Alternative Splicing of NOX4 in the Failing Human Heart

Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX) isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions) were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5'-3' end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure

Isolated hypercholesterolemia leads to steatosis in the liver without affecting the pancreas

Abstract Background Lipid accumulation in the liver and pancreas is primarily caused by combined hyperlipidemia. However, the effect of isolated hypercholesterolemia without hypertriglyceridemia is not fully described. Therefore, our aim was to investigate whether hypercholesterolemia alone leads to alterations both in hepatic and pancreatic lipid panel and histology in rats. Methods Male Wistar rats were fed with 2% cholesterol +0.25% cholate-supplemented diet or standard chow for 12 weeks. Blood was collected at weeks 0, 4, 8 and 12 to measure serum cholesterol and triglyceride levels. At week 12, both the pancreas and the liver were isolated for further histological and biochemical analysis. Hepatic and plasma fatty acid composition was assessed by gas chromatography. Expression of mRNA of major enzymes involved in saturated/unsaturated fatty acid synthesis was analyzed by qPCR. In separate experiments serum enzyme activities and insulin levels were measured at week 9. Results At week 12, rats fed with 2% cholesterol +0.25% cholate-supplemented diet were characterized by elevated serum cholesterol (4.09 ± 0.20 vs. 2.89 ± 0.22 mmol/L, *p < 0.05) while triglyceride (2.27 ± 0.05 vs. 2.03 ± 0.03 mmol/L) and glucose levels (5.32 ± 0.14 vs. 5.23 ± 0.10 mmol/L) remained unchanged. Isolated hypercholesterolemia increased hepatic lipid accumulation, hepatic cholesterol (5.86 ± 0.22 vs. 1.60 ± 0.15 ng/g tissue, *p < 0.05) and triglyceride contents (19.28 ± 1.42 vs. 6.78 ± 0.71 ng/g tissue, *p < 0.05), and hepatic nitrotyrosine level (4.07 ± 0.52 vs. 2.59 ± 0.31 ng/mg protein, *p < 0.05). The histology and tissue lipid content of the pancreas was not affected. Serum total protein level, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities remained unchanged in response to isolated hypercholesterolemia while serum alkaline phosphatase activity (ALP) significantly increased. Plasma insulin levels did not change in response to isolated hypercholesterolemia suggesting an intact endocrine function of the pancreas. Isolated hypercholesterolemia caused a significantly increased hepatic and serum fatty acid level associated with a marked alteration of fatty acid composition. Hepatic expression of Δ9-desaturase (SCD1) was increased 4.92×, while expression of Δ5-desaturase and Δ6-desaturase were decreased (0.447× and 0.577×, respectively) due to isolated hypercholesterolemia. Conclusions Isolated hypercholesterolemia leads to hepatic steatosis and marked alterations in the hepatic lipid profile without affecting the pancreas. Altered fatty acid profile might mediate harmful effects of cholesterol in the liver

Transcriptomic alterations in the heart of non-obese type 2 diabetic Goto-Kakizaki rats

BACKGROUND: There is a spectacular rise in the global prevalence of type 2 diabetes mellitus (T2DM) due to the worldwide obesity epidemic. However, a significant proportion of T2DM patients are non-obese and they also have an increased risk of cardiovascular diseases. As the Goto-Kakizaki (GK) rat is a well-known model of non-obese T2DM, the goal of this study was to investigate the effect of non-obese T2DM on cardiac alterations of the transcriptome in GK rats. METHODS: Fasting blood glucose, serum insulin and cholesterol levels were measured at 7, 11, and 15 weeks of age in male GK and control rats. Oral glucose tolerance test and pancreatic insulin level measurements were performed at 11 weeks of age. At week 15, total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41,012 genes, and then expression of selected genes was confirmed by qRT-PCR. Gene ontology and protein-protein network analyses were performed to demonstrate potentially characteristic gene alterations and key genes in non-obese T2DM. RESULTS: Fasting blood glucose, serum insulin and cholesterol levels were significantly increased, glucose tolerance and insulin sensitivity were significantly impaired in GK rats as compared to controls. In hearts of GK rats, 204 genes showed significant up-regulation and 303 genes showed down-regulation as compared to controls according to microarray analysis. Genes with significantly altered expression in the heart due to non-obese T2DM includes functional clusters of metabolism (e.g. Cyp2e1, Akr1b10), signal transduction (e.g. Dpp4, Stat3), receptors and ion channels (e.g. Sln, Chrng), membrane and structural proteins (e.g. Tnni1, Mylk2, Col8a1, Adam33), cell growth and differentiation (e.g. Gpc3, Jund), immune response (e.g. C3, C4a), and others (e.g. Lrp8, Msln, Klkc1, Epn3). Gene ontology analysis revealed several significantly enriched functional inter-relationships between genes influenced by non-obese T2DM. Protein-protein interaction analysis demonstrated that Stat is a potential key gene influenced by non-obese T2DM. CONCLUSIONS: Non-obese T2DM alters cardiac gene expression profile. The altered genes may be involved in the development of cardiac pathologies and could be potential therapeutic targets in non-obese T2DM

The effect of a preparation of minerals, vitamins and trace elements on the cardiac gene expression pattern in male diabetic rats

BACKGROUND: Diabetic patients have an increased risk of developing cardiovascular diseases, which are the leading cause of death in developed countries. Although multivitamin products are widely used as dietary supplements, the effects of these products have not been investigated in the diabetic heart yet. Therefore, here we investigated if a preparation of different minerals, vitamins, and trace elements (MVT) affects the cardiac gene expression pattern in experimental diabetes. METHODS: Two-day old male Wistar rats were injected with streptozotocin (i.p. 100 mg/kg) or citrate buffer to induce diabetes. From weeks 4 to 12, rats were fed with a vehicle or a MVT preparation. Fasting blood glucose measurement and oral glucose tolerance test were performed at week 12, and then total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41012 oligonucleotides. RESULTS: Significantly elevated fasting blood glucose concentration and impaired glucose tolerance were markedly improved by MVT-treatment in diabetic rats at week 12. Genes with significantly altered expression due to diabetes include functional clusters related to cardiac hypertrophy (e.g. caspase recruitment domain family, member 9; cytochrome P450, family 26, subfamily B, polypeptide; FXYD domain containing ion transport regulator 3), stress response (e.g. metallothionein 1a; metallothionein 2a; interleukin-6 receptor; heme oxygenase (decycling) 1; and glutathione S-transferase, theta 3), and hormones associated with insulin resistance (e.g. resistin; FK506 binding protein 5; galanin/GMAP prepropeptide). Moreover the expression of some other genes with no definite cardiac function was also changed such as e.g. similar to apolipoprotein L2; brain expressed X-linked 1; prostaglandin b2 synthase (brain). MVT-treatment in diabetic rats showed opposite gene expression changes in the cases of 19 genes associated with diabetic cardiomyopathy. In healthy hearts, MVT-treatment resulted in cardiac gene expression changes mostly related to immune response (e.g. complement factor B; complement component 4a; interferon regulatory factor 7; hepcidin). CONCLUSIONS: MVT-treatment improved diagnostic markers of diabetes. This is the first demonstration that MVT-treatment significantly alters cardiac gene expression profile in both control and diabetic rats. Our results and further studies exploring the mechanistic role of individual genes may contribute to the prevention or diagnosis of cardiac complications in diabetes

A new role of AMP-activated protein kinase in regulating proliferation of mesenchymal stem cells

Purpose: Natriuretic peptides (NPs) administered during early reperfusion are protective in models of myocardial infarction. A previous study examining the endogenous components of B-type natriuretic peptide (BNP) protection of reperfused myocardium, implicated both sarcolemmal (s) KATP and mitochondrial (m) KATP channels. The indirect evidence characterising the relationship between BNP signalling and KATP was obtained using sulphonylurea receptor inhibitors in a rat isolated heart model of ischaemia-reperfusion injury. Here we seek to further examine the relationship between NPs and sKATP openings using single channel electrophysiology. Given our previous findings and the overarching consensus that cardioprotective autacoids open KATP channels, it was hypothesised that NPs elicit sKATP opening. Methods: Cardiomyocyte isolation. Left ventricular cardiomyocytes were isolated from male Sprague-Dawley rat hearts subjected to enzymatic digestion with Liberase Blendzyme DL. Cardiomyocytes were cultured overnight in Medium 199, prior to patch clamp. Single channel patch clamp. Single channel recordings at room temperature (22°C) were made from cell attached patches bathed in Na+ Locke, pH 7.2. The recording pipette contained high KCl (140 mM), pH 7.2. Recordings (45 sec) were made over a range of patch potentials (0, -30, -60, -90, -120 mV), in the absence (control) and in the presence of bath applied BNP (10, 100 nM and 1 µM), pinacidil (200 µM) or pinacidil vehicle (DMSO, 0.25%). Recordings were also made with BNP and pinacidil applied concomitantly. Data are mean ± S.E.M. Results: The current voltage relationship of sKATP under control conditions was linear at –ve patch potentials, the mean conductance being 52.9 ± 1.8 pS (n = 18 hearts, n = 35 cells). Pinacidil caused a four fold increase in sKATP open probability compared to control. Mean channel conductance in the presence of pinacidil was 59.9 ± 1.9 pS (n = 16 hearts, n = 44 cells). Interestingly BNP at all concentrations had negligible effects on sKATP open probability and unitary conductance. However, BNP at all concentrations and patch potentials inhibited pinacidil induced sKATP openings, restoring channel open probability to baseline. Conclusion: These data illustrate the inhibitory effect of NP signalling on sKATP function in the cardiomyocyte under normoxia. They are concordant with the inhibitory effect of atrial NP on KATP in the pancreatic beta cell, but are in apparent conflict with the current cardioprotection paradigm. However, differential effects on sKATP and mKATP and the effects of hypoxia-reoxygenation require further exploration