/ERK/ESE1 axis in OA with regard to MMP-13 expression and activity will merit further investigation.

186 TOLL-LIKE RECEPTORS ACTIVATE THE ESE-1 PROMOTER IN CHONDROCYTES BY A MECHANISM PARTIALLY DEPENDENT UPON NF-κB

ing insulin sensitivity. Recently, adiponectin has also been found to regulate immune responses and inflammation. Adiponectin is found in OA joints but its role in the pathogenesis of OA and in cartilage metabolism is not clear. In the present study, we investigated the relation of circulating adiponectin and biomarkers of cartilage degradation (COMP and MMP-3) in patients with OA, and the effects of adiponectin on human OA cartilage. Methods: Blood samples were collected from 38 male OA patients (BMI 29.5±0.8 kg/m2) undergoing knee replacement surgery because of severe OA, and adiponectin, COMP and MMP-3 concentrations were measured by immunoassay. Cartilage samples collected from OA patients under total knee arthroplasty were placed in tissue culture and exposed to adiponectin. Results: Plasma adiponectin (2.5±0.2 μg/ml) correlated positively with serum COMP (r=0.55, p=0.001) and plasma MMP-3 (r=0.34, p=0.046). In tissue culture experiments, adiponectin increased the expression of iNOS, and production of nitric oxide, interleukin-6 (IL6) and MMP-3 in human OA cartilage. The effects of adiponectin of NO and IL-6 production were mediated through MAP kinases Erk1/2, p38 and JNK, and p38 pathway was involved in the adiponectin-induced MMP-3 production. Conclusions: Circulating adiponectin concentrations correlated positively with the measured biomarkers of cartilage degradation, i.e. COMP and MMP-3 in male OA patients; and adiponectin was found to increase the production of catabolic/proinflammatory mediators MMP-3, nitric oxide and IL-6 in human OA cartilage. The findings introduce adiponectin as a catabolic factor in OA.