Proteomic and transcriptomic analysis of heart failure due to volume overload in a rat aorto-caval fistula model provides support for new potential therapeutic targets - monoamine oxidase A and transglutaminase 2

<p>Abstract</p> <p>Background</p> <p>Chronic hemodynamic overloading leads to heart failure (HF) due to incompletely understood mechanisms. To gain deeper insight into the molecular pathophysiology of volume overload-induced HF and to identify potential markers and targets for novel therapies, we performed proteomic and mRNA expression analysis comparing myocardium from Wistar rats with HF induced by a chronic aorto-caval fistula (ACF) and sham-operated rats harvested at the advanced, decompensated stage of HF.</p> <p>Methods</p> <p>We analyzed control and failing myocardium employing iTRAQ labeling, two-dimensional peptide separation combining peptide IEF and nano-HPLC with MALDI-MS/MS. For the transcriptomic analysis we employed Illumina RatRef-12v1 Expression BeadChip.</p> <p>Results</p> <p>In the proteomic analysis we identified 2030 myocardial proteins, of which 66 proteins were differentially expressed. The mRNA expression analysis identified 851 differentially expressed mRNAs.</p> <p>Conclusions</p> <p>The differentially expressed proteins confirm a switch in the substrate preference from fatty acids to other sources in the failing heart. Failing hearts showed downregulation of the major calcium transporters SERCA2 and ryanodine receptor 2 and altered expression of creatine kinases. Decreased expression of two NADPH producing proteins suggests a decreased redox reserve. Overexpression of annexins supports their possible potential as HF biomarkers. Most importantly, among the most up-regulated proteins in ACF hearts were monoamine oxidase A and transglutaminase 2 that are both potential attractive targets of low molecular weight inhibitors in future HF therapy.</p

Valuable Secondary Habitats or Hazardous Ecological Traps? Environmental Risk Assessment of Minor and Trace Elements in Fly Ash Deposits across the Czech Republic

Deposits of coal combustion wastes, especially fly ash, are sources of environmental and health risks in industrial regions. Recently, fly ash deposits have been reported as habitat surrogates for some threatened arthropods in Central Europe. However, the potential environmental risks of fly ash have not yet been assessed in the region. We analysed concentrations of 19 minor and trace elements in 19 lignite combustion waste deposits in the Czech Republic. We assessed their environmental risks by comparison with the national and EU legislation limits, and with several commonly used indices. Over 50% of the samples exceeded the Czech national limits for As, Cu, V, or Zn, whilst only V exceeded the EU limits. For some studied elements, the high-risk indices were detected in several localities. Nevertheless, the measured water characteristics, the long-term presence of fly ash, previous leaching by acid rains, and the low amount of organic matter altogether can infer low biological availability of these elements. We presume the revealed high concentrations of some heavy metals at some studied sites can be harmful for some colonising species. Nevertheless, more ecotoxicological research on particular species is needed for final decision on their conservation potential for terrestrial and freshwater biota.info:eu-repo/semantics/publishedVersio

The Role of Renal Vascular Reactivity in the Development of Renal Dysfunction in Compensated and Decompensated Congestive Heart Failure

Background/Aims: Reduction of renal blood flow (RBF) is commonly thought to be a causative factor of renal dysfunction in congestive heart failure (CHF), but the exact mechanism of the renal hypoperfusion is not clear. Apart from the activation of neurohormonal systems controlling intrarenal vascular tone, the cause might be altered reactivity of the renal vasculature to endogenous vasoactive agents. Methods: To evaluate the role of this mechanism, we assessed by an ultrasonic transient-time flow probe maximum RBF responses to renal artery infusion of angiotensin II (ANG II), norepinephrine (NE) and acetylcholine (Ach) in healthy male rats and animals with compensated and decompensated CHF. CHF was induced by volume overload achieved by the creation of the aorto-caval fistula (ACF) in Hannover Sprague-Dawley rats. Results: Maximum responses in RBF to ANG II were similar in rats studied five weeks (compensated phase) and 20 weeks (decompensated phase) after ACF creation when compared to sham-operated rats. On the other hand, NE elicited larger maximum decreases in RBF in rats with CHF (five and 20 weeks post-ACF) than in sham-operated controls. We observed greater maximum vasodilatory responses to Ach only in rats with a compensated stage of CHF (five weeks post-ACF). Conclusion: Greater renal vasoconstrictor responsiveness to ANG II or reduced renal vasodilatation in response to Ach do not play a decisive role in the development of renal dysfunction in ACF rats with compensated and decompensated CHF. On the other hand, exaggerated renal vascular responsiveness to NE may be here a contributing causative factor, active in either CHF phase

Deployment and Measurement of Quality of Service Parameters for Triple Play Services in Optical Access Networks

Broadband telecommunication networks are the future of communication and distribution of multimedia services. Thanks to the high transmission potential and capacity of optical fibres, the optical networks are predestined to be more widely used in telecommunications considering the higher data rates through Triple Play services. This paper describes the impact of selected quality parameters on multimedia services defined as Triple Play within an optical network based on the EPON standard. The Triple Play services are then evaluated with measuring instruments and software applications according to QoS requirements and using evaluation methods based on generally defined standards. These are mainly subjective (listening and conversational) and objective (MSE, PSNR, SSIM) methods, or parameters (MOS) and factors (bitrate, delay, packet loss rate, jitter, BER) that are defined for the respective service. The values measured during the experimental tests were related to the limiting parameters of optical topology based on EPON networks for individual services according to their QoS requirements and objective user evaluation

The impact of phosphodiesterase‐5 inhibition or angiotensin‐converting enzyme inhibition on right and left ventricular remodeling in heart failure due to chronic volume overload

Abstract While phosphodiesterase‐5 inhibition (PED5i) may prevent hypertrophy and failure in pressure‐overloaded heart in an experimental model, the impact of PDE5i on volume‐overload (VO)‐induced hypertrophy is unknown. It is also unclear whether the hypertrophied right ventricle (RV) and left ventricle (LV) differ in their responsiveness to long‐term PDE5i and if this therapy affects renal function. The goal of this study was to elucidate the effect of PDE5i treatment in VO due to aorto‐caval fistula (ACF) and to compare PDE5i treatment with standard heart failure (HF) therapy with angiotensin‐converting enzyme inhibitor (ACEi). ACF/sham procedure was performed on male HanSD rats aged 8 weeks. ACF animals were randomized for PDE5i sildenafil, ACEi trandolapril, or placebo treatments. After 20 weeks, RV and LV function (echocardiography, pressure‐volume analysis), myocardial gene expression, and renal function were studied. Separate rat cohorts served for survival analysis. ACF led to biventricular eccentric hypertrophy (LV: +68%, RV: +145%), increased stroke work (LV: 3.6‐fold, RV: 6.7‐fold), and reduced load‐independent systolic function (PRSW, LV: −54%, RV: −51%). Both ACF ventricles exhibited upregulation of the genes of myocardial stress and glucose metabolism. ACEi but not PDE5i attenuated pulmonary congestion, LV remodeling, albuminuria, and improved survival (median survival in ACF/ACEi was 41 weeks vs. 35 weeks in ACF/placebo, p = .02). PDE5i increased cyclic guanosine monophosphate levels in the lungs, but not in the RV, LV, or kidney. PDE5i did not improve survival rate and cardiac and renal function in ACF rats, in contrast to ACEi. VO‐induced HF is not responsive to PDE5i therapy

Pollen anthropogenic indicators revisited using large-scale pollen and archaeological datasets: 12,000 years of human-vegetation interactions in central Europe

Tracing human-vegetation interactions that occurred in the past has always been one of the key topics of paleoecology. Here we use the pollen and archaeological databases available for the Czech Republic to determine links between individual pollen taxa and archaeological data and search for the spatial scales of comparability. The datasets include 1,500 pollen samples and 65,000 archaeological components covering the period from 12,000 to 700 cal. BP, divided into time windows of 250 years. Spearman’s rank correlation was used to measure the link between pollen and archaeological data at different sites. Using generalized additive models for the whole dataset, we explained the variance of pollen by archaeologically registered human activities and by two environmental variables. The first was the overall trend for each taxon in the Holocene representing the long-term dynamics of the species, the second was the elevation of pollen sites. Both factors affect species representation over the whole period studied or/and the area and cannot be statistically separated from human-induced changes. Both decrease the indicative strength of anthropogenic pollen; however, elevation did so more than the Holocene trend, since past human activities and elevation are strongly correlated and account for the first main gradient. The pollen taxa with a positive correlation with the level of past human activity, indicated by all methods, are: Plantago lanceolata, Artemisia and Amaranthaceae, resprouting edible trees that tolerate fire and pruning (Quercus) and pioneer trees (Pinus). Probability indicating the presence or absence of archaeological evidence when pollen of these species is present or absent is high (0.56–0.76). However, explained variability by the full model is low (0.01–0.09). Fagus, Carpinus and Abies expand during the late-successional stages after human disturbance, therefore their relationships to past human activity are negative when considering a 250-year time window. Secale does not correlate at the level of individual sites due to its late appearance during the Holocene. We ascribe the weak relationship between archaeological data and pollen of Cerealia to inconsistent determinations. The radius of comparability of pollen and archaeological evidence is around tens of kilometres due to the spatial resolution of archaeology is the area of a parish, but lower for herbaceous plants (15–20 km) than for trees (30–40 km). This critical comparison delimits overlaps and gaps between widely-used assumptions and data-based evidence

MELENOVSKY V: Effect of metformin therapy on cardiac function and survival in a volume-overload model of heart failure in rats.

Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg · kg − 1 of body weight · day − 1 , 0.5 % in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n = 6-12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n = 10-90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified &apos;free&apos; fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial 14 C-glucose and 14 C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2 + − 0.7 μg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF. Key words: AMP-activated protein kinase (AMPK), energy metabolism, heart failure, metformin, survival, volume overload. Abbreviations: ACC, acetyl-CoA carboxylase; ACF, aortocaval fistula; AMPK, AMP-activated protein kinase; HF, heart failure; i.p., intraperitoneally; KEGG, Kyoto Encyclopedia of Genes and Genomes; LVEDP, left ventricular end-diastolic pressure; LVEF, left ventricular ejection fraction; MET, metformin; NEFA, non-esterified &apos;free&apos; fatty acid; OCT, organic cation transporter; oGTT, oral glucose tolerance test; pACC, phosphorylated ACC; pAMPK, phosphorylated AMPK; PLAX, parasternal long-axis; PPAR, peroxisome-proliferator-activated receptor; PGC-1α, PPAR-γ coactivator-1α; PSAX, parasternal short-axis; tACC, total ACC; tAMPK, total AMPK. Correspondence: Dr Jan Benes (email [email protected]). INTRODUCTION Advanced HF (heart failure) is characterized not only by a depression of heart mechanical performance, but also by altered myocardial metabolism, attenuated expression of fatty acid oxidation genes [1,2] and by diminished oxidation of long-chain fatty acids [1,[3][4][5], which may contribute to diminished metabolic flexibility and to energetic deficiency that further promotes worsening of HF [6]. Targeting energetic substrate metabolism might thus serve as a target for novel therapeutic approaches to HF [7,8]. MET (metformin), a widely used antihyperglycaemic drug with insulin-sensitizing properties, could be a suitable candidate for metabolic HF therapy. MET lowers serum glucose by inhibiting liver gluconeogenesis, lowers circulating NEFAs (non-esterified &apos;free&apos; fatty acids) and improves insulin sensitivity. Some effects of MET can be explained by an activation of AMPK (AMPactivated protein kinase) [9], the enzyme that senses and regulates cellular energetic homoeostasis, but it is not likely to be the only mechanism of MET effects [10,11]. Administration of MET might also favourably affect mitochondrial function and increase mitochondrial biogenesis by activating PPAR (peroxisome-proliferatoractivated receptor)-α/PGC-1α (PPAR-γ coactivator-1α) [12]. Although MET is one of the most widely prescribed medications in human medicine, its effects on the heart are not well characterized. Until recently, MET use in patients with HF was contraindicated due to a theoretical risk of lactic acidosis. Non-randomized observational studies had suggested that MET-treated diabetics with HF may have lower mortality than those on other antidiabetic regimes [13,14]. Because non-diabetic HF patients also have insulin resistance [15] and NEFA elevation [16], MET might be helpful in the wider HF population. The use of MET for metabolic therapy of HF needs to be established in experimental settings. Volume overload represents a clinically relevant condition leading to HF, for example in aortic or mitral valve insufficiency. The rat model of chronic HF due to volume overload induced by ACF (aortocaval fistula) has been well characterized previously [17][18][19]. It shares many similarities with the natural course of human HF, including gradual development of the disease that proceeds through a stage of compensated hypertrophy followed by gradual decompensation into overt HF [19], neurohumoral activation, cardiac output redistribution [20], fluid retention with pulmonary congestion and impairment of myocardial efficiency [21]. On the other hand, volume-overload-induced HF has several features distinct from other HF models, including a lack of myocardial fibrosis and inflammation [22,23] and involvement of different signalling pathways (upregulation of Akt and Wnt signalling) compared with experimental myocardial infarction or pressure overload [23]. The aim of the present study was to test the hypothesis that chronic MET therapy would correct HFinduced metabolic abnormalities and improve cardiac performance and survival in the volume-overload HF rat model. MATERIALS AND METHODS Animal HF model HF was induced by volume overload from ACF using a needle technique [17,18]. Further details of the methods used can be found in the Supplementary Materials and methods section at http://www.clinsci. org/cs/121/cs1210029add.htm. Sham-operated controls underwent a similar procedure but without the creation of ACF. MET groups received 0.5 % MET (Teva Pharmaceuticals) mixed into the standard diet (normal salt/protein diet; 0.45 % NaCl, 19-21 % protein; SEMED), placebo (PL) groups received an identical diet but without MET. The study examined three rat cohorts, and each cohort had four randomly allocated groups: SH+PL (sham-operated without MET), SH+MET (sham-operated with MET), ACF+PL (ACF-without MET), ACF+MET (ACF with MET). The first cohort (n = 6-10/group) served for cardiac and mitochondrial function assessment, the second cohort (n = 6-8/group) served for organ metabolic studies and both cohorts were killed at week 21 after the ACF procedure. The third cohort (n = 10/SH groups, n = 90/ACF groups) was left free of any procedures and served for a survival analysis until week 52. The investigation conformed to the National Institutes of Health &apos;Guide for the care and use of laboratory animals &apos; (NIH Publication no. 85-23, 1996) and Animal protection law of the Czech Republic (311/1997), and was approved by the ethics committee at IKEM. Echocardiography and haemodynamics Animals were anaesthetized i.p. (intraperitoneally) with a ketamine/midazolam injection (50 mg and 5 mg/kg of body weight). Echocardiography was performed using a 7.5 MHz probe (Vivid System 5, GE), and end-systolic and end-diastolic sizes of the left ventricle together with wall thicknesses were measured in PLAX (parasternal long-axis) and PSAX (parasternal short-axis) projection, the size of the right ventricle in A4C (apical fourchamber) projection. Invasive haemodynamic evaluation was performed by F2 Millar catheter inserted into the aorta and left ventricle via the carotid artery. After the haemodynamic assessment, rats were killed by exsanguination, the coronary tree was flushed with icecold cardioplegic solution and left ventricle free wall samples were instantly flash frozen in liquid nitrogen for C The Authors Journal compilation C 2011 Biochemical Society Metformin therapy in volume-overload heart failure in rats 31 biochemical analyses or used for mitochondrial function assessment or electron microscopy. Myocardial biochemistry and ultrastructure Myocardial ATP content was measured in flash-frozen tissue using HPLC Mitochondrial function In the myocardial tissue homogenate, the maximal ADP-stimulated oxidative capacity of mitochondria was determined as the oxygen consumption rate with palmitoylcarnitine (12.5 μM)+malate (3 mM)+glutamate (10 mM)+succinate (10 mM) using a high-resolution oxygraph-2k (OROBOROS) Myocardial gene expression Total RNA was isolated by RNeasy Micro Kit (Qiagen), and 200 ng of total RNA was used for the amplification procedure and 1.5 μg of amplified RNA was hybridized on the chip according to the manufacturer&apos;s procedure. Microarray analysis The raw data (.TIFF image files) were analysed using &apos;beadarray&apos; package [31] of the &apos;Bioconductor&apos; [32] within the R environment (http://www.r-project.org) Systemic and organ metabolic analyses MET serum level was checked in tail-vein serum at week 11 in the ACF+MET (n = 12) and SH+MET (n = 18) groups. The MET level was measured using an HPLC method with separation on a silica column (ThermoQuest) with spectrophotometric detection. oGTTs (oral glucose tolerance tests) were performed in all groups at week 20 using an oral glucose load of 300 mg/100 g of body weight by gavage after overnight fasting. Blood was drawn from the tail without anaesthesia before the glucose load (0-min time point) and at 30, 60 and 120 min thereafter. Serum glucose was measured by the glucoseoxidase assay and serum NEFAs were determined using a colorimetric assay (Roche). Serum insulin was determined using a rat insulin ELISA kit (Mercodia). Tissue triacylglycerols were measured in liquid nitrogenpowdered tissues after chloroform/methanol extraction using the enzymatic assay (Pliva-Lachema); this assay was also used for serum triacylglycerols. The glycogen in the heart was measured after KOH extraction Glycogen synthesis and glucose oxidation in the heart and muscle Basal and insulin-stimulated 14 C-glucose incorporation into glycogen and CO 2 was determined ex vivo in isolated diaphragm Fatty acid oxidation in the heart Fatty acid oxidation in the heart tissue muscles and heart slices was determined by measuring the incorporation of 14 C-palmitic acid into CO 2 Statistics Two-way ANOVA with Bonferroni post-hoc adjustment was used to compare the effects of surgery and MET treatment. Survival analysis was performed using the Gehan-Breslow-Wilcoxon test. P values &lt;0.05 were considered statistically significant. RESULTS MET serum assessment MET serum level at week 11 was 2.2 + − 0.7 μg/ml (13 + − 4.15 nmol/ml) in the ACF+MET group (n = 12) and 1.9 + − 2.7 μg/ml (11.6 + − 16.1 nmol/ml) in the Organ morphometry, haemodynamics and echocardiography All groups had similar body weights and tibial lengths. Both ACF groups had marked heart hypertrophy ( ACF animals had marked enlargement of both ventricles Metabolic assessment Glucose and glycogen metabolism When assessed using oGTTs, all the groups showed similar glucose levels throughout the test and preserved postprandial glycaemic regulation ( Lipid metabolism Serum and liver triacylglycerols were similar in all groups Mitochondrial function Cytochrome c oxidase (complex IV) and citrate synthase activities ( Electron microscopy showed no apparent structural abnormalities, and the proportions occupied by myofibrils, mitochondria and cytosol were similar in all groups (Supplementary AMPK signalling To characterize the activity of the AMPK-regulatory cascade, total content and phosphorylation of both AMPK and its target ACC were assessed by Western blotting. At the level of AMPK, ACF animals showed significantly higher contents of both tAMPK and pAMPK than sham groups. However, the ratio between pAMPK and tAMPK (pAMPK/tAMPK) was similar, independent of ACF procedure or MET treatment ( Myocardial gene expression analysis Out of 23 401 detected transcripts, we observed no difference between ACF+MET and ACF+PL, which was in striking contrast with fistula-induced transcriptional changes (ACF+PL compared with SH+PL), where 128 transcripts were differentially expressed (99 up-regulated and 29 down-regulated; Storey&apos;s q value &lt;0.05 and 2-fold or greater change in intensity). A heatmap with all differentially expressed transcripts is shown in Supplementary Survival None of the control animals died throughout the study. The first deaths in the ACF groups occurred between weeks 10 and 15, and 77.2 % of the ACF+PL (80.5 % of ACF+MET) animals were dead by the end of the study. Median survival was 45.5 weeks in the ACF+PL group and 44.5 weeks in the ACF+MET group. MET therapy had no effect on survival in ACF animals ( DISCUSSION The present study shows that chronic volume overloadinduced HF is associated with lower glycogen synthesis in the skeletal muscle (diaphragm), lower heart triacylglycerol content, higher plasma NEFAs, lower plasma insulin level and depressed myocardial glucose and palmitate oxidation. Long-term administration of the antihyperglycaemic drug MET normalized elevated NEFAs, further decreased myocardial glucose oxidation and increased myocardial palmitate oxidation, but had no effect on myocardial AMPK activation, ATP content, mitochondrial function or morphology. No relevant improvement in cardiac performance or long-term survival was observed in MET-treated HF animals. Despite several recent studies reported beneficial effect of MET in other non-diabetic HF models Peripheral and systemic MET effects At the systemic level, MET lowered basal and postprandial circulating NEFAs due to increased NEFA utilization and perhaps also due to diminished NEFA release from adipose tissue because of known inhibitory effects of MET on catecholamine-stimulated lipolysis Metformin therapy in volume-overload heart failure in rats Figure 6 Survival analysis insulin-mediated glycogen synthesis in skeletal muscle, which is a measure of insulin sensitivity. Cardiac effects of MET In the heart, MET treatment significantly increased the palmitate oxidation that was attenuated in the ACF+PL group. Diminished oxidation of long-chain fatty acids and down-regulation of enzymes of fatty acid oxidation in the heart have been repeatedly described both in HF patients [1] and in animal HF models [3,4, Comparison with other HF studies The absence of benefit of MET on cardiac function or survival in ACF-induced HF is in contrast with other recently published studies in other HF models. Gundewar et al. [44] examined the effect of very low dose MET (125 μg · kg − 1 of body weight · day − 1 , i.p.) on cardiac function and survival in mice subjected to LAD (left anterior descending coronary artery) ligation. MET extended the survival at 4 weeks by 47 %, improved left ventricular remodelling and corrected MI (myocardial infarction)-induced defects in mitochondrial respiration and ATP synthesis. Despite the fact that the administered MET dose was lower by three orders of magnitude than in our present study (i.e. 300 mg of MET · kg − 1 of body weight · day − 1 ) or than is normally used in humans, authors were able to detect increased phosphorylation of AMPK, eNOS (endothelial NO synthase) and increased expression of PGC-1α in the heart. In another study, Sasaki et al. [42] examined the effect of 4-week oral MET therapy (100 mg · kg − 1 of body weight · day − 1 ) in the tachypacing HF model in dogs. Compared with placebo, MET improved LVEF, slowed HF progression and decreased myocardial apoptosis via an AMPKdependent mechanism Lack of a protecting effect of MET in a volume-overload HF model The mechanism of MET action is still incompletely understood. One possibility suggests an activation of AMPK that turns on energy-providing and turns off energy-consuming metabolic pathways [9, [44], we did not find any increase in AMPK activity or decrease in oxygen consumption rate or respiratory control index. It appears that in contrast with pressure overload, volume overload does not sufficiently alter resting mitochondrial function [23], and thus, it may lack the substrate for MET action. Finally, no insulin resistance was observed in our volume-overload HF model, so the lack of insulin resistance might also imply a missing substrate for MET action. Despite all these specifics of the model, we should be aware that HF is a nonuniform syndrome, and it should be studied in subsets. Volume overload is a clinically important condition, and its most common form (mitral insufficiency) often complicates other heart diseases and independently increases mortality C The Authors Journal compilation C 2011 Biochemical Society Metformin therapy in volume-overload heart failure in rats 39 Metabolic abnormalities in the ACF HF model The ACF-induced HF model showed several specific features. Despite gene expression analysis showing an extensive down-regulation of the β-oxidation pathway and several respiratory chain components in ACF, the ATP-generating capacity of mitochondria in surplus oxygen and substrates was preserved. This might be explained by a redundancy in enzyme activities and longer half-life [4, [58] who showed normal myocardial oxidative capacity in compensated ACF-induced HF (week 15), but marked sensitivity of the heart to hypoxia, indicating preserved ATP levels at rest, but attenuated energetic reserve during increased stress. Low myocardial triacylglycerol content in ACF hearts, also reported for the first time, is probably related to limited re-esterification of triacylglycerols due to low availability of NADPH In conclusion, the results of the present study show that long-term MET therapy in rats with HF due to volume overload decreases circulating NEFAs, decreases myocardial glucose oxidation and increases myocardial palmitate oxidation, but these effects have neutral impact on cardiac performance and survival in HF. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. Prolonged exposure of a large group of severely symptomatic HF animals to highdose MET led to no apparent increase in mortality, which provides robust data regarding the toxicology of ME