Alternative Splicing of NOX4 in the Failing Human Heart

Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX) isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions) were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5'-3' end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure

Hypercholesterolemia downregulates autophagy in the rat heart

Background: We have previously shown that efficiency of ischemic conditioning is diminished in hypercholesterolemia and that autophagy is necessary for cardioprotection. However, it is unknown whether isolated hypercholesterolemia disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways. Therefore, we investigated whether isolated hypercholesterolemia modulates cardiac autophagy-related pathways or programmed cell death mechanisms such as apoptosis and necroptosis in rat heart. Methods: Male Wistar rats were fed either normal chow (NORM; n=9) or with 2% cholesterol and 0.25% cholic acid-enriched diet (CHOL; n=9) for 12 weeks. CHOL rats exhibited a 41% increase in plasma total cholesterol level over that of NORM rats (4.09mmol/L vs. 2.89mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out. Left ventricles were snap-frozen for determination of markers of autophagy, mTOR pathway, apoptosis, and necroptosis by Western blot. Results: Isolated hypercholesterolemia was associated with a significant reduction in expression of cardiac autophagy markers such as LC3-II, Beclin-1, Rubicon and RAB7 as compared to controls. Phosphorylation of ribosomal S6, a surrogate marker for mTOR activity, was increased in CHOL samples. Cleaved caspase-3, a marker of apoptosis, increased in CHOL hearts, while no difference in the expression of necroptotic marker RIP1, RIP3 and MLKL was detected between treatments. Conclusions: This is the first comprehensive analysis of autophagy and programmed cell death pathways of apoptosis and necroptosis in hearts of hypercholesterolemic rats. Our data show that isolated hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an activated mTOR pathway. Reduced autophagy was accompanied by increased apoptosis, while cardiac necroptosis was not modulated by isolated hypercholesterolemia. Decreased basal autophagy and elevated apoptosis may be responsible for the loss of cardioprotection reported in hypercholesterolemic animals

Primary care obesity management in Hungary: evaluation of the knowledge, practice and attitudes of family physicians

BACKGROUND: Obesity, a threatening pandemic, has an important public health implication. Before proper medication is available, primary care providers will have a distinguished role in prevention and management. Their performance may be influenced by many factors but their personal motivation is still an under-researched area. METHOD: The knowledge, attitudes and practice were reviewed in this questionnaire study involving a representative sample of 10% of all Hungarian family physicians. In different settings, 521 practitioners (448 GPs and 73 residents/vocational trainees) were questioned using a validated questionnaire. RESULTS: The knowledge about multimorbidity, a main consequence of obesity, was balanced.Only 51% of the GPs were aware of the diagnostic threshold for obesity; awareness being higher in cities (60%) and the highest among residents (90%). They also considered obesity an illness rather than an aesthetic issue.There were wider differences regarding attitudes and practice, influenced by the the doctors' age, gender, known BMI, previous qualification, less by working location.GPs with qualification in family medicine alone considered obesity management as higher professional satisfaction, compared to physicians who had previously other board qualification (77%vs68%). They measured their patients' waist circumference and waist/hip ratio (72%vs62%) more frequently, provided the obese with dietary advice more often, while this service was less frequent among capital-based doctors who accepted the self-reported body weight dates by patients more frequently / commonly. Similar reduced activity and weight-measurement in outdoor clothing were more typical among older doctors.Diagnosis based on BMI alone was the highest in cities (85%). Consultations were significantly shorter in practices with a higher number of enrolled patients and were longer by female providers who consulted longer with patients about the suspected causes of developing obesity (65%vs44%) and offered dietary records for patients significantly more frequently (65%vs52%). Most of the younger doctors agreed that obesity management was a primary care issue.Doctors in the normal BMI range were unanimous that they should be a model for their patients (94%vs81%). CONCLUSION: More education of primary care physicians, available practical guidelines and higher community involvement are needed to improve the obesity management in Hungary

Acute hyperglycemia abolishes cardioprotection by remote ischemic perconditioning

BACKGROUND: Remote ischemic perconditioning (RIPerC) has a promising therapeutic insight to improve the prognosis of acute myocardial infarction. Chronic comorbidities such as diabetes are known to interfere with conditioning interventions by modulating cardioprotective signaling pathways, such as e.g., mTOR pathway and autophagy. However, the effect of acute hyperglycemia on RIPerC has not been studied so far. Therefore, here we investigated the effect of acute hyperglycemia on cardioprotection by RIPerC. METHODS: Wistar rats were divided into normoglycemic (NG) and acute hyperglycemic (AHG) groups. Acute hyperglycemia was induced by glucose infusion to maintain a serum glucose concentration of 15-20 mM throughout the experimental protocol. NG rats received mannitol infusion of an equal osmolarity. Both groups were subdivided into an ischemic (Isch) and a RIPerC group. Each group underwent reversible occlusion of the left anterior descending coronary artery (LAD) for 40 min in the presence or absence of acute hyperglycemia. After the 10-min LAD occlusion, RIPerC was induced by 3 cycles of 5-min unilateral femoral artery and vein occlusion and 5-min reperfusion. After 120 min of reperfusion, infarct size was measured by triphenyltetrazolium chloride staining. To study underlying signaling mechanisms, hearts were harvested for immunoblotting after 35 min in both the NG and AHG groups. RESULTS: Infarct size was significantly reduced by RIPerC in NG, but not in the AHG group (NG + Isch: 46.27 +/- 5.31 % vs. NG + RIPerC: 24.65 +/- 7.45 %, p < 0.05; AHG + Isch: 54.19 +/- 4.07 % vs. 52.76 +/- 3.80 %). Acute hyperglycemia per se did not influence infarct size, but significantly increased the incidence and duration of arrhythmias. Acute hyperglycemia activated mechanistic target of rapamycine (mTOR) pathway, as it significantly increased the phosphorylation of mTOR and S6 proteins and the phosphorylation of AKT. In spite of a decreased LC3II/LC3I ratio, other markers of autophagy, such as ATG7, ULK1 phopsphorylation, Beclin 1 and SQSTM1/p62, were not modulated by acute hyperglycemia. Furthermore, acute hyperglycemia significantly elevated nitrative stress in the heart (0.87 +/- 0.01 vs. 0.50 +/- 0.04 microg 3-nitrotyrosine/mg protein, p < 0.05). CONCLUSIONS: This is the first demonstration that acute hypreglycemia deteriorates cardioprotection by RIPerC. The mechanism of this phenomenon may involve an acute hyperglycemia-induced increase in nitrative stress and activation of the mTOR pathway

In vivo MRI and ex vivo histological assessment of the cardioprotection induced by ischemic preconditioning, postconditioning and remote conditioning in a closed-chest porcine model of reperfused acute myocardial infarction: importance of microvasculature

BACKGROUND: Cardioprotective value of ischemic post- (IPostC), remote (RIC) conditioning in acute myocardial infarction (AMI) is unclear in clinical trials. To evaluate cardioprotection, most translational animal studies and clinical trials utilize necrotic tissue referred to the area at risk (AAR) by magnetic resonance imaging (MRI). However, determination of AAR by MRI' may not be accurate, since MRI-indices of microvascular damage, i.e., myocardial edema and microvascular obstruction (MVO), may be affected by cardioprotection independently from myocardial necrosis. Therefore, we assessed the effect of IPostC, RIC conditioning and ischemic preconditioning (IPreC; positive control) on myocardial necrosis, edema and MVO in a clinically relevant, closed-chest pig model of AMI. METHODS AND RESULTS: Acute myocardial infarction was induced by a 90-min balloon occlusion of the left anterior descending coronary artery (LAD) in domestic juvenile female pigs. IPostC (6 x 30 s ischemia/reperfusion after 90-min occlusion) and RIC (4 x 5 min hind limb ischemia/reperfusion during 90-min LAD occlusion) did not reduce myocardial necrosis as assessed by late gadolinium enhancement 3 days after reperfusion and by ex vivo triphenyltetrazolium chloride staining 3 h after reperfusion, however, the positive control, IPreC (3 x 5 min ischemia/reperfusion before 90-min LAD occlusion) did. IPostC and RIC attenuated myocardial edema as measured by cardiac T2-weighted MRI 3 days after reperfusion, however, AAR measured by Evans blue staining was not different among groups, which confirms that myocardial edema is not a measure of AAR, IPostC and IPreC but not RIC decreased MVO. CONCLUSION: We conclude that IPostC and RIC interventions may protect the coronary microvasculature even without reducing myocardial necrosis

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

River water quality modelling: I. state of the art

River water quality models are used extensively in research as well as in the design and assessment of water quality management measures. The application of mathematical models for that purpose dates back to the initial studies of oxygen depletion due to organic waste pollution. Since then, models have been constantly refined and updated to meet new and emerging problems of surface water pollution, such as eutrophication, acute and chronic toxicity, etc. In order to handle the complex interactions caused by the increased influence of human activities in rivers it is today mandatory to couple river water quality models with models describing emissions from the drainage and sewerage system (such as the IAWQ Activated Sludge model No.1). In this paper—which is the first of a three-part series by the IAWQ Task Group on River Water Quality Modelling— the state of the art is summarized with the above aim in mind. Special attention is given here to the modelling of conversion processes but also the methods and tools to work with the models, i.e. parameter estimation, measurement campaign design, and simulation software, are discussed

River water quality modelling: II.:Problems of the art

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River water quality modelling: III. Future of the art.

This paper is the third of a three-part series summarizing the background to and objectives of the activity of the IAWQ Task Group on River Water Quality Modelling (RWQM). On the basis of the two other papers and a comparison between the best known state of the art river model, QUAL2, and the IAWQ Activated Sludge Model (ASM) No. 1, the Task Group proposes to develop improved conversion models for inclusion in a river water quality model. The model should describe the cycling of oxygen, nitrogen, and phosphorus in both water and sediment, and should be compatible with the ASM to support the development of integrated emission reduction strategies. The model should be particularly well suited to handle problems characterized by significant temporal and spatial influences (e.g. CSOs and NPSs). It should serve for research, education, improved communication, knowledge transfer, regulatory applications such as catchment planning, and improved data collection. Anticipated results of the Task Group effort include (i) standardized conversion sub-models; (ii) a decision support tool to guide model construction and usage; and (iii) case study applications. The model development, which is not intended to result in a software product, is intended to be an open-ended and flexible process to encourage the participation of interested professionals