Distinct cardiotoxic effects by venoms of a spitting cobra (Naja pallida) and a rattlesnake (Crotalus atrox) revealed using an ex vivo Langendorff heart model.

Here we describe the acute myocardial effects of an elapid (red spitting cobra, Naja pallida) and a viper (western diamondback rattlesnake, Crotalus atrox) venom using an ex vivo heart model. Our results reveal two different pathophysiological trajectories that influence heart function and morphology. While cobra venom causes a drop in contractile force, rattlesnake venom causes enhanced contractility and frequency that coincides with differences in myocellular morphology. This highlights the medical complexity of snake venom-induced cardiotoxicity.

Challenging Future Generations: A Qualitative Study of Students' Attitudes toward the Transition to Animal-Free Innovations in Education and Research.

In 2016, the Dutch government declared its commitment to phasing out animal experiments by 2025. Although a high number of animal experiments are still performed and the 2025 target will not be met, the commitment remains. Efforts are being made to identify levers that might foster the transition to animal-free science. Education has been found to play a key role in the future of animal-free science and young generations are increasingly seen as key stakeholders. However, their attitudes toward the transition to animal-free innovations have not been investigated. The present article focuses on the values and beliefs held by students, who in 2022, participated in the course 'Replacing Animal Testing' (RAT) Challenge, organized by a consortium of Dutch universities. Contextually, students' motivations to follow the course were investigated. The research was based on a qualitative study, including semi-structured interviews and a literature review. Our analysis of the findings revealed that students feel aligned with the social, ethical, and scientific reasons that support the transition to animal-free innovations. Moreover, the participants identified a series of regulatory, educational, cultural, and political obstacles to the transition that align with those identified in recent literature. From the discussion of these findings, we extrapolated six fundamental challenges that need to be addressed to foster the transition to animal-free science in an acceptable and responsible way.

Absence of fatty acid transporter CD36 protects against Western-type diet-related cardiac dysfunction following pressure overload in mice.

Cardiac patients often are obese and have hypertension, but in most studies these conditions are investigated separately. Here, we aimed at 1) elucidating the interaction of metabolic and mechanophysical stress in the development of cardiac dysfunction in mice and 2) preventing this interaction by ablation of the fatty acid transporter CD36. Male wild-type (WT) C57Bl/6 mice and CD36(-/-) mice received chow or Western-type diet (WTD) for 10 wk and then underwent a sham surgery or transverse aortic constriction (TAC) under anesthesia. After a 6-wk continuation of the diet, cardiac function, morphology, lipid profiles, and molecular parameters were assessed. WTD administration affected body and organ weights of WT and CD36(-/-) mice, but it affected only plasma glucose and insulin concentrations in WT mice. Cardiac lipid concentrations increased in WT mice receiving WTD, decreased in CD36(-/-) on chow, and remained unchanged in CD36(-/-) receiving WTD. TAC induced cardiac hypertrophy in WT mice on chow but did not affect cardiac function and cardiac lipid concentrations. WTD or CD36 ablation worsened the outcome of TAC. Ablation of CD36 protected against the WTD-related aggravation of cardiac functional and structural changes induced by TAC. In conclusion, cardiac dysfunction and remodeling worsen when the heart is exposed to two stresses, metabolic and mechanophysical, at the same time. CD36 ablation prevents the metabolic stress resulting from a WTD. Thus, metabolic conditions are a critical factor for the compromised heart and provide new targets for metabolic manipulation in cardioprotection.

Diabetic cardiomyopathy in Zucker diabetic fatty rats: the forgotten right ventricle.

BACKGROUND: In patients with myocardial infarction or heart failure, right ventricular (RV) dysfunction is associated with death, shock and arrhythmias. In patients with type 2 diabetes mellitus, structural and functional alterations of the left ventricle (LV) are highly prevalent, however, little is known about the impact of diabetes on RV characteristics. The purpose of the present study was to investigate whether LV changes are paralleled by RV alterations in a rat model of diabetes. METHODS: Zucker diabetic fatty (ZDF) and control (ZL) rats underwent echocardiography and positron emission tomography (PET) scanning using [18F]-2-fluoro-2-deoxy-D-glucose under hyperinsulinaemic euglycaemic clamp conditions. Glucose, insulin, triglycerides and fatty acids were assessed from trunk blood. Another group of rats received an insulin or saline injection to study RV insulin signaling. RESULTS: ZDF rats developed hyperglycaemia, hyperinsulinaemia and dyslipidaemia (all p < 0.05). Echocardiography revealed depressed LV fractional shortening and tricuspid annular plane systolic excursion (TAPSE) in ZDF vs. ZL rats (both p < 0.05). A decrease in LV and RV insulin-mediated glucose utilisation was found in ZDF vs. ZL rats (both p < 0.05). LV associated with RV with respect to systolic function (r = 0.86, p < 0.05) and glucose utilisation (r = 0.74, p < 0.05). TAPSE associated with RV MRglu (r = 0.92, p < 0.05) and M-value (r = 0.91, p < 0.0001) and RV MRglu associated with M-value (r = 0.77, p < 0.05). Finally, reduced RV insulin-stimulated phosphorylation of Akt was found in ZDF vs. ZL (p < 0.05). CONCLUSIONS: LV changes were paralleled by RV alterations in insulin-stimulated glucose utilisation and RV systolic function in a rat model of diabetes, which may be attributed to ventricular interdependence as well as to the uniform effect of diabetes. Since diabetic patients are prone to develop diabetic cardiomyopathy and myocardial ischaemia, it might be suggested that RV dysfunction plays a central role in cardiac abnormalities in this population.

Altered myocardial substrate metabolism is associated with myocardial dysfunction in early diabetic cardiomyopathy in rats: studies using positron emission tomography.

BACKGROUND: In vitro data suggest that changes in myocardial substrate metabolism may contribute to impaired myocardial function in diabetic cardiomyopathy (DCM). The purpose of the present study was to study in a rat model of early DCM, in vivo changes in myocardial substrate metabolism and their association with myocardial function. METHODS: Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats underwent echocardiography followed by [11C]palmitate positron emission tomography (PET) under fasting, and [18F]-2-fluoro-2-deoxy-D-glucose PET under hyperinsulinaemic euglycaemic clamp conditions. Isolated cardiomyocytes were used to determine isometric force development. RESULTS: PET data showed a 66% decrease in insulin-mediated myocardial glucose utilisation and a 41% increase in fatty acid (FA) oxidation in ZDF vs. ZL rats (both p < 0.05). Echocardiography showed diastolic and systolic dysfunction in ZDF vs. ZL rats, which was paralleled by a significantly decreased maximal force (68%) and maximal rate of force redevelopment (69%) of single cardiomyocytes. Myocardial functional changes were significantly associated with whole-body insulin sensitivity and decreased myocardial glucose utilisation. ZDF hearts showed a 68% decrease in glucose transporter-4 mRNA expression (p < 0.05), a 22% decrease in glucose transporter-4 protein expression (p = 0.10), unchanged levels of pyruvate dehydrogenase kinase-4 protein expression, a 57% decreased phosphorylation of AMP activated protein kinase alpha1/2 (p < 0.05) and a 2.4-fold increased abundance of the FA transporter CD36 to the sarcolemma (p < 0.01) vs. ZL hearts, which are compatible with changes in substrate metabolism. In ZDF vs. ZL hearts a 2.4-fold reduced insulin-mediated phosphorylation of Akt was found (p < 0.05). CONCLUSION: Using PET and echocardiography, we found increases in myocardial FA oxidation with a concomitant decrease of insulin-mediated myocardial glucose utilisation in early DCM. In addition, the latter was associated with impaired myocardial function. These in vivo data expand previous in vitro findings showing that early alterations in myocardial substrate metabolism contribute to myocardial dysfunction.

RhoA-ROCK signaling is involved in contraction-mediated inhibition of SERCA2a expression in cardiomyocytes.

In neonatal ventricular cardiomyocytes (NVCM), decreased contractile activity stimulates sarco-endoplasmic reticulum Ca(2+)-ATPase2a (SERCA2a), analogous to reduced myocardial load in vivo. This study investigated in contracting NVCM the role of load-dependent RhoA-ROCK signaling in SERCA2a regulation. Contractile arrest of NVCM resulted in low peri-nuclear localized RhoA levels relative to contracting NVCM. In arrested NVCM, ROCK activity was decreased (59%) and paralleled a loss in F-actin levels. Y-27632-induced ROCK inhibition in contracting NVCM increased SERCA2a messenger RNA expression by 150%. This stimulation was transcriptional, as evident from transfections with the SERCA2a promoter. A reciprocal effect of Y-27632 treatment on the promoter activity of atrial natriuretic factor was observed. SERCA2a transcription was not altered by co-transfection of the RhoA-ROCK-dependent serum response factor (SRF) alone or in combination with myocardin. Furthermore, GATA4, another ROCK-dependent transcription factor, induced rather than repressed SERCA2a transcription. This study shows that contractile activity suppresses SERCA2a gene expression via RhoA-ROCK-dependent transcription modulation. This modulation is likely to be accomplished by a transcription factor other than SRF, myocardin, or GATA4.

Insulin-mediated phosphorylation of the proline-rich Akt substrate PRAS40 is impaired in insulin target tissues of high-fat diet-fed rats.

Clinical insulin resistance is associated with decreased activation of phosphatidylinositol 3'-kinase (PI3K) and its downstream substrate protein kinase B (PKB)/Akt. However, its physiological protein substrates remain poorly characterized. In the present study, the effect of in vivo insulin action on phosphorylation of the PKB/Akt substrate 40 (PRAS40) was examined. In rat and mice, insulin stimulated PRAS40-Thr246 phosphorylation in skeletal and cardiac muscle, the liver, and adipose tissue in vivo. Physiological hyperinsulinemia increased PRAS40-Thr246 phosphorylation in human skeletal muscle biopsies. In cultured cell lines, insulin-mediated PRAS40 phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002. Immunohistochemical and immunofluorescence studies showed that phosphorylated PRAS40 is predominantly localized to the nucleus. Finally, in rats fed a high-fat diet (HFD), phosphorylation of PRAS40 was markedly reduced compared with low-fat diet-fed animals in all tissues examined. In conclusion, the current study identifies PRAS40 as a physiological target of in vivo insulin action. Phosphorylation of PRAS40 is increased by insulin in human, rat, and mouse insulin target tissues. In rats, this response is reduced under conditions of HFD-induced insulin resistance.

SIH--a novel lipophilic iron chelator--protects H9c2 cardiomyoblasts from oxidative stress-induced mitochondrial injury and cell death.

Recent evidence suggests that oxidative stress is a common denominator in many aspects of cardiovascular pathogenesis. Free cellular iron plays a crucial catalytic role in the formation of highly toxic hydroxyl radicals, and thereby it may aggravate the contribution of oxidative stress to cardiovascular disease. Therefore, iron chelation may be an effective therapeutic approach, but the progress in this area is hindered by the lack of effective agents. In this study, using the rat heart myoblast-derived cell line H9c2, we aimed to investigate whether the novel lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) protects the cells against hydrogen peroxide (H2O2)-induced cytotoxicity. Exposure of cells to 100 micromol/l H2O2 has within 4 h induced a complete dissipation of their mitochondrial membrane potential (DeltaPsim). Co-treatment with SIH dose-dependently reduced (EC50=0.8 micromol/l) or even completely abolished (3 micromol/l) this collapse. Furthermore, the latter SIH concentration was capable to fully prevent alterations in cell morphology, and inhibited both apoptosis (annexin-V staining, nuclear chromatin shrinkage, TUNEL positivity) and necrosis (propidium iodide staining), even 24 h after the H2O2 exposure. In comparison, deferoxamin (a commercially available hydrophilic iron chelator used in clinical practice and most previous studies) was cytoprotective only at three-order higher and clinically unachievable concentrations (EC50=1300 micromol/l). Thus, in this study, we present iron chelation as a very powerful tool by which oxidative stress-induced myocardial damage can be prevented.

Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular cardiomyocytes.

OBJECTIVE: Downregulation of sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) expression is a critical marker of pathological myocardial hypertrophy. The effects of calcium-dependent signaling and of contractile activity on the regulation of myocardial SERCA2a expression remain unclear. The present study dissociates effects of calcium-dependent signaling through calcineurin (CN) and calmodulin dependent protein kinase-II (CAMK-II), from effects of contractile activity in spontaneously contracting rat neonatal ventricular cardiomyocytes (NVCM) using 2,3-butanedione monoxime (BDM), which arrests contractions but maintains calcium fluxes. METHODS: SERCA2a mRNA expression was analysed using Northern hybridisation in spontaneously contracting NVCM (control) and in NVCM treated with either BDM, L-type Ca2+-channel blocker (verapamil), CN-blocker (cyclosporin A; CsA), CAMK-II blocker (KN-93), or combinations thereof. Transient transfection of the CN-dependent transcription factor nuclear factor of activated T-lymphocytes (NFATc), coupled to GFP, was used to detect NFAT nuclear translocation. The effects of CN/CAMK-II-dependent signaling were further dissected into effects of the transcription factors NFATc4 and myocyte enhancer factor 2c (MEF2c) on the activity of various SERCA2a promoter fragments using transient transfection assays. RESULTS: Treatment with BDM induced a 2.5-fold rise in SERCA2a mRNA, which was abolished by addition of verapamil and was reduced by addition of CsA (-40%) and KN-93 (-20%). NFAT nuclear translocation was similar in control and BDM-treated NVCM. SERCA2a promoter activity was stimulated by NFATc4 and MEF2c, but only when both factors were co-transfected. CONCLUSION: Following contractile arrest with BDM, upregulation of SERCA2a mRNA expression by CN/CAMK-II signaling becomes evident. This upregulation is likely the result of synergistic stimulation of SERCA2a promoter activity by NFATc4 and MEF2c. Contractile activity opposes this upregulation through distinct and independent pathways.

The role of the Ferric Uptake Regulator (Fur) in regulation of Helicobacter pylori iron uptake.

BACKGROUND: Availability of the essential nutrient iron is thought to vary greatly in the gastric mucosa, and thus the human gastric pathogen Helicobacter pylori requires regulatory responses to these environmental changes. Bacterial iron-responsive regulation is often mediated by Ferric Uptake Regulator (Fur) homologs, and in this study we have determined the role of H. pylori Fur in regulation of H. pylori iron uptake. METHODS: Wild-type H. pylori and fur mutant derivatives were compared after growth in iron-restricted and iron-replete conditions. Iron-uptake was measured using 55Fe-labeled iron, whereas gene expression was monitored at the transcriptional level using Northern hybridization and lacZ reporter gene fusions. RESULTS: Iron-uptake and total cellular iron content were approximately five-fold increased in the fur mutant compared with the wild-type strain, which indicated that in the fur mutant iron-uptake is not repressed by excess iron. A comprehensive screening of all H. pylori genes encoding putative iron-uptake proteins indicated that some of these H. pylori genes are constitutively expressed, while others are iron- and Fur-regulated. CONCLUSIONS: Iron uptake in H. pylori is in part differently regulated compared with other bacteria, since in H. pylori some iron-uptake systems are constitutively expressed. However, other iron uptake systems of H. pylori display the iron- and Fur-mediated repression that is common in bacteria. Taken together, this Fur-mediated modulation of iron-uptake capacity may be a specific adaptation to the conditions in the human stomach, where iron starvation and iron overload can be encountered in relatively short time intervals.

Discordance of genetic alterations between primary head and neck tumors and corresponding metastases associated with mutational status of the TP53 gene.

Ample molecular data are available on the progression from normal mucosa to invasive head and neck squamous cell carcinoma (HNSCC), but information on further genetic progression to metastatic disease is scarce. To obtain insight into the metastatic process, we compared 23 primary HNSCCs with 25 corresponding lymph node metastases (LNMs) and 10 corresponding distant metastases (DMs) with respect to TP53 mutations and patterns of loss of heterozygosity (LOH) based on 26 microsatellite markers on six chromosome arms (3p, 9p, 17p, 13q, 8p, and 18q). In 18 of the 23 patients, a TP53 mutation was detected in the primary tumor, and in all cases the same TP53 mutation was present in the corresponding LNM or DM. In nine of 20 patients with LNMs and three of seven patients with DMs, the LOH pattern of metastasis differed from that of the corresponding primary tumor by at least one marker. Microsatellite markers located on chromosome arms 13q, 8p, and 18q were most frequently discordant, providing evidence that alterations at these chromosomes occur late in HNSCC carcinogenesis. Moreover, evidence was found that DMs had developed directly from the primary tumor and not from LNMs. Remarkably, we observed that the mutational status of the TP53 gene is associated significantly with the degree of genetic differences between primary HNSCCs and corresponding metastases. All patients with TP53 wild-type primary tumors showed significantly more discordant LOH patterns in the corresponding LNMs and DMs than patients with TP53-mutated tumors. The percentages were 100% versus 27% (LNMs) and 100% versus 0% (DMs), respectively (P = 0.008 and P = 0.029; two-sided Fisher exact test). This finding suggests that TP53-mutated tumors need fewer additional genetic alterations to develop metastases compared with TP53 wild-type primary tumors.