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1.  Effects of perhexiline-induced fuel switch on the cardiac proteome and metabolome 
Perhexiline is a potent anti-anginal drug used for treatment of refractory angina and other forms of heart disease. It provides an oxygen sparing effect in the myocardium by creating a switch from fatty acid to glucose metabolism through partial inhibition of carnitine palmitoyltransferase 1 and 2. However, the precise molecular mechanisms underlying the cardioprotective effects elicited by perhexiline are not fully understood. The present study employed a combined proteomics, metabolomics and computational approach to characterise changes in murine hearts upon treatment with perhexiline. According to results based on difference in-gel electrophoresis, the most profound change in the cardiac proteome related to the activation of the pyruvate dehydrogenase complex. Metabolomic analysis by high-resolution nuclear magnetic resonance spectroscopy showed lower levels of total creatine and taurine in hearts of perhexiline-treated mice. Creatine and taurine levels were also significantly correlated in a cross-correlation analysis of all metabolites. Computational modelling suggested that far from inducing a simple shift from fatty acid to glucose oxidation, perhexiline may cause complex rebalancing of carbon and nucleotide phosphate fluxes, fuelled by increased lactate and amino acid uptake, to increase metabolic flexibility and to maintain cardiac output. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".
Graphical abstract
Highlights
► Mice were fed perhexiline to achieve steady state concentrations. ► Hearts were analysed using a combined proteomic and metabolomic approach. ► Computer modelling was used to cross-validate the findings. ► Perhexiline has more wide-ranging and complex metabolic effects than previously thought.
doi:10.1016/j.yjmcc.2012.12.014
PMCID: PMC3573230  PMID: 23277191
CPT, carnitine palmitoyltransferase; DIGE, difference in-gel electrophoresis; FCS, foetal calf serum; FDR, false discovery rate; GO, Gene ontology; 1H NMR, proton nuclear magnetic resonance spectroscopy; LC-MS/MS, liquid chromatography tandem mass spectrometry; TCA, tricarboxylic acid; Metabolomics; Proteomics; Cardioprotection; Metabolism; Heart failure
2.  Novel Role of ADAMTS-5 Protein in Proteoglycan Turnover and Lipoprotein Retention in Atherosclerosis* 
The Journal of Biological Chemistry  2012;287(23):19341-19345.
Background: In atherosclerosis, proteoglycan accumulation results in increased lipoprotein retention.
Results: ADAMTS-5 is reduced in aortas of apolipoprotein E-null mice. ADAMTS-5 deficiency impairs processing of vascular proteoglycans, and ADAMTS-5 activity affects proteoglycan-mediated lipoprotein retention.
Conclusion: ADAMTS-5 regulates vascular proteoglycan catabolism and alters lipoprotein retention.
Significance: This is the first study implicating ADAMTS-5 proteolytic activity in atherosclerosis.
Atherosclerosis is initiated by the retention of lipoproteins on proteoglycans in the arterial intima. However, the mechanisms leading to proteoglycan accumulation and lipoprotein retention are poorly understood. In this study, we set out to investigate the role of ADAMTS-5 (a disintegrin and metalloprotease with thrombospondin motifs-5) in the vasculature. ADAMTS-5 was markedly reduced in atherosclerotic aortas of apolipoprotein E-null (apoE−/−) mice. The reduction of ADAMTS-5 was accompanied by accumulation of biglycan and versican, the major lipoprotein-binding proteoglycans, in atherosclerosis. ADAMTS-5 activity induced the release of ADAMTS-specific versican (DPEAAE441) and aggrecan (374ALGS) fragments as well as biglycan and link protein from the aortic wall. Fibroblast growth factor 2 (FGF-2) inhibited ADAMTS-5 expression in isolated aortic smooth muscle cells and blocked the spontaneous release of ADAMTS-generated versican and aggrecan fragments from aortic explants. In aortas of ADAMTS-5-deficient mice, DPEAAE441 versican neoepitopes were not detectable. Instead, biglycan levels were increased, highlighting the role of ADAMTS-5 in the catabolism of vascular proteoglycans. Importantly, ADAMTS-5 proteolytic activity reduced the LDL binding ability of biglycan and released LDL from human aortic lesions. This study provides the first evidence implicating ADAMTS-5 in the regulation of proteoglycan turnover and lipoprotein retention in atherosclerosis.
doi:10.1074/jbc.C112.350785
PMCID: PMC3365970  PMID: 22493487
Atherosclerosis; Cardiovascular Disease; Extracellular Matrix; Lipoprotein; Protease; Proteoglycan; ADAMTS-5
3.  The interrelationship of mycophagous small mammals and ectomycorrhizal fungi in primeval, disturbed and managed Central European mountainous forests 
Oecologia  2012;170(2):395-409.
Small forest dwelling mammals are considered to be major consumers and vectors of hypogeous ectomycorrhizal (ECM) fungi, which have lost the ability of active spore discharge. Fungal spore dispersal by mycophagy is deemed an important process involved in forest regeneration, resilience and vitality, primarily based on evidence from Australia and the Pacific Northwestern USA, but is poorly known for Central European mountainous forests thus far. Small mammal mycophagy was investigated by live trapping and microscopical analysis of faecal samples. All small mammal species recorded (Myodes glareolus, Microtus agrestis, Pitymys subterraneus, Apodemus spp., Glis glis, Sorex spp.) had ingested spores of ECM fungi, albeit in varying amounts. My. glareolus was found to be the most important vector of ECM fungal spores, both in quantity and diversity. Species of the genus Sorex seem to play a hitherto underestimated role as dispersers of fungal spores. Glis glis is likely to be an important vector owing to its large home range. Hypogeous ECM basidiomycetes accounted for most spores found in the faecal samples. The frequency of various genera of hypogeous ECM ascomycetes and ECM epigeous fungi was much lower. Comparison with null models indicated a non-random structure of the mycophagy network similar to other mutualistic bipartite networks. Mycophagy can be considered (1) to contribute to nutrition of small forest mammals, (2) to play a pivotal role for forest regeneration and functioning by providing mycorrhizal inoculum to tree seedlings and (3) to be vital for reproduction and diversity of the still poorly known hypogeous fungi.
Electronic supplementary material
The online version of this article (doi:10.1007/s00442-012-2303-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s00442-012-2303-2
PMCID: PMC3439606  PMID: 22466900
Rodents; Shrews; Truffles; Mutualism; Nestedness
4.  Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution* 
Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were “skinned,” and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.
doi:10.1074/mcp.M900275-MCP200
PMCID: PMC2849712  PMID: 20037178
5.  Exacerbated vein graft arteriosclerosis in protein kinase Cδ–null mice 
Journal of Clinical Investigation  2001;108(10):1505-1512.
Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCδ knockout mice and performed vein bypass grafts on these animals. PKCδ–/– mice developed normally and were fertile. Vein segments from PKCδ–/– mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCδ+/+ vein grafts. Arteriosclerotic lesions in PKCδ–/– mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCδ–/– mice. SMCs derived from PKCδ–/– aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCδ–/– relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCδ maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.
PMCID: PMC209416  PMID: 11714742

Results 1-5 (5)