The landscape of DNA repeat elements in human heart failure.

BACKGROUND: The epigenomes of healthy and diseased human hearts were recently examined by genome-wide DNA methylation profiling. Repetitive elements, heavily methylated in post-natal tissue, have variable methylation profiles in cancer but methylation of repetitive elements in the heart has never been examined. RESULTS: We analyzed repetitive elements from all repeat families in human myocardial samples, and found that satellite repeat elements were significantly hypomethylated in end-stage cardiomyopathic hearts relative to healthy normal controls. Satellite repeat elements are almost always centromeric or juxtacentromeric, and their overexpression correlates with disease aggressiveness in cancer. Similarly, we found that hypomethylation of satellite repeat elements correlated with up to 27-fold upregulation of the corresponding transcripts in end-stage cardiomyopathic hearts. No other repeat family exhibited differential methylation between healthy and cardiomyopathic hearts, with the exception of the Alu element SINE1/7SL, for which a modestly consistent trend of increased methylation was observed. CONCLUSIONS: Satellite repeat element transcripts, a form of non-coding RNA, have putative functions in maintaining genomic stability and chromosomal integrity. Further studies will be needed to establish the functional significance of these non-coding RNAs in the context of heart failure

Physiological response to lipid peroxidation in ischemia and reperfusion during carotid endarterectomy

<p>Abstract</p> <p>Background</p> <p>In this study we aimed to assess lipid peroxidation during carotid endarterectomy by the formation of PUFA hydroperoxides (PUFAHP) and isoprostanes (IP) and concomitant peroxisomal beta-oxidation as a physiological mechanism to limit their concentration. Two markers of peroxisomal beta oxidation have been evaluated, formation of 2,3 dinor from IP and conjugated esadecadienoic acid (CD 16:2) from peroxisomal beta-oxidation of conjugated linoleic acid (CLA), an unusual fatty acid present in small concentration in our diet and preferentially beta-oxidised in peroxisomes.</p> <p>The study was conducted on 30 patients undergoing carotid endarterectomy. Blood samplings were performed before, during endarterectomy in the "ischemic phase", and 30 seconds, 30 minutes and 2 hours after reperfusion.</p> <p>Results</p> <p>The results showed that PUFAHP increased significantly after 30 min of reperfusion in patients with controlateral stenosis > 50%, and steeply decreased after 2 hour of reperfusion. Interestingly, IP increased in a similar fashion of PUFAHP but never significantly. Both ratios CD16:2/CLA and DIN/IP also increased significantly after 30 min of reperfusion to decrease thereafter.</p> <p>Conclusions</p> <p>Our data show that lipid peroxidation takes place only in patients with high controlateral stenosis and within 2 hours occurs a physiological response aimed to decrease IP and PUFAHP by increasing their catabolism in peroxisomes.</p

Krill oil significantly decreases 2-arachidonoylglycerol plasma levels in obese subjects

We have previously shown that krill oil (KO), more efficiently than fish oil, was able to downregulate the endocannabinoid system in different tissues of obese zucker rats

Effect of acute administration of Pistacia lentiscus L. essential oil on rat cerebral cortex following transient bilateral common carotid artery occlusion

<p>Abstract</p> <p>Background</p> <p>Ischemia/reperfusion leads to inflammation and oxidative stress which damages membrane highly polyunsaturated fatty acids (HPUFAs) and eventually induces neuronal death. This study evaluates the effect of the administration of <it>Pistacia lentiscus </it>L. essential oil (E.O.), a mixture of terpenes and sesquiterpenes, on modifications of fatty acid profile and endocannabinoid (eCB) congener concentrations induced by transient bilateral common carotid artery occlusion (BCCAO) in the rat frontal cortex and plasma.</p> <p>Methods</p> <p>Adult Wistar rats underwent BCCAO for 20 min followed by 30 min reperfusion (BCCAO/R). 6 hours before surgery, rats, randomly assigned to four groups, were gavaged either with E.O. (200 mg/0.45 ml of sunflower oil as vehicle) or with the vehicle alone.</p> <p>Results</p> <p>BCCAO/R triggered in frontal cortex a decrease of docosahexaenoic acid (DHA), the membrane highly polyunsaturated fatty acid most susceptible to oxidation. Pre-treatment with E.O. prevented this change and led further to decreased levels of the enzyme cyclooxygenase-2 (COX-2), as assessed by Western Blot. In plasma, only after BCCAO/R, E.O. administration increased both the ratio of DHA-to-its precursor, eicosapentaenoic acid (EPA), and levels of palmytoylethanolamide (PEA) and oleoylethanolamide (OEA).</p> <p>Conclusions</p> <p>Acute treatment with E.O. before BCCAO/R elicits changes both in the frontal cortex, where the BCCAO/R-induced decrease of DHA is apparently prevented and COX-2 expression decreases, and in plasma, where PEA and OEA levels and DHA biosynthesis increase. It is suggested that the increase of PEA and OEA plasma levels may induce DHA biosynthesis via peroxisome proliferator-activated receptor (PPAR) alpha activation, protecting brain tissue from ischemia/reperfusion injury.</p

Distinct epigenomic features in end-stage failing human hearts

Background— The epigenome refers to marks on the genome, including DNA methylation and histone modifications, that regulate the expression of underlying genes. A consistent profile of gene expression changes in end-stage cardiomyopathy led us to hypothesize that distinct global patterns of the epigenome may also exist. Methods and Results— We constructed genome-wide maps of DNA methylation and histone-3 lysine-36 trimethylation (H3K36me3) enrichment for cardiomyopathic and normal human hearts. More than 506 Mb sequences per library were generated by high-throughput sequencing, allowing us to assign methylation scores to ≈28 million CG dinucleotides in the human genome. DNA methylation was significantly different in promoter CpG islands, intragenic CpG islands, gene bodies, and H3K36me3-enriched regions of the genome. DNA methylation differences were present in promoters of upregulated genes but not downregulated genes. H3K36me3 enrichment itself was also significantly different in coding regions of the genome. Specifically, abundance of RNA transcripts encoded by the DUX4 locus correlated to differential DNA methylation and H3K36me3 enrichment. In vitro, Dux gene expression was responsive to a specific inhibitor of DNA methyltransferase, and Dux siRNA knockdown led to reduced cell viability. Conclusions— Distinct epigenomic patterns exist in important DNA elements of the cardiac genome in human end-stage cardiomyopathy. The epigenome may control the expression of local or distal genes with critical functions in myocardial stress response. If epigenomic patterns track with disease progression, assays for the epigenome may be useful for assessing prognosis in heart failure. Further studies are needed to determine whether and how the epigenome contributes to the development of cardiomyopathy

Dietary Conjugated Linoleic Acid-Enriched Cheeses Influence the Levels of Circulating n-3 Highly Unsaturated Fatty Acids in Humans

peer-reviewedn-3 highly unsaturated fatty acids (n-3 HUFA) directly and indirectly regulate lipid metabolism, energy balance and the inflammatory response. We investigated changes to the n-3 HUFA score of healthy adults, induced by different types and amounts of conjugated linoleic acid (CLA)-enriched (ENCH) cheeses consumed for different periods of time, compared to dietary fish oil (FO) pills (500 mg, each containing 100 mg of eicosapentaenoic and docosahexaenoic acids—EPA+DHA) or α-linolenic acid (ALA)-rich linseed oil (4 g, containing 2 g of ALA). A significant increase in the n-3 HUFA score was observed, in a dose-dependent manner, after administration of the FO supplement. In terms of the impact on the n-3 HUFA score, the intake of ENCH cheese (90 g/day) for two or four weeks was equivalent to the administration of one or two FO pills, respectively. Conversely, the linseed oil intake did not significantly impact the n-3 HUFA score. Feeding ENCH cheeses from different sources (bovine, ovine and caprine) for two months improved the n-3 HUFA score by increasing plasma DHA, and the effect was proportional to the CLA content in the cheese. We suggest that the improved n-3 HUFA score resulting from ENCH cheese intake may be attributed to increased peroxisome proliferator-activated receptor alpha (PPAR-α) activity. This study demonstrates that natural ENCH cheese is an alternative nutritional source of n-3 HUFA in humans

Shoc2 Is Targeted to Late Endosomes and Required for Erk1/2 Activation in EGF-Stimulated Cells

Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF) receptor (EGFR), we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S) mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module

LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts

(1) Background: The LEO1 (Left open reading frame 1) protein is a conserved subunit of the PAF1C complex (RNA polymerase II-associated factor 1 complex). PAF1C has well-established mechanistic functions in elongation of transcription and RNA processing. We previously showed, in fission yeast, that LEO1 controls histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for the proper regulation of gene expression during cellular quiescence. Human fibroblasts enter a reversible quiescence state upon serum deprivation in the growth media. Here we investigate the function of LEO1 in human fibroblasts. (2) Methods: We knocked out the LEO1 gene using CRISPR/Cas9 methodology in human fibroblasts and verified that the LEO1 protein was undetectable by Western blot. We characterized the phenotype of the ΔLEO1 knockout cells with FACS analysis and cell growth assays. We used RNA-sequencing using spike-in controls to measure gene expression and spike-in controlled ChIP-sequencing experiments to measure the histone modification H3K9me2 genome-wide. (3) Results: Gene expression levels are altered in quiescent cells, however factors controlling chromatin and gene expression changes in quiescent human cells are largely unknown. The ΔLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild-type cells. ΔLEO1 cells showed a slower entry into quiescence and a different morphology compared to wild-type cells. Gene expression was generally reduced in quiescent wild-type cells. The downregulated genes included genes involved in cell proliferation. A small number of genes were upregulated in quiescent wild-type cells including several genes involved in ERK1/ERK2 and Wnt signaling. In quiescent ΔLEO1 cells, many genes were mis-regulated compared to wild-type cells. This included genes involved in Calcium ion transport and cell morphogenesis. Finally, spike-in controlled ChIP-sequencing experiments demonstrated that the histone modification H3K9me2 levels are globally increased in quiescent ΔLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control of H3K9me2 levels, and gene expression in human fibroblasts

Hepatic retinol secretion and storage are altered by dietary CLA: common and distinct actions of CLA c9,t11 and t10,c12 isomers[S]

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid obtained from ruminant products. Previous studies in rats and pigs showed that a dietary equimolar mixture of c9,t11 and t10,c12 CLA isomers induces changes in serum and tissue levels of retinoids (vitamin A derivatives). However, the mechanism(s) responsible for these actions remain(s) unexplored. Given the numerous crucial biological functions regulated by retinoids, it is key to establish whether the perturbations in retinoid metabolism induced by dietary CLA mediate some of the beneficial effects associated with intake of this fatty acid or, rather, have adverse consequences on health. To address this important biological question, we began to explore the mechanisms through which dietary CLA alters retinoid metabolism. By using enriched preparations of CLA c9,t11 or CLA t10,c12, we uncoupled the effects of these two CLA isomers on retinoid metabolism. Specifically, we show that both isomers induce hepatic retinyl ester accumulation. However, only CLA t10,c12 enhances hepatic retinol secretion, resulting in increased serum levels of retinol and its specific carrier, retinol-binding protein (RBP). Dietary CLA t10,c12 also redistributes retinoids from the hepatic stores toward the adipose tissue and possibly stimulates hepatic retinoid oxidation. Using mice lacking RBP, we also demonstrate that this key protein in retinoid metabolism mediates hepatic retinol secretion and its redistribution toward fat tissue induced by CLA t10,c12 supplementation