System identification to analyse changed kinetics of SERCA in intact rat heart

\u3cp\u3e A mechanistic-based model has been derived of calcium handling in the intact heart. This model incorporates the quantitatively most important processes involved in beat-to-beat calcium homeostasis. Based on a priori physiological information the model has been reduced to yield (kinetic) parameters that could be estimated using time-series data of the free calcium concentration in the sarcoplasma. Observations of the dynamics of the overall system were translated into the underlying mechanisms. Experiments in which the most important calcium extrusion pump (Sarcoplasmic Reticulum Ca \u3csup\u3e2+\u3c/sup\u3e -ATPase, SERCA) was disturbed have been successfully analysed and interpreted using model and identification. \u3c/p\u3

Evolution of the family of intracellular lipid binding proteins in vertebrates

Members of the family of intracellular lipid binding proteins (iLBPs) have been implicated in cytoplasmic transport of lipophilic ligands, such as long-chain fatty acids and retinoids. iLBPs are low molecular mass proteins (14-16kDa) sharing a common structural fold. The iLBP family likely arose through duplication and diversification of an ancestral iLBP gene. Phylogenetic analysis undertaken in the present study indicates that the ancestral iLBP gene arose after divergence of animals from fungi and plants. The first gene duplication was dated around 930 millions of years ago, and subsequent duplications in the succeeding 550 millions of years gave rise to the 16 iLBP types currently recognized in vertebrates. Four clusters of proteins, each binding a characteristic range of ligands, are evident from the phylogenetic tree. Evolution of different binding properties probably allowed cytoplasmic trafficking of distinct ligands. It is speculated that recruitment of an iLBP during evolution of animals enabled the mitochondrial oxidation of long-chain fatty acid

Intra-section analysis of human coronary arteries reveals a potential role for micro- calcifications in macrophage recruitment in the early stage of atherosclerosis

Background\u3cbr/\u3e\u3cbr/\u3eVascular calcification is associated with poor cardiovascular outcome. Histochemical analysis of calcification and the expression of proteins involved in mineralization are usually based on whole section analysis, thereby often ignoring regional differences in atherosclerotic lesions. At present, limited information is available about factors involved in the initiation and progression of atherosclerosis.\u3cbr/\u3e\u3cbr/\u3eAim of This Study\u3cbr/\u3e\u3cbr/\u3eThis study investigates the intra-section association of micro-calcifications with markers for atherosclerosis in randomly chosen section areas of human coronary arteries. Moreover, the possible causal relationship between calcifying vascular smooth muscle cells and inflammation was explored in vitro.\u3cbr/\u3e\u3cbr/\u3eTechnical Approach\u3cbr/\u3e\u3cbr/\u3eTo gain insights into the pathogenesis of atherosclerosis, we performed analysis of the distribution of micro-calcifications using a 3-MeV proton microbeam. Additionally, we performed systematic analyses of 30 to 40 regions of 12 coronary sections obtained from 6 patients including histology and immuno-histochemistry. Section areas were classified according to CD68 positivity. In vitro experiments using human vascular smooth muscle cells (hVSMCs) were performed to evaluate causal relationships between calcification and inflammation.\u3cbr/\u3e\u3cbr/\u3eResults\u3cbr/\u3e\u3cbr/\u3eFrom each section multiple areas were randomly chosen and subsequently analyzed. Depositions of calcium crystals at the micrometer scale were already observed in areas with early pre-atheroma type I lesions. Micro-calcifications were initiated at the elastica interna concomitantly with upregulation of the uncarboxylated form of matrix Gla-protein (ucMGP). Both the amount of calcium crystals and ucMGP staining increased from type I to IV atherosclerotic lesions. Osteochondrogenic markers BMP-2 and osteocalcin were only significantly increased in type IV atheroma lesions, and at this stage correlated with the degree of calcification. From atheroma area type III onwards a considerable number of CD68 positive cells were observed in combination with calcification, suggesting a pro-inflammatory effect of micro-calcifications. In vitro, invasion assays revealed chemoattractant properties of cell-culture medium of calcifying vascular smooth muscle cells towards THP-1 cells, which implies pro-inflammatory effect of calcium deposits. Additionally, calcifying hVSMCs revealed a pro-inflammatory profile as compared to non-calcifying hVSMCs.\u3cbr/\u3e\u3cbr/\u3eConclusion\u3cbr/\u3e\u3cbr/\u3eOur data indicate that calcification of VSMCs is one of the earliest events in the genesis of atherosclerosis, which strongly correlates with ucMGP staining. Our findings suggest that loss of calcification inhibitors and/or failure of inhibitory capacity is causative for the early precipitation of calcium, with concomitant increased inflammation followed by osteochondrogenic transdifferentiation of VSMCs.\u3cbr/\u3

Acute stimulation of glucose transport by histamine in cardiac microvascular endothelial cells

AbstractThe purpose of the present work was to study the acute regulation of glucose uptake in cultured cardiac endothelial cells (CEC). Two types of potential stimuli were considered: (1) agents that are known to acutely stimulate glucose transport (i.e., within minutes) in fat and muscle tissues and (2) agents that influence endothelial cell function. Among the former agents, neither insulin, nor catecholamines (adrenaline, dopamine, phenylephrine), nor serotonin affected the rate of glucose transport in CEC, while SH-group reagents (phenylarsine oxide, diamide or menadione) were inhibitory. Among the factors of the second group that were tested (heparin, ADP, histamine, bradykinin), histamine was found to stimulate glucose transport in CEC by 10–50%. This effect was concentration-dependent (with an EC50 value ⋟ 12 μM) and reached a maximum within 5 min upon histamine addition. This stimulation of glucose transport was suppressed by pyrilamine (100 nM), a specific H1-receptor antagonist, but not by cimetidine (100 μM), a H2-selective antagonist. Northern blot and Western blot analysis of CEC extracts revealed the presence of the ubiquitous glucose transporter isoform GLUT1 mRNA and protein, but not of the ‘insulin-regulatable’ isoform GLUT4. In conclusion, this is the first report on an acute stimulation of glucose transport in cardiac endothelial cells, in particular, and in an insulin-unresponsive cell type, in general. The effect of histamine is most likely mediated by H1-receptors and cannot be accounted for by a recruitment of GLUT4

Hypertension Is a Conditional Factor for the Development of Cardiac Hypertrophy in Type 2 Diabetic Mice

<div><p>Background</p><p>Type 2 diabetes is frequently associated with co-morbidities, including hypertension. Here we investigated if hypertension is a critical factor in myocardial remodeling and the development of cardiac dysfunction in type 2 diabetic db/db mice.</p><p>Methods</p><p>Thereto, 14-wks-old male db/db mice and non-diabetic db/+ mice received vehicle or angiotensin II (AngII) for 4 wks to induce mild hypertension (n = 9–10 per group). Left ventricular (LV) function was assessed by serial echocardiography and during a dobutamine stress test. LV tissue was subjected to molecular and (immuno)histochemical analysis to assess effects on hypertrophy, fibrosis and inflammation.</p><p>Results</p><p>Vehicle-treated diabetic mice neither displayed marked myocardial structural remodeling nor cardiac dysfunction. AngII-treatment did not affect body weight and fasting glucose levels, and induced a comparable increase in blood pressure in diabetic and control mice. Nonetheless, AngII-induced LV hypertrophy was significantly more pronounced in diabetic than in control mice as assessed by LV mass (increase +51% and +34%, respectively, p<0.01) and cardiomyocyte size (+53% and +31%, p<0.001). This was associated with enhanced LV mRNA expression of markers of hypertrophy and fibrosis and reduced activation of AMP-activated protein kinase (AMPK), while accumulation of Advanced Glycation End products (AGEs) and the expression levels of markers of inflammation were not altered. Moreover, AngII-treatment reduced LV fractional shortening and contractility in diabetic mice, but not in control mice.</p><p>Conclusions</p><p>Collectively, the present findings indicate that type 2 diabetes in its early stage is not yet associated with adverse cardiac structural changes, but already renders the heart more susceptible to hypertension-induced hypertrophic remodeling.</p></div

Cardiac phospho-AMPK levels are decreased in hypertensive, diabetic mice.

<p>Western blot analysis of LV pAMPK levels in non-diabetic (Cn) and diabetic (DM) mice treated with vehicle or AngII (Ang) for 4 weeks. In the top panel, the quantification of LV pAMPK levels from 4–6 animals per group is shown. Data are analyzed by one-way ANOVA with Bonferroni post-hoc testing and expressed as mean±SEM. In the bottom panel, representative examples of pAMPK and GAPDH signals are shown for 2 animals per group. * refers to effect of AngII in non-diabetic and diabetic mice (* P<0.05, ** P<0.01, *** P<0.001) and # refers to effect of diabetes in vehicle-treated and AngII-treated mice (# P<0.05, ## P<0.01, ### P<0.001).</p