Le syndrome du QT long (LQTs) et le syndrome de Brugada (BS) sont deux maladies cardiaques héréditaires, pouvant causer la mort subite en relation avec des torsades de pointe dues à une fibrillation ventriculaire. SCN5A est le gène codant pour la sous-unité a du canal Na4 dépendant du voltage, exprimé dans le cœur humain. Objectif : Dans la présente étude, deux mutations (R1193Q et F1344S) dans le gène SCN5A ont été identifiées. La mutation R1193Q a été trouvée chez deux patients après le test de provocation à la procainamide par voie intraveineuse. Un des patients est atteint de BS et l'autre est atteint à la fois de BS et de LQTs. La mutation F1344S a été trouvée chez un autre patient atteint uniquement de BS. Méthode : Les canaux mutants ont été exprimés dans un système d'expression de mammifère (lignée cellulaire tsA 201) et les propriétés biophysiques ont été étudiées avec la technique de patch clamp en configuration cellule entière. Résultats : L'analyse des séquences montre un changement de G à A à la position 3587 sur l'exon 20. La mutation RI 193Q montre un déplacement négatif de 5mV de l'inactivation et un courant Na+ persistant. Le déplacement vers des potentiels négatifs de l'inactivation est responsable d'une perte de fonction causant l'élévation du segment ST chez le patient atteint de BS. La présence du courant persistant est responsable du gain de fonction causant l'augmentation de l'intervalle QT chez le patient avec LQTs. La mutation F1344S montre une manifestation typique de BS sur l'électrocardiogramme pendant une période de fièvre. Le séquençage a révélé un changement de T à C à la position 4031 sur l'exon 23. L'analyse biophysique montre une perte de fonction due au déplacement de la courbe d'activation vers des potentiels positifs à 23°C et ce déplacement est exacerbé à 40.5°C avec une pente plus lente. Conclusion : Les différentes manifestations cliniques de ces deux mutations sont la conséquence des anormalités électrophysiologiques distinctes du canal Na+ cardiaque mutant décrites dans cette étude. Pour les patients porteurs de R1193Q, la prescription de quelques drogues pouvant provoquer le LQTs et le BS doit être limitée. Pour éviter les épisodes de fibrillation ventriculaire, l'hyperthermie doit être contrôlée chez les patients porteurs de la mutation F1344S. / Long QT syndrome (LQTs) and Brugada syndrome (BS) are two distinct hereditary cardiac diseases, causing sudden cardiac death related to torsade de pointes and ventricular tachycardia (VT) / ventricular fibrillation (VF). SCN5A is the gene encoding the principal voltage-gated Na+ channel a-subunit, which is only expressed in the human heart. Objective: In present study, two mutations (R1193Q and F1344S) in SCN5A have been identified. RI 193Q mutation was found in two patients, one with BS and another with an overlap of BS and LQTs after the intravenous procainamide test. F1344S mutation was found in another patient with BS. Method: The mutant channels were expressed in a mammalian expression System (tsA 201 cell Une) and the biophysical properties were studied by the patch clamp technique with whole cell configuration. Results: The sequence analysis showed a G to A base change at position 3587 in exon 20. The R1193Q mutation produced a negative shift for 5 mV of inactivation and a persistent Na+ current. The negative shift of inactivation is responsible for loss of the function, leading to ST segment elevation in BS. The persistent Na+ current is responsible for gain of function, causing QT interval prolongation in LQTs. The F1344S mutation presented a typical BS manifestation on ECG during fever period. The sequencing analysis revealed a T to C base change at position 4031 in exon 23. The biophysical analysis showed loss of function due to significantly positive shift of activation at both 23°C and 40.5°C, but the shift was more important at 40.5°C with a slower slope factor. Conclusion: The different clinical manifestations of these two mutations probably derive from the distinct electrophysiological abnormalities of the mutant cardiac Na+ channels reported in this study. For patients with RI 193Q mutation, some drugs that likely precipitate LQTs and BS should be limited to prescribe. To prevent from VT/VF events, hyperthermia should be effectively controlled in the patients with F1344S mutation.

Les médicaments qui provoquent un allongement de la repolarisation cardiaque, mesuré sur l'électrocardiogramme (ECG) par la prolongation de l'intervalle QT, ont été associés à une augmentation du risque pro-arythmique, et plus particulièrement à la survenue de Torsades de pointes, une tachycardie ventriculaire polymorphe potentiellement mortelle. Les méthodes d'analyse du QT conventionnelles se restreignent à l'extraction de quelques complexes ECG. Cette pratique se traduit par de nombreuses limitations. L'inclusion de tous les battements enregistrés sur 24h et mesurés par des méthodes automatiques de mesure ECG a le potentiel de résoudre ces inconvénients. Ce travail de thèse a démontré que les méthodes de mesure ECG automatisées et les analyses continues peuvent supplanter les méthodes conventionnelles pour l'analyse de la prolongation du QT lors des essais cliniques. Des recommandations ont été établis afin de permettre une utilisation optimale des méthodes d'analyse ECG continues.

Background. Acquired long QT syndrome (aLQTS) is a reversible condition characterized by a pathological prolongation of the QT interval that can lead to a polymorphic ventricular tachycardia known as Torsades de Pointe and sudden cardiac death. Identifying the incidence, onset, and risk factors for aLQTS in intensive care init (ICU) populations has not been studied and may help clinicians develop safe monitoring guidelines to identify patients early preventing devastating outcomes. Objective. The objective of this study was to determine the frequency, temporal onset of occurrence, frequency of medications and host risk factors for aLQTS in an ICU. Method. In a retrospective chart review of 88 subjects, hourly electrocardiographic data collected in an ICU were analyzed for baseline, first long, longest, and final corrected QT intervals (QTc) using Bazett's formula. aLQTS was defined as a QTc interval ≥ 500 milliseconds (ms) or a change in QTc of ≥ 60 ms from baseline. Host risk factors were collected from the physician's dictated history and physicals and nursing admission databases. Names and timing of each medication administered were collected from the medication record. Results. aLQTS occurred in 52.3% of the ICU sample. All subjects positive for aLQTS (n=46) had a mean onset of 7.4 ± 9.4 hours from ICU admission. Subjects who developed aLQTS after ICU admission (n=32) had a mean onset of 10.6 ± 9.5 hours; 14 were positive on ICU admission. A statistically significant difference was noted in subjects receiving QT prolonging medications positive for aLQTS (63.5%, n=33) compared with subjects negative for aLQTS (36.5%, n=19), (X²[1] = 6.38, p = .012). Thirteen subjects (28.3%) developed aLQTS in the absence of a known QT interval prolonging medication. No host risk factors were found to have a significant difference between groups positive and negative for aLQTS. Conclusions. aLQTS was present in approximately one-half of the sample. Approximately a quarter of the subjects developed aLQTS in the absence of a known QT prolonging medication, indicating the importance of frequent QTc monitoring in all patients in ICUs. Larger studies to determine common host risk factors associated with aLQTS in ICU populations are warranted.

The Hedonic Price Method (HPM), also known as hedonic regression, is used for estimating the value of a commodity or the demand for a commodity. The HPM has been extensively used in real estate and housing market research in the recent past. In this paper, we discuss theoretical and methodological developments related to hedonic regression and undertake an examination of use of this methodology in the recent real estate and housing literature. We first define the HPM, and explain the fundamentals behind the methodology. The idea behind the HPM is that the commodities are characterized by their constitute properties, hence the value of a commodity can be calculated by adding up the estimated values of its separate properties. In the second part of the paper, we emphasise that the heterogeneous nature of real estate property justifies the use of HPM for estimating their value or demand. We also take a stock of most cited empirical studies on real estate and housing using the HPM, and classify those into several categories. The classification indicates that neighbourhood characteristics of real estate are relatively over-researched as a determinant of price or rent. It also shows that implicit value of structural characteristics is under-researched. In general, implicit value of environmental amenities in the neighbourhood and air pollution are relatively overresearched. The effect of social factors, i.e. racial segregation and crimes on real estate value is under-researched. (authors's abstract) / Series: SRE - Discussion Papers

This thesis investigated the impact of sodium acetate infusion on muscle fuel metabolism during low and high intensity exercise and demonstrated a decrease in fat oxidation during the former and a decrease in muscle lactate production during the latter. We propose that the rate of fat oxidation decreased during low intensity exercise due to acetate being preferentially metabolised over fat and possibly reduced muscle free carnitine availability. During high intensity exercise, muscle lactate accumulation decreased due to higher acetyl-CoA supply to the TCA cycle. This thesis also investigated the impact of chronic carnitine supplementation on overall glucose disposal and demonstrated a decrease in blood glucose and serum insulin concentrations following an OGTT in the carnitine group post-supplementation compared to baseline, coupled with a decrease in muscle 2-deoxyglucose accumulation in the carnitine group post-supplementation compared to control. We propose that carnitine-induced increased fat oxidation caused a decrease in hepatic fat deposition which in turn resulted in increasing glucose disposal by the liver. We also propose that increased hepatic glucose disposal resulted in reduced hepatic glucose release and hence diminished serum insulin concentrations. The impact of a chronic lifestyle intervention protocol involving either oral supplementation with carnitine (carnitine group) or placebo (control group) combined with carbohydrate and protein, together with regular exercise and a prescribed diet on muscle fuel metabolism and body composition in overweight volunteers was investigated in this thesis. This study demonstrated that increased muscle carnitine content caused an increase in fasting fat oxidation and a decrease in carbohydrate oxidation at rest. The carnitine group did not show any significant difference in body mass and body fat mass losses compared to the control group. Whether increased carnitine content, in combination with caloric restriction and exercise, has any impact on body composition seems likely but needs further investigations.

Series: Research Paper Series of the Research Focus Managing Sustainability

<div>The human ether-a-go-go-related gene (hERG, KCNH2) potassium channel has been implicated in diverse physiological and pathological processes. The KCNH2 gene encodes a rectifier voltage-gated potassium channel (Kv 11.1) that governs the chief repolarizing current, IKr, which is essential for normal electrical activity in excitable cells such as cardiomyocytes. It is also involved in cell growth and apoptosis regulation in non-excitable cells, such as tumor cells. Dysfunction of hERG is associated with potentially lethal complications, including diseases and sudden death under certain circumstances. While the mechanisms regulating KCNH2 expression remain unclear, recent data suggested that microRNAs (miRNAs) are involved, particularly in the context of several pathologic effects. </div><div>miRNA is a class of RNA defined by its conserved, short, non-coding nature. miRNAs are important regulators of gene expression at the post-transcriptional level that bind through complimentary annealing to the 3’ untranslated regions (3’ UTRs) of target mRNAs, resulting in mRNA destabilization and translational repression. The primary objectives of this research were to 1) identify miRNAs regulating KCNH2 expression in cancer, 2) investigate the potential association between miR-362-3p expression and risk of drug-induced QT interval lengthening, and 3) identify miRNAs potentially regulating KCNH2 expression and function in cardiac cells. </div><div>Through bioinformatics approaches, five miRNAs were identified to potentially regulate KCNH2 expression and function in breast cancer cells. The five identified miRNAs were validated through a Dual-Luciferase Assay using the KCNH2 3′ UTR. Only miR-362-3p was validated to bind to the KCNH2 3’ UTR, decreasing luciferase activity by 10% ± 2.3 (P < 0.001, n = 3) when compared to cells transfected with luciferase plasmid alone. miR-362-3p was also the only miRNA that its expression positively correlated with overall survival of patients with breast cancer from The Cancer Genome Atlas-Cancer Genome (TCGA) database by log-rank test (HR: 0.39, 95% CI: 0.18 to 0.82, P = 0.012). Cell proliferation was assessed by MTS assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) 48 hours following transfection in breast cancer cell lines, including SK-BR-3 and MCF-7. miR-362-3p significantly decreased proliferation of SK-BR-3 and MCF-7 cells by 23% ± 8.7 (P = 0.014, n = 3) and 11.7% ± 1.0 (P < 0.001, n = 3), respectively. Cell cycle phases in SK-BR-3 and MCF-7 cells were differentiated by flow cytometry 48 hours following transfection. miR-362-3p and hERG siRNA (positive control) significantly increased the accumulation of cells in G0/G1 phase in MCF-7 by 11.7% (from 51.1% ± 0.64 to 57.1 ± 0.96, P = 0.002, n = 3) and 10% (from 51.1% ± 0.64 to 56.8 ± 0.96, P < 0.001, n = 3), respectively. </div><div>The demonstrated ability of miR-362-3p to regulate hERG in breast cancer cells coupled with previously published data that indicated an alteration of miR-362-3p expression during HF and a potential association between its expression and QT interval prolongation suggesting an important role for this miRNA in regulation of hERG function during HF. Therefore, the contribution of miR-362-3p to hERG function was investigated in patients administered the QT prolonging drug ibutilide, known to inhibit hERG. A total of 22 patients completed a prospective, parallel-group comparative study during which they received subtherapeutic doses (0.003 mg/kg) of ibutilide. The study was originally designed to investigate the influence of heart failure with preserved ejection fraction (HFpEF) on response to drug-induced QT prolongation. Blood for determination of serum Ibutilide concentrations and miR-362-3p expression, along with electrocardiograms (ECGs) were serially collected over a span of 12 hours. ΔΔ-Fridericia-heart rate corrected QT (ΔΔ QTF) intervals were utilized for all analyses to account for baseline and diurnal variation. </div><div>To assess the ability of miR-362-3p to predict ibutilide QT-induced ΔΔQTF changes, nonlinear mixed effects pharmacokinetic/ pharmacodynamic (PKPD) modeling was performed to assess the contribution of miR-362-3p to drug-induced QT interval lengthening. The model that best fit serum ibutilide concentrations versus time was a 3-compartment model with first order elimination and proportional residual errors, while the model that best described the ibutilide concentration- ΔΔQTF relationship was an Emax model with an effect compartment. In addition to miR-362-3p expression, several demographic and clinical data were evaluated as potential covariates on PK and PD parameter estimates. Of tested covariates, heart failure (HF) status on Emax (ΔOFV = -4.1; P < 0.05), and miR-362-3p expression on EC50 (ΔOFV = -9.9; P < 0.05) were incorporated in the final PKPD model. The mean individual Emax was significantly higher in HF patients when compared to non-HF patients (P = 0.015), while EC50 was negatively correlated with miR-362-3p expression (P < 0.0001, R2 0.93). </div><div>Previous evidence indicates that miR-362-3p is altered in patients with HF. In addition, several miRNAs commonly regulate the same ion channel. Therefore, we have developed a large-scale high-throughput bioassay (HT-bioassay) to explore and identify other miRNAs potentially involved in KCNH2 expression and function in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) during sustained β-adrenergic receptor (βAR) stimulation or overexpression of activated calcium/calmodulin-dependent protein kinase 2 (CaMKII), which are classical consequences of HF. </div><div>Through bioinformatic approaches, putative miRNA binding sites (n=327) were identified in the KCNH2 3′ UTR. Fragments containing these putative binding sites were synthesized, cloned into linearized plasmids, and amplified. The plasmid pool was transfected into hiPS-CM cells either treated with βAR stimulation or overexpressing CaMKII. Next-generation sequencing was performed to identify: 1) expression of putative miRNA binding sites and 2) endogenous miRNAs versus control. Eight predicted binding sites were found to be significantly downregulated in the CAMKII group (P <0.05, log fold change -0.287 to -0.59), and six significantly downregulated in the sustained βAR group (P <0.05, log fold change -0.29 to -0.72). Two binding sites were significantly reduced in both treatment groups (P < 0.05, log fold change between -0.38 and -0.61).</div><div>Thirty-one miRNAs were predicted to bind to the 16 binding sites identified from the bioassay. Of these, seven were selected for further screening using dual luciferase assays. None of the putative miRNAs reduced luciferase activity. However, hERG expression was assessed by immunoblot analysis following transfection of the seven miRNAs into HEK293 cells stably expressing hERG (HEK293-hERG). Six of the seven miRNA mimics reduced hERG protein expression. An additional validation step was performed by assessing hERG-related current density by whole cell electrophysiology, in which three of the six miRNAs inhibited hERG protein transfected into HEK293-hERG cells. Those same three miRNA mimics significantly decreased Ikr current (P <0.05). </div><div>Finally, expression of the miRNAs identified by HT-bioassay was examined in the patients enrolled in the clinical trial in which genome-wide next generation sequencing was performed on miRNAs extracted from whole blood samples. Of the 31 miRNAs identified from HT-bioassay, six were found to be expressed in patients (n = 12). A correlation analysis was performed between levels of the expressed miRNAs and corresponding QTF interval lengthening with ibutilide. Of the six miRNAs, only miR-4665-5p was significantly associated with QTF interval (P = 0.0379). </div><div>In summary, miR-362-3p was identified to regulate hERG, and reduces proliferation of breast cancer cells through a mechanism that may be partially mediated by hERG inhibition. While miR-362-3p may have modest effects in cancer, in Aim 2 we demonstrated that it along with HF status accounts for a significant amount of variability in QTF prolongation following ibutilide administration. However, it is common for several miRNAs to regulate a single ion channel. Therefore, an HT-bioassay was developed to identify all miRNAs that potentially regulate KCNH2 during HF. In addition to miR-362-3p, thirty-one miRNAs were predicted to regulate KCNH2; one miRNA (miR-4665-5p) was significantly associated with QTF prolongation. The potential for miR-362-3p and HT-bioassay-identified miRNAs to reduce hERG-related current and influence susceptibility to drug-induced QT interval prolongation warrants further investigation. </div><div><br></div>

Commonly prescribed small molecule drugs can have net-positive and well-understood safety profiles when prescribed individually, but unexpected consequences when taken at the same time. Detection of these drug-drug interactions (DDIs) continues to be a critical and unmet area of translational research. The Centers for Disease Control and Prevention (CDC) estimate that one third of Americans are concurrently taking two or more prescription drugs, and DDIs are estimated to be responsible for 17% of all drug adverse events. The consequences of DDIs can be relatively minor (headache, skin rash) or much more severe (bleeding, liver toxicity). At a cellular level, DDIs can occur as a result of both drugs competing for metabolism (known as pharmacokinetic interactions) or targeting the same protein target or biological pathway (pharmacodynamic interactions). Clinical trials typically focus on the effects of individual drugs, leaving DDIs to usually be discovered only after the drugs have been approved. One of the most carefully studied drug adverse events is long QT syndrome (LQTS), an unexpected change in the heart's electrical activity that can lead to a potentially fatal ventricular tachycardia known as torsades de pointes (TdP). Some patients have genetic mutations that lead to congenital forms of LQTS, while drug-induced LQTS typically occurs via block of the hERG potassium channel (KCNH2) responsible for ventricular repolarization. After a number of high profile drugs were withdrawn from the market due to discovered risk of TdP, the FDA issued guidelines so that pharmaceutical companies could anticipate and test for this side effect before a new drug is approved. These recommendations have helped prevent new QT-prolonging drugs from entering the market, but nonetheless over 180 approved drugs have been associated with drug-induced LQTS. While information on individual QT-prolonging drugs is thus readily available to clinicians, little has remained known about DDIs (QT-DDIs). There are many more commonly prescribed drugs that are safe when given individually but could increase TdP risk when administered together. This troubling situation is compounded by the fact that traditional post-market surveillance algorithms are poorly equipped to sensitively and specifically detect DDIs. Data science – the application of rigorous analytical methods to large datasets – offers an opportunity for predicting previously unknown QT-DDIs. Some biomedical datasets (such as drug-target binding affinities and experiments to determine protein-protein interactions) have been collected explicitly for research, while other valuable datasets (such as electronic health records) were initially recorded for billing purposes. Each data modality has its own important set of advantages and disadvantages, and integrative data science approaches can incorporate multiple types of data to help account for these limitations. In this thesis we develop new data sciences techniques that combine clinical, biological, chemical, and genetic data. These approaches are explicitly designed to be robust to biased and missing data. We apply these new methodologies to (1) predict new QT-DDIs, (2) validate them experimentally, and (3) investigate their molecular and genetic mechanisms. We exemplify this approach in the discovery of a previously unknown QT-DDI between ceftriaxone (cephalosporin antibiotic) and lansoprazole (proton pump inhibitor); importantly, both drugs have no cardiac indications and are safe when given individually. The clinical data mining, drug target prediction, biological network analysis, genetic ancestry prediction, and experimental validation methods described in this thesis form the basis for a comprehensive pipeline to predict QT-DDIs rapidly and robustly. They also provide an opportunity for further enriching our understanding of LQTS biology and ultimately enabling the design of safer drugs.

Reproduction de : Thèse de doctorat : Médecine. Aspects moléculaires et cellulaires de la biologie : Nantes : 2008. / Bibliogr.

Reproduction de : Thèse de doctorat : Médecine. Physiologie : Nantes : 2007. / Bibliogr.