Methods
Chronic heart failure model from volume overload
Heart failure was induced by volume overload from aorto-caval fistula (ACF) created using needle technique [2;7;10]. Male Wistar rats (Anlabsro, Czech Republic) weighting 300-350g were anesthetized with ketamine/midazolam mixture and midline abdominal laparotomy was performed to expose infrarenal aorta and vena cava.18-gauge needle (diameter 1.2 mm, Becton-Dickinson) was inserted into the abdominal aorta and advanced through the medial wall into the vena cava to create ACF as described previously[2;7;10]. The aorta above puncture was than temporarily clamped, needle withdrawn and the puncture site was sealed with cyanacrylate glue. Creation of ACF was confirmed by observing pulsatile, bright flow in the inferior vena cava. Abdominal cavity was closed by absorbable suture. The ACF procedure was associated with 13% early (≤ 7 days) mortality, occurring mostly within first 48 hours. An additional 5% of ACF animals died till the end of experiment and those were excluded from the analyses. The animals were kept on a 12/12-hour light/dark cycle, fed normal salt/protein diet (0.45% NaCl, 19-21% protein, SEMED, CR) and were weighted weekly till the end of the experiment on 22nd week. The investigation conformed to the NIH Guide for the care and use of laboratory animals (NIH Publication No. 85-23, 1996), Animal protection law of the CR (311/1997) and was approved by the ethics committee of IKEM.
Echocardiography and hemodynamics
Echocardiography was performed at 10th week after ACF. Animals were be anesthetized with halothane inhalational anesthesia, shaved and ventricular thickness and dimensions were measured in triplicate by experienced echocardiographer using 10 MHz US probe (Vivid System 5, GE, USA). End-systolic and end-diastolic LV volume were derived by cubic equation[3] and stroke volume as their difference. All other measurements besides echocardiography, were performed at the study end at 22th week. After induction of anesthesia (ketamine 50 mg/kg and midazolam 5 mg/kg), invasive hemodynamics evaluation was performed by Millar micro-manometer catheter inserted into aorta and LV via carotid artery. Pressure and 3-lead ECG signal were digitalized at 1 kHz and recorded using Powerlab 8 platform for off-line analysis with LabChart software (ADInstruments, Germany). Relaxation constant tau was calculated using Blood Pressure Module by fitting a monoexponential function and assuming zero asymptote ([5]. The presence of pulsatile AV fistula was then verified from midline laparotomy and the animals were killed by rapid exsanguination. Beating heart was excised and coronary tree was promptly ortogradely perfused with 10 ml ice-cold St.Thomascardioplegia solution. Basal third of the ventricles were used for morphology. Mid-ventricular samples of LV free wall were immediately harvested into RNA-Later solution (Ambion Inc., Austin, TE, USA) and stored in -80°C. Apical part of the LV served for biochemical analysis of lipids in the myocardium. Wet weights of liver, kidney, lungs were measured in wet state and normalized to body weight and tibia length.
Myocardial morphology
Basal portions of the left ventricle were fixed in 4% paraformaldehyde in PBS overnight and then prepared for embedding into TissuTek through series of graded saccharose solutions. Frozen sections were cut at 12 microns and subjected to standard histological staining (hematoxyllin-eosin with alcian blue), Sudan Black staining for lipids and picrosirius red for collagen. Immunohistochemical staining with anti-alpha actinin antibody (Sigma) detected by rhodamine red-conjugated secondary (Jackson Imnmuno) together with Alexa488-conjugated wheat germ aggluttinin (Invitrogen) and Hoechst nuclear counterstaining was used to evaluate myocyte size and proportion of fibrosis. Images were acquired on Olympus BX51 microscope fitted with Olympus DP70 CCD camera (transmitted and polarized light) and Leica SPE confocal system (immunofluorescence). Images were further processed for presentation in Adobe Photoshop (background and levels adjustment, Unsharp Mask filtering); quantitative analysis was done on raw data using ImageJ (National Institutes of Health) image analysis software (percentage of fibrosis, densitometry of Sudan staining).
Gene expression and GSEA pathway analysis
RNA analysis: Total RNA was isolated by RNeasy Micro Kit (QIAGEN Inc., Valencia CA, USA) according to the procedure for fibrous tissue (cardiomyocytes) and lipid tissue (adipocytes, RNA was purified after Qiazol extraction). Quantity of the RNA was measured on NanoDrop ND-1000 (NanoDrop Technologies LLC, Wilmington, DE, USA). RNA integrity was assessed on Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA). All RNA samples had RNA integrity number (RIN) 8. Illumina RatRef-12 v1 Expression BeadChip (Illumina Inc., San Diego USA) was used for the microarray analysis following the standard protocol: 200 ng of total RNA was amplified with IlluminaTotalPrep RNA Amplification Kit (Ambion Inc., Austin, TE, USA) and 1.5 μg of amplified RNA was hybridized on the chip according to the manufacturer procedure. All analyses were done in ≥6 individual animals per group.
Microarray analysis: The raw data (.TIFF image files) was analyzed using beadarray package[6] of the Bioconductor[8] within the R environment ( All hybridizations passed quality control. The data was background corrected, variance-stabilized by logarithmic transformation and normalized with the probe level quantile method. Before detection of differential expression, 50% of the least varying probes based on IQR (inter-quantile range) were disregarded, however, the probes with detection p-value < 0.05 in at least one sample (null model: negative controls of the BeadChip) were kept. Analysis of differential expression was performed with the limma package[11]. Annotation of differentially expressed transcripts was done against the manifest provided by Illumina (RatRef_12_V1_0_R3_11222119_A.bgx, Illumina Inc., San Diego USA). Only transcripts with Storey´s q-value <0.05[12] and fold-change <0.5 or >2 were reported. The data was deposited to the ArrayExpress database (accession # E-MTAB-190).
Pathway analysis: The gene set enrichment analysis (GSEA) was performed on gene sets defined by the KEGG pathways[9]. Lists of genes assigned to the KEGG pathways were downloaded from the KEGG (release 57.0) directly and the probes were assigned to KEGG pathways accordingly. Two methods of GSEA were applied: first, we performed simple Fischer test on overrepresentation of upregulated (resp. downregulated, significantly changed) probes in a selected KEGG pathway against overall ratio of upregulated (downregulated, significantly changed) probes in the complete dataset. Second, we performed GSEA according to Tian[13]. Only pathways with false discovery rate[1]< 0.05 were reported here.
Quantitative real-time PCR
RNA for analysis was harvested from LV tissue and epididymal fat as described in Gene expression and GSEA pathway analysis. Reverse transcription was performed by QuantiTect® Reverse Transcription Kit (QIAGEN Inc., USA) according to manufacturers’ instructions (starting with 50 ng/µl RNA). Final cDNA was diluted six times and 2 µl aliquot was used as a template for quantitative real-time PCR (RT-qPCR). The qRT-PCR was performed on LightCycler 2.0 System using LightCycler® 480 DNA SYBR Green I Master kit (Roche Diagnostics, Germany). PCR reactions (5 µl) were run according to the standard manufacturer’s protocol, cycled 45 times. Target genes (Nppa, Postn_predicted, Thbs4, Wisp2, Eno3, Acat1, Hadh, Gipr, Vwf, Aox3, Nol5) and housekeeping genes (Rps9, Eaf1a1, Tbp, Ppia, Eif3k) were measured under same conditions from the same cDNA (the list of amplicons / primers is provided in the supplementary file 3). Results were analyzed by LightCycler software and crossing point values were further determined using the R environment (R Development Core Team 2007). Every examined tissue was performed in 1 RT and 2 technical replicates. Stability measurement of housekeeping genes and normalization to housekeeping genes was carried out using method of Vandesompele et al.[14]. Statistical significance of changes in mRNA level of target genes between different samples was calculated by anunpaired t-test.
Biochemical analyses
Oral Glucose Tolerance Testing—Oral glucose tolerance tests (OGTT) were performed using a glucose load of 300 mg/100 g body weight after overnight fasting. Blood was drawn from the tail without anesthesia before the glucose load (0-min time point) and at 30, 60, and 120 min thereafter.
Biochemical Analyses—Blood glucose levels were measured by the glucose oxidase assay (Pliva-Lachema, Brno, Czech Republic) using tail vein blood drawn into 5% trichloroacetic acid and promptly centrifuged. Serum free (non-esterified) fatty acid (FFA) levels were determined using an acyl-CoAoxidase based colorimetric kit (Roche Diagnostics GmbH, Mannheim, Germany). Serum triglyceride concentrations were measured by standard enzymatic methods (Pliva-Lachema, Brno, Czech Republic). Serum insulin concentrations were determined using the Mercodia Rat Insulin ELISA kit (Mercodia AB, Uppsala, Sweden).
Tissue Triglyceride Measurements—For determination of triglycerides in the heart, tissue were powdered under liquid N2 and extracted for 16 h in chloroform/methanol, after which 2% KH2PO4 was added and the solution was centrifuged. The organic phase was removed and evaporated under N2. The resulting pellet was dissolved in isopropyl alcohol, and triglyceride content was determined by enzymatic assay (Pliva-Lachema, Brno, Czech Republic).
The activity of SOD was analyzed by the reaction of nitrotethrazolium blue reduction and nitrophormasane formation[4]. The reduced (GSH) and oxidized form of glutathione (GSSG) was determined by high performance liquid chromatography (HPLC) method with fluorescent detection using HPLC kit (Chromsystems, Germany). The level of thiobarbituric acid-reactive substances (TBARS) was determined by the reaction with thiobarbituric acid[16].
Metabolic assessment of isolated tissue
Basal and epinephrine-stimulated lipolysis in isolated intraabdominal fat tissue-The measurement of adrenaline-stimulated lipolysis was performed as described earlier[15]. The distal parts of epididymal adipose tissue were incubated in Krebs–Ringer phosphate buffer (118 mmol.l-1 NaCl, 4.7 mmol.l-1 KCl, 2.5 mmol.l-1 CaCl2, 1.2 mmol.l-1 MgSO4, 1.2 mmol.l-1 KH2PO4 and 25mmol.l-1 NaHCO3, pH 7.4), containing 3% bovine serum albumin (Fraction V, Armour Pharmaceutical Co., Chicago, IL, USA), at 37 °C with or without epinephrine (0.25 mg.ml-1). The adipose tissue was incubated for 2 h and the concentrations of FFA in the medium were determined.
Basal and insulin-stimulated lipogenesis in isolated intraabdominal fat tissue-Distal parts of epididymal adipose tissue were rapidly dissected and incubated for 2 h in Krebs–Ringer bicarbonate buffer with 5 mmol.l-1 glucose, 0.1 µCi14C-U-glucose per ml (UVVR, Prague, Czech Republic) and 2% bovine serum albumin, gaseous phase 95% O2 and 5% CO2, in the presence or absence of insulin (250 µU.ml-1) in incubation media. All incubations were performed at 37 °C in sealed vials in a shaking water bath. The estimation of 14C-glucose incorporation into neutral lipids was performed. Adipose tissue was removed from incubation medium, rinsed in saline and immediately put into chloroform. The pieces of tissue were dissolved using a Teflon pestle homogenizer, methanol was added (chloroform/methanol, 2:1) and lipids were extracted at 4°C overnight. The remaining tissue was removed, KH2PO4 was added and a clear extract was taken for further analysis. An aliquot was evaporated, reconstituted in scintillation liquid and the radioactivity measured by scintillation counting. Incremental glucose utilization was calculated as the difference between the insulin-stimulated and basal incorporation of glucose into neutral lipids.
Statistics
Data from animals that died during the course of the experiment (18% total mortality including ACF operation), were excluded from the analyses. Sample size for echocardiography was 12-14/group, for other methods 5-8/group. Data are expressed as means±SD, in graphs as means±SE. Student´s t-test statistics was used for comparisons and p value <0.05 was considered significant. Gene expression differences were compared with false discovery rate (FDR) and described with Storey´s q-value.
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