We recently demonstrated early metabolic alterations in the dystrophin-deficient mdx heart that precede overt cardiomyopathy and may represent an early "subclinical" signature of a defective nitric ...oxide (NO)/cGMP pathway. In this study, we used genetic and pharmacological approaches to test the hypothesis that enhancing cGMP, downstream of NO formation, improves the contractile function, energy metabolism, and sarcolemmal integrity of the mdx heart. We first generated mdx mice overexpressing, in a cardiomyocyte-specific manner, guanylyl cyclase (GC) (mdx/GC⁺/⁰). When perfused ex vivo in the working mode, 12- and 20-week-old hearts maintained their contractile performance, as opposed to the severe deterioration observed in age-matched mdx hearts, which also displayed two to three times more lactate dehydrogenase release than mdx/GC⁺/⁰. At the metabolic level, mdx/GC⁺/⁰ displayed a pattern of substrate selection for energy production that was similar to that of their mdx counterparts, but levels of citric acid cycle intermediates were significantly higher (36 ± 8%), suggesting improved mitochondrial function. Finally, the ability of dystrophin-deficient hearts to resist sarcolemmal damage induced in vivo by increasing the cardiac workload acutely with isoproterenol was enhanced by the presence of the transgene and even more so by inhibiting cGMP breakdown using the phosphodiesterase inhibitor sildenafil (44.4 ± 1.0% reduction in cardiomyocyte damage). Overall, these findings demonstrate that enhancing cGMP signaling, specifically downstream and independent of NO formation, in the dystrophin-deficient heart improves contractile performance, myocardial metabolic status, and sarcolemmal integrity and thus constitutes a potential clinical avenue for the treatment of the dystrophin-related cardiomyopathies.
Introduction La leucine est un acide aminé branché capable d’induire une résistance à l’insuline dans le muscle et le tissu adipeux. Le mécanisme proposé pour expliquer l’action inhibitrice de la ...leucine implique la voie mTOR/p70S6K/IRS-1. Cette voie peut être activée par la leucine et est impliquée dans l’activation d’une boucle de rétrocontrôle négatif de l’insuline. Sachant que l’insulino-résistance participe à l’établissement d’une cardiomyopathie diabétique, nous avons étudié l’effet inhibiteur de la leucine dans le cardiomyocyte. Matériels et méthodes Des cardiomyocytes de rats adultes en culture primaire ont été prétraités avec différentes concentrations de leucine (entre 1 et 10 mM) pendant différentes périodes d’incubation avant d’être exposés à l’insuline (3×10−9 M, 30 min). Résultats En absence de leucine, l’insuline induit une augmentation du captage de glucose (0,31 ± 0,04 vs. 0,05 ± 0,01 μmoles/mg. h). Celle-ci corrèle avec l’augmentation de la phosphorylation de PKB et AS160, connus pour réguler le captage de glucose en aval de l’insuline. Une pré-incubation de 1 h en leucine active la voie mTOR/p70S6K résultant en l’inhibition d’IRS-1 situé en amont dans la voie de signalisation insulinique. Ceci s’accompagne d’une diminution significative de la phosphorylation de PKB et AS160. Etonnamment, le captage de glucose stimulé par l’insuline est préservé malgré cette inhibition (0,31 ± 0,05 μmoles/mg. h). D’autre part, une pré-incubation plus longue (14 h) en leucine induit une diminution drastique du captage de glucose (0,056 ± 0,01 μmoles/mg. h). La rapamycine, un inhibiteur de mTOR/p70S6K, n’empêche pas cette inhibition. En outre, un analogue non métabolisable de la leucine (BCH) stimule la voie mTOR/p70S6K sans avoir d’effet sur le captage de glucose. En revanche, les intermédiaires du catabolisme de la leucine, l’α- cétoisocaproate, l’acétoacétate, le β-hydroxybutyrate, inhibent le transport de glucose de manière similaire à la leucine. Conclusion Le catabolisme de la leucine réduit le transport de glucose indépendamment de la signalisation insulinique.
There has been a resurgence of interest for the field of cardiac metabolism catalysed by the increased need for new therapeutic targets for patients with heart failure. The primary focus of research ...in this area to date has been on the impact of substrate selection for oxidative energy metabolism; however, anaplerotic metabolism also has significant interest for its potential cardioprotective role. Anaplerosis refers to metabolic pathways that replenish the citric acid cycle intermediates, which are essential to energy metabolism; however, our understanding of the role and regulation of this process in the heart, particularly under pathophysiological conditions, is very limited. Therefore, the goal of this article is to provide a foundation for future directions of research on cardiac anaplerosis and heart disease. We include an overview of anaplerotic metabolism, a critical evaluation of current methods available for its quantitation in the intact heart, and a discussion of its role and regulation both in health and disease as it is currently understood based mostly on animal studies. We also consider genetic diseases affecting anaplerotic pathways in humans and acute intervention studies with anaplerotic substrates in the clinics. Finally, as future perspectives, we will share our thoughts about potential benefits and practical considerations on modalities of interventions targeting anaplerosis in heart disease, including heart failure.
Abstract only
Background & Objective
The integration of genome wide association studies (GWAS) with metabolomics, termed mGWAS, offers a tremendous opportunity to gain insights into the genetic ...control of metabolism. A current bottleneck of mGWAS is the biological interpretation of the large amount of generated data, which include associations between SNP‐annotated genes and metabolites. This project aimed at developing a robust bioinformatic package to quantitatively annotate gene‐metabolite association pairs through metabolic pathway mapping.
Methods & Results
A R package, PathQuant, has been developed following Bioconductor guidelines to ensure reproducibility of results and easy growth. The current version of PathQuant uses as input a list of gene‐metabolite associations pairs and enables: (i) gene classification into enzymatic
vs.
non‐enzymatic category using the Enzyme Commission number (EC) as annotation; (ii) mapping of metabolic gene‐metabolite pairs on a graph model of human KEGG metabolic pathway maps, where genes are edges and metabolites are nodes, and (iii) calculation of shortest reactional distances between gene‐metabolite pairs with either graphical visualization or textual tables as outputs. As a proof‐of concept, PathQuant was used to map mGWAS gene‐metabolite associations data from Shin
et al.
(2014) using all KEGG metabolism pathway maps, which include the metabolism reconstruction overview map and specific individual pathway maps. We applied the method for 86 reported associations between 50 enzymatic genes and 66 metabolites measured in plasma. When mapped to KEGG metabolism overview (
Fig. 1
), these associations are mostly found in “Energy” (purple), “Amino acids” (orange) and “Nucleotides” (green) pathway classes. PathQuant annotated finite numerical distances between 28 genes and 31 metabolites involved in 38 associations of which 36 had a short distance, between 0 and 5, which indicates that the reaction catalyzed by the gene encoded enzyme was not more than 5 reactions apart from that involving its associated metabolite. For 17 genes and 27 metabolites, representing 27 pairs, we were unable to calculate finite numerical distances using PathQuant, which is attributed to current limitations of human KEGG pathway maps, such as (i) missing enzymes annotated in humans creating disconnected subgraphs within the maps, (ii) the presence of a gene and a metabolite from a given pair on different maps and (iii) limited coverage of lipid metabolic diversity in KEGG pathway maps (
Fig. 1
). While improvement of the tool's capacity for annotation could address limitations (i) and (ii), there were, however, 4 genes and 12 metabolites (21 pairs) that were not present on any KEGG pathways.
Conclusion
PathQuant provides a high‐throughput approach to link and objectively annotate gene‐metabolite pairs. Future work aims at upgrading PathQuant by refining the annotation and improving coverage of pathway classes by including other pathway databases than KEGG as well as to expand the annotation to genes involved in cell signaling pathways.
Support or Funding Information
Genome Canada, Genome Québec, Genome British Columbia, Agilent Technologies, CIHR, Crohn's and Colitis Canada, Government of Canada.
BACKGROUND: To improve the prevention, treatment and risk prediction of cardiovascular diseases, genetic markers and gene–diet interactions are currently being investigated. The Montreal Heart ...Institute (MHI) Biobank is suitable for such studies because of its large sample size (currently, n = 17 000), the availability of biospecimens, and the collection of data on dietary intakes of saturated (SFAs) and n‐3 and n‐6 polyunsaturated (PUFAs) fatty acids estimated from a 14‐item food frequency questionnaire (FFQ). We tested the validity of the FFQ by correlating dietary intakes of these fatty acids with their red blood cell (RBC) content in MHI Biobank participants. METHODS: Seventy‐five men and 75 women were selected from the Biobank. We successfully obtained RBC fatty acids for 142 subjects using gas chromatography coupled to mass spectrometry. Spearman correlation coefficients were used to test whether SFA scores and daily intakes (g day⁻¹) of n‐3 and n‐6 PUFAs correlate with their RBC content. RESULTS: Based on covariate‐adjusted analyses, intakes of n‐3 PUFAs from vegetable sources were significantly correlated with RBC α‐linolenic acid levels (ρ = 0.23, P = 0.007), whereas n‐3 PUFA intakes from marine sources correlated significantly with RBC eicosapentaenoic acid (ρ = 0.29, P = 0.0008) and docosahexaenoic acid (ρ = 0.41, P = 9.2 × 10–⁷) levels. Intakes of n‐6 PUFAs from vegetable sources correlated with RBC linoleic acid (ρ = 0.18, P = 0.04). SFA scores were not correlated with RBC total SFAs. CONCLUSIONS: The MHI Biobank 14‐item FFQ can appropriately estimate daily intakes of n‐3 PUFAs from vegetable and marine sources, as well as vegetable n‐6 PUFAs, which enables the possibility of using these data in future studies.
ABSTRACT
Although absence of the cytoskeletal protein dystrophin leads to dilated cardiomyopathy in humans, the functional role of dystrophin in cardiac muscle remains undefined. We have addressed ...the hypothesis that dystrophin could help protect the heart against injury and contractile dysfunction induced by mechanical stress. In normal and dystrophin‐deficient (mdx) mice, cardiac mechanical stress was first manipulated ex vivo in a perfused working heart preparation. Despite an afterload level in the normal physiologic range, ex vivo perfused mdx hearts developed severe contractile dysfunction and nonischemic tissue damage, as is shown by excessive LDH release without a rise in coronary lactate. Injury to dystrophin‐deficient hearts was significantly correlated with cardiac work, and reducing the afterload level improved contractility and prevented injury in mdx hearts studied ex vivo. The response to mechanical stress in vivo was also assessed by using the vital dye Evans blue, which penetrates into cardiomyocytes with a disrupted sarcolemma. In the mdx group only, cardiomyocyte injury was increased markedly by acute elevations of mechanical stress induced by isoproterenol or brief aortic occlusion. Strikingly accelerated mortality and cardiac necrosis were also observed in the mdx group subjected to chronically increased cardiac mechanical stress via subtotal aortic constriction. Taken together, our results provide the first direct evidence that dystrophin serves to protect cardiomyocytes from mechanical stress and workload‐induced damage. Accordingly, reducing cardiac work in patients with dystrophin deficiency could be beneficial not only in treating established cardiomyopathy, but also in preventing the onset of cardiac disease.
Over the past 20 years, stable isotopes combined with isotopomer analysis have proven to be a powerful approach to probe the dynamics of metabolism in various biological systems, including the heart. ...The aim of this paper is to demonstrate how isotopomer analysis of metabolic fluxes can provide novel insights into the myocardial phenotype. Specifically, building on our past experience using NMR spectroscopy and GC-MS as applied to investigations of cardiac energy metabolism, we highlight specific complex metabolic networks that would not be predicted by classical biochemistry or by static measurements of metabolite, protein and mRNA levels.
Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC), a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase ...(COX) and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol), or modulate fatty acid (L-carnitine) or Krebs cycle metabolism (propionate) are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol) exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise questions about the nature of the diets, particularly excess fat intake, as well as on the use of antioxidants in patients with LSFC and, possibly, other COX defects.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Increasing evidence indicates that protein-aldehyde adducts involving mostly 4-hydroxynonenal could be causally involved in both pathophysiological and adaptive events following an oxidative stress ...insult such as ischemia/reperfusion. The goal of this study was to assess if isotope dilution chromatography-mass spectrometry can be used to quantitate changes in the cardiac levels of 4-hydroxynonenal and 1,4-dihydroxynonene, one of its major metabolites, bound to thiol proteins during ischemia/reperfusion. For this purpose, we modified a previously published method
1 to include treatment with Raney Nickel, which specifically cleaves thioether linkages. Our study model was the isolated Langendorff-perfused rat heart subjected to various ischemia/reperfusion protocols. Hearts perfused under normoxia contained small amounts of protein-bound 4-hydroxynonenal and 1,4-dihydroxynonene (1.38 ± 0.29 and 2.69 ± 0.17 nmol/g wet weight, respectively). The accumulation of these adducts after global ischemia depended on the severity of the ischemic insult up to a plateau and was not exacerbated by reperfusion. In conclusion, our method allows the quantification of time-dependent changes in 4-hydroxynonenal and 1,4-dihydroxynonene bound to proteins via thioether linkage in ischemic/reperfused heart tissues. The presence of protein-bound 1,4-dihydroxynonene in heart tissues suggests that this organ can detoxify protein-bound 4-hydroxynonenal.
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