Thèses sur le sujet « LQT1 »

Introduzione: Recentemente, varianti minori di SNP del gene NOS1AP sono stati associati a prolungamento del QT e aumentata incidenza di morte improvvisa in pazienti LQT1. Il gene NOS1AP codifica per la proteina CAPON, che localizza NOS1 in prossimità del reticolo sarcoplasmico (SR). L' attività di NOS1 è importante per la modulazione mediate da NO di ICaL, dei canali RyR2 e di SERCA, interferendo così con la regolazione dell’handling del Ca2+ e la stabilità del SR. Perciò abbiamo ipotizzato che gli SNPs di NOS1AP possano alterare la localizzazione/funzione di NOS1 diminuendo la stabilità del SR. In questo contesto, il prolungamento del QT indotto da mutazione, potrebbe indurre un sovraccarico di Ca2+, il cui effetto pro-aritmico potrebbe essere smascherato da un’alterata localizzazione/funzione di NOS1. Scopo: Valutare l’effetto di cambiamenti nell’attività di NOS1 a livello della stabilità funzionale del SR, della ripolarizzazione e aritmogenesi in un contesto di perdita di IKs (LQT1). Metodi: In miociti ventricolari di cavia soggetti a blocco di IKs (per riprodurre il fenotipo LQT1) e a stimolazione adrenergica (Isoproterenolo, ISO), abbiamo misurato l’attività elettrica, le correnti di membrane e il Ca2+ intracellulare, in condizione basale e dopo inibizione selettiva di NOS1 (SMTC 3µM). Risultati: In condizioni basali, l’inibizione di NOS1 prolunga la durata del PA (APD) (152.6  11.7 ms vs 96.1  9.0 ms; 58.8%. p<0.01), aumenta la densità di ICaL (SMTC vs CTRL: -16.61.2 pA/pF vs -13.51.0 pA/pF; p<0.05) ma non altera ne IKs (SMTC vs CTRL: 2.50.4 pA/pF vs 2.60.2 pA/pF) nè IKr (SMTC vs CTRL: 0.840.04 pA/pF vs 0.910.05 pA/pF). L’isoproterenolo, agonista -adrenergico (ISO, 1nM), induce delayed afterdepolarizations (DADs), un indice di instabilità del SR, in una percentuale significativamente maggiore di cellule trattate con SMTC rispetto a quelle di controllo (93% per SMTC vs 22% per CTRL, p<0.01). Inoltre, il tempo medio di comparsa delle DADs è significativamente ridotto in miociti trattati con SMTC rispetto a quelli di CTRL (25.8 ± 3.8 s e 61.5 ± 15.3 s rispettivamente, p<0.01). In aggiunta, la durata del PA è importante per il verificarsi di questi eventi, poiché il passaggio, in AP clamp, da un PA lungo (140 ms) a uno corto (100 ms) durante applicazione di ISO, abolisce le “transient inward currents” (ITI). Conclusioni: Questi risultati indicano che la mancanza di NOS1 può contribuire al prolungamento dell’APD e aumentare l’influsso di Ca2+; questi effetti compromettono la stabilità del SR in presenza di stimolazione adrenergica. Gli effetti dell’inibizione di NOS1 sono tali da poter spiegare l’effetto aritmogenico dei polimorfismi di NOS1AP.
Background: Recently, minor SNP variants of the NOS1AP gene have been reported to be associated with QT prolongation and increased incidence of sudden death in LQT1 patients. The NOS1AP gene encodes for CAPON protein, that localizes NOS1 close to the sarcoplasmic reticulum (SR). NOS1 activity accounts for NO-mediated modulation of ICaL, RyR2 channels and SERCA, thus interfering with regulation of Ca2+ handling and SR stability. Therefore we hypothesize that NOS1AP SNPs might affect NOS1 localization/function to decrease SR stability. In this setting, mutation-induced QT prolongation would induce Ca2+ overload, whose proarrhythmic effect would be unveiled by abnormal NOS1 localization/function. Aim: To evaluate the effect of changes in NOS1 activity on SR functional stability, repolarization and arrhythmogenesis in the context of IKs deficiency (LQT1). Methods: In guinea-pig ventricular myocytes subjected to IKs blockade (to reproduce the LQT1 phenotype) and adrenergic stimulation (Isoproterenol, ISO), we measured electrical activity, membrane currents and intracellular Ca2+, in basal condition and under selective inhibition of NOS1 (SMTC 3µM). Results: Under basal conditions, NOS1 inhibition prolonged AP duration (APD) (152.6  11.7 ms vs 96.1  9.0 ms; 58.8%. p<0.01), enhanced ICaL density (peak current density at +10 mV, SMTC vs CTRL: -16.61.2 pA/pF vs -13.51.0 pA/pF; p<0.05) and did not affect IKs (SMTC vs CTRL: 2.50.4 pA/pF vs 2.60.2 pA/pF) and IKr (SMTC vs CTRL: 0.840.04 pA/pF vs 0.910.05 pA/pF). The -adrenergic agonist isoproterenol (ISO, 1nM) induced delayed afterdepolarizations (DADs), an index of SR instability, in a significantly greater percentage of SMTC treated cells, compared to control ones (93% for SMTC vs 22% for CTRL, p<0.01). Moreover, the average time of DADs appearance was significantly different between SMTC and CTRL myocytes, with a earlier rise after NOS1 inhibition (25.8 ± 3.8 s and 61.5 ± 15.3 s respectively, p<0.01). Furthermore, the duration of the AP is important for the occurrence of these events, as switching from a long AP (140 ms) to a short AP (100 ms) waveform under ISO application in AP clamp mode, transient inward currents (Iti) were abolished. Conclusions: These results indicate that NOS1 deficiency may contribute to APD prolongation and enhance Ca2+ influx; these effects compromise SR stability in the presence of adrenergic stimulation. The effects of NOS1 inhibition are such as to account for the arrhythmogenic effect of NOS1AP polymorphism.

Cardiac arrhythmias arise when the myocardium is electrically unstable. They represent a life-threatening phenomena with more than 300,000 people dying every year for arrhythmic sudden cardiac death (SCD) only in the United States. One of the most important and well described arrhythmic condition is the Long QT Syndrome (LQTS). LQTS is an heterogeneous disorder of myocardial repolarization characterized by a prolongation of QT interval on the electrocardiogram and clinically manifested with syncopal episodes and SCD. Mutations in 13 genes regulating cardiac action potential (AP) have been found directly responsible for LQTS. In addition, genes regulating Ca2+-handling are found associated with a LQTS phenotype. Furthermore, many modifier genes were identified as responsible in shaping the phenotype of LQTS mutations. The most recent experimental model available for in vitro studies of cardiac arrhythmias is represented by human induced pluripotent stem cells (hiPSC). The subsequent differentiation generates functional cardiomyocytes (hiPSC-CMs) with the same genotype of patients. The aims of this study were: 1) to characterized the functional features underlying an asymptomatic or symptomatic phenotype derived from the same KCNQ1 mutation; 2) to assess the functional effect of a new de novo mutation in CALM1. Functional studies were performed on hiPSC-CMs with the patch clamp technique in voltage- and current-clamp configurations. In addition, an in silico IK1 conductance was injected with dynamic-clamp to provide for the low expression of native IK1, which is characteristic of immature cells. Field potential duration (FPD) was assessed by 256-electrodes MultiElectrode Array in hiPSC-CMs embryoid bodies. The first study was focused on the mutation Y111C on KCNQ1 gene, encoding for Kv7.1 responsible for the repolarizing current IKs. Two relatives with the same mutation were involved: the symptomatic (S) son, with a markedly prolonged corrected QT interval (QTc), and the asymptomatic (AS) father, with a borderline QTc. Control, AS and S hiPSC-CMs lines were generated. Results show that: 1) Y111C mutation induced a prolongation in AP duration (APD) and FPD in S but not in AS cells. 2) IKs density was diminished in S and a partial rescue was evidenced in AS. 3) A different IKs density was evidenced between AS and S hiPSC-CMs when transfected with wild type KCNQ1. These results suggest a likely trafficking defect of Kv7.1 to cell membrane; in addition, we hypothesized the presence of a rescue mechanism, likely a modifier gene, involving protein folding and degradation in AS, blunting the effect of Y111C mutation. The second study was focused on F142L mutation in CALM1 gene, which has been recently found directly related to a very severe form of LQTS. CALM1 encodes for calmodulin, an ubiquitous Ca2+-binding protein mainly involved in the regulation of the electrical activity of cardiomyocytes. Two hiPSC-CMs lines were generated from healthy donors and F142L patient. Results show that F142L mutation caused: 1) a prolongation in APD and FPD; 2) a strong reduction in Ca2+-current (ICaL) Ca2+-dependent inactivation. 3) an incomplete ICaL steady-state inactivation with an increased window current. 4) an increased rate of arrhythmogenic events under β-adrenergic stimulation. We can conclude that F142L mutation is the responsible of the severe phenotype evidenced in patients by strongly impairing ICaL biophysical properties. Overall, hiPSC-CMs totally recapitulate the clinical phenotype and allows the study of cardiac arrhythmias of genetic origin with an high-level translational relevance. Furthermore, they represent an excellent starting point for the generation of mutation-specific and patient-specific pharmacological therapies.

Orientador: Márcia Miguel Castro Ferreira
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química
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Resumo: O planejamento de fármacos com o auxílio do computador é uma área de pesquisa de extrema importância em química e áreas correlatas. O conjunto de ferramentas disponíveis para tal fim consiste, dentre outras, em programas para geração de descritores e construção e validação de modelos matemáticos em QSAR (do inglês, Quantitative Structure-Activity Relationship). Com o objetivo de tornar esse estudo mais acessível para a comunidade científica, novas metodologias e programas para geração de descritores e construção e validação de modelos QSAR foram desenvolvidos nessa tese. Uma nova metodologia de QSAR 4D, conhecida com LQTA-QSAR, foi desenvolvida com o objetivo de gerar descritores espaciais levando em conta os perfis de amostragem conformacional das moléculas em estudo obtidos a partir de simulações de dinâmica molecular. A geração desses perfis é feita com o software livre GROMACS e os descritores são gerados a partir de um novo software desenvolvido nesse trabalho, chamado de LQTAgrid. Os resultados obtidos com essa metodologia foram validados comparando-os com resultados obtidos para conjuntos de dados disponíveis na literatura. Um outro software de fácil uso, e que engloba as principais ferramentas de construção e validação de modelos em QSAR, foi desenvolvido e chamado de QSAR modeling. Esse software implementa o método de seleção de variáveis OPS, desenvolvido em nosso laboratório, e utiliza PLS (do inglês Partial Least Squares) como método de regressão. A escolha do algoritmo PLS implementado no programa foi feita com base em um estudo sobre o desempenho e a precisão no erro de validação dos principais algoritmos PLS disponíveis na literatura. Além disso, o programa QSAR modeling foi utilizado em um estudo de QSAR 2D para um conjunto de 20 flavonóides com atividade anti-mutagênica contra 3-nitrofluoranteno (3-NFA)
Abstract: Computer aided drug design is an important research field in chemistry and related areas. The available tools used in such studies involve software to generate molecular descriptors and to build and validate mathematical models in QSAR (Quantitative Structure-Activity Relationship). A new set of methodologies and software to generate molecular descriptors and to build and validate QSAR models were developed aiming to make these kind of studies more accessible to scientific community. A new 4DQSAR methodology, known as LQTA-QSAR, was developed with the purpose to generate spatial descriptors taking into account conformational ensemble profile obtained from molecular dynamics simulations. The generation of these profiles is performed by free software GROMACS and the descriptors are generated by a new software developed in this work, called LQTAgrid. The results obtained with this methodology were validated comparing them with results available in literature. Another user friendly software, which contains some of the most important tools used to build and validate QSAR models was developed and called QSAR modeling. This software implements the OPS variable selection algorithm, developed in our laboratory, and uses PLS (Partial Least Squares) as regression method. The choice of PLS algorithm implemented in the program was performed by a study about the performance and validation precision error involving the most important PLS algorithms available in literature. Further, QSAR modeling was used in a 2D QSAR study with 20 flavonoid derivatives with antimutagenic activity against 3-nitrofluoranthene (3-NFA)
Doutorado
Físico-Química
Doutor em Ciências

Orientador: Márcia Miguel Castro Ferreira
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química
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Resumo: LQTA-QSAR é uma metodologia computacional para QSAR-4D desenvolvida pelo Laboratório de Quimiometria Teórica e Aplicada implementada em um software de acesso livre. O método permite considerar simultaneamente as vantagens da representação molecular multiconformacional e os descritores de campos de interação. Esta tese apresenta a evolução da proposta inicial da metodologia LQTA-QSAR independente de receptores para uma abordagem dependente de receptores. Sua aplicação é demonstrada na construção de modelos de QSAR-4D para a previsão da atividade inibitória de compostos fenotiazínicos da enzima tripanotiona redutase. Foi obtido um modelo com bom poder de previsão (Qprev = 0,78) e com descritores de fácil interpretação. Tal modelo pode ser usado para a proposição de compostos que poderão vir a ser usados para o tratamento da doença de chagas. Para a filtragem e seleção de descritores foi necessário o desenvolvimento de um protocolo completamente distinto daquele disponível na literatura. Foi proposto um procedimento automatizado para identificar e eliminar descritores irrelevantes quando a correlação e um algoritmo que elimina descritores com distribuição díspar em relação à atividade biológica. Foram introduzidos também testes de validação de modelos QSAR nunca antes usados para modelos que utilizam descritores de campo de interação. O protocolo completo foi testado em três conjuntos de dados e os modelos obtidos tiveram capacidade de previsão superior aos da literatura. Os modelos mostraram ser bastante simples e robustos quando submetidos aos testes leave-N-out e y-randomization
Abstract: The New Receptor-Dependent LQTA-QSAR approach is proposed as a new 4D-QSAR method. The RD-LQTA-QSAR is an evolution to the receptor independent LQTA-QSAR. This approach make use of the simulation package GROMACS to carry out molecular dynamics simulations and generate a conformational ensemble profile for each compound. Such ensemble is used to build molecular interaction field based QSAR models, as in CoMFA. To verify the usefulness of the methodology it was chosen some phenothiazine derivatives that are specific competitive T. cruzi trypanothione reductase inhibitors. Using a combination of molecular docking and molecular dynamics simulations the binding mode of 38 phenotiazine derivatives was evaluated in a simulated induced fit approach. The ligands¿ alignment, necessary to the methodology, was performed using both ligand and binding site atoms hereafter enabling unbiased alignment. The obtained models were extensively validated by Leave-N-out cross-validation and y-randomization techniques to test robustness and absence of chance correlation. The final model presented Q LOO of 0.87 and R of 0.92 and suitable external prediction = 0.78. It is possible to use the obtained adapted binding site of to perform virtual screening and ligand structures based design, as well as using models descriptors to design new inhibitors. In the process of QSAR modeling, the relevance of correlation and distribution profiles were tested in order to improve prediction power. A set of tools to filter descriptors prior to variable selection and a protocol for molecular interaction field descriptors selection and models validation are proposed. The algorithms and protocols presents are quite simple to apply and enable a different and powerful way to build LQTA-QSAR models
Doutorado
Físico-Química
Doutor em Ciências

In this paper the higgs mechanism for the standard model is constructed in steps. First by considering spontaneous breaking of discrete and continuous global gauge invariance. Then spontaneous breaking of local gauge invariance. These results are then used to construct the electroweak part of the standard model through application of the higgs mechanism. Finally, the LQT-upper bound of 1 TeV for the higgs mass is calculated through unitarity constraints.
I denna artikel konstrueras higgsmekanismen i standardmodellen stegvis. Först genom att beakta spontant symmetribrott av diskreta samt kontinuerliga globala gaugeinvarianser. Därefter spontant symmetribrott av lokala gaugeinvarianser. Dessa resultat används sedan för att konstruera den elektrosvaga delen av standardmodellen genom tillämpning av higgsmekanismen. Slutligen beräknas en övre gräns för higgsmassan, den så kallade LQT-gränsen, via unitaritetsbegränsingar.

This diploma thesis deals with study of the rate-dependence of the magnitude of a current through the heart channel that conducts slowly activating component of delayed rectifier outward current (IKs). This property is very important for the IKs channel function. When other repolarizing currents are insufficient, but also when the heart rate accelerates, especially during elevated sympathetic tone, IKs provides so-called repolarization reserve, which prevents excessive lengthening of cardiac action potential repolarization. The IKs channel structure is encoded by the KCNQ1 (pore-forming -subunit) and KCNE1 (modulatory -subunit) genes. Mutations in these genes disrupt the physiological function of the IKs channel and cause inherited arrhythmogenic syndromes, especially long QT syndrome (LQTS). Such mutations include the c.926C>T (p.T309I) mutation in the KCNQ1 gene, which results in LQTS type 1 in heterozygous carriers. The theoretical part of the thesis provides basic information about the IKs channel and the patch clamp technique, this knowledge is necessary for the practical part. The experimental part is focused on cultivation of the CHO cell line and its transient transfection for subsequent electrophysiological measurements by whole-cell patch clamp technique to study the dependence of the IKs magnitude on stimulation frequency, both in the wild type channels (i.e. without mutation) and in those with cotransfected wild type and T309I subunits.

Thesis (DMed (Medicine. Internal Medicine))--University of Stellenbosch, 2007.
Background Although great progress has been made in defining genes conferring the majority of genetic risk in Long QT Syndrome (LQTS) patients, there remains a substantial challenge to explain the widely observed variability in disease expression and phenotype severity, even among family members, sharing the same mutation. Identifying clinical and genetic variables capable of influencing/predicting the clinical phenotype of LQTS patients would allow a more accurate risk stratification, important for determining prognosis, selecting patients for the most appropriate therapy, and counseling asymptomatic mutation carriers (MCs). To address these questions an Italian LQT2 family and a South African Founder LQT1 population have been used. Methods and Results Italian LQT2 family. The proband, a 44-yr-old white woman, presented with ventricular fibrillation and cardiac arrest. Intermittent QT prolongation was subsequently observed and LQT2 was diagnosed following the identification of a missense KCNH2 mutation (A1116V). The proband also carried the common KCNH2 polymorphism K897T on the non-mutant allele. Relatives who carried A1116V without K897T were asymptomatic but some exhibited transient mild QTc prolongation suggesting latent disease. Expression studies in Chinese Hamster Ovary (CHO) cells, demonstrated that the presence of KCNH2-K897T is predicted to exaggerate the IKr reduction caused by the A1116V mutation. These data explain why symptomatic LQTS occurred only in the proband carrying both alleles. South African LQT1 population. The study population involved 320 subjects, 166 MCs and 154 non mutation carriers (NMCs). Off ß-blocker therapy, MCs had a wide range of QTc values (406-676 ms) and a QTc>500 ms was associated with increased risk for cardiac events (OR=4.22; 95%CI 1.12-15.80; p=0.033). We also found that MCs with a heart rate <73 bpm were at significantly lower risk (OR=0.23; 95%CI 0.06-0.86; p=0.035). In a subgroup of patients Baroreflex Sensitivity (BRS) was determined both in presence and absence of ß-blocker therapy. BRS, analyzed in subjects in the 2nd and 3rd age quartiles (age 26-47) to avoid the influence of age, was lower among asymptomatic than symptomatic MCs (11.8±3.5 vs 20.1±10.9 ms/mmHg, p<0.05). A BRS in the lower tertile carried a lower risk of cardiac events (OR 0.13, 95%CI 0.02-0.96; p<0.05). This study also unexpectedly determined that KCNQ1-A341V was associated with greater risk than that reported for large databases of LQT1 patients: A341V MCs were more symptomatic by age 40 (79% vs 30%) and became symptomatic earlier (7±4 vs 13±9 years), both p<0.001. Accordingly, functional studies of KCNQ1-A341V in CHO cells with KCNE1, identified a dominant negative effect of the mutation on wild-type channels. Conclusion Our findings indicate that risk stratification for LQTS patients must be more individually tailored and may have to take into account the specific mutation and probably additional clinical and genetic variables capable of influencing/predicting the clinical phenotype of LQTS patients. As a matter of fact, we have provided evidence that a common KCNH2 polymorphism may modify the clinical expression of a latent LQT2 mutation and the availability of an extended kindred with a common mutation allowed us to highlight that KCNQ1-A341V is associated with an unusually severe clinical phenotype and to identify two autonomic markers, HR and BRS, as novel risk factors.

[ES] Las enfermedades cardiovasculares siguen siendo la principal causa de muerte en Europa. Las arritmias cardíacas son una causa importante de muerte súbita, pero sus mecanismos son complejos. Esto denota la importancia de su estudio y prevención. La investigación sobre electrofisiología cardíaca ha demostrado que las anomalías eléctricas causadas por mutaciones que afectan a canales cardíacos pueden desencadenar arritmias. Sorprendentemente, se ha descubierto una gran variedad de fármacos proarrítmicos, incluidos aquellos que usamos para prevenirlas. Las indicaciones de uso de fármacos actuales intentaron solucionar este problema diseñando una prueba para identificar aquellos fármacos que podían ser peligrosos basado en el bloqueo de un solo canal iónico. El estudio de las interacciones fármaco-canal ha revelado la existencia no sólo de compuestos que bloquean múltiples canales, sino también una gran complejidad en esas interacciones. Esto podría explicar por qué algunos medicamentos pueden mostrar efectos muy diferentes en la misma enfermedad. Existen dos desafíos importantes con respecto a los efectos de los fármacos en la electrofisiología cardíaca. Por un lado, las empresas y entidades reguladoras están buscando una herramienta de alto rendimiento que mejore la detección del potencial proarrítmico durante el desarrollo de fármacos. Por otro lado, los pacientes con anomalías eléctricas a menudo requieren tratamientos personalizados más seguros. Las simulaciones computacionales contienen un poder sin precedentes para abordar fenómenos biofísicos complejos. Deberían ser de utilidad a la hora de determinar las características que definen tanto los efectos beneficiosos como no deseados de los fármacos mediante la reproducción de datos experimentales y clínicos. En esta tesis doctoral, se han utilizado modelos computacionales y simulaciones para dar respuesta a estos dos desafíos. El estudio de los efectos de los fármacos sobre la actividad cardíaca se dividió en el estudio de su seguridad y de su eficacia, respectivamente. Para dar respuesta al primer desafío, se adoptó un enfoque más amplio y se generó un nuevo biomarcador fácil de usar para la clasificación del potencial proarrítmico de los fármacos utilizando modelos del potencial de acción de células y tejidos cardíacos humanos. Se integró el bloqueo de múltiples canales a través de IC50 y el uso de concentraciones terapéuticas con el fin de mejorar el poder predictivo. Luego, se entrenó el biomarcador cuantificando el potencial proarrítmico de 84 fármacos. Los resultados obtenidos sugieren que el biomarcador podría usarse para probar el potencial proarrítmico de nuevos fármacos. Respecto al segundo desafío, se adoptó un enfoque más específico y se buscó mejorar la terapia de pacientes con anomalías eléctricas cardíacas. Por lo tanto, se creó un modelo detallado de la mutación V411M del canal de sodio, causante del síndrome de QT largo, reproduciendo datos clínicos y experimentales. Se evaluaron los posibles efectos beneficiosos de ranolazina, a la par que se aportó información sobre los mecanismos que impulsan la efectividad de la flecainida. Los resultados obtenidos sugieren que, si bien ambos fármacos mostraron diferentes mecanismos de bloqueo de los canales de sodio, un tratamiento con ranolazina podría ser beneficioso en estos pacientes.
[CA] Les malalties cardiovasculars continuen sent la principal causa de mort a Europa. Les arrítmies cardíaques són una causa important de mort sobtada, però els seus mecanismes són complexos. Això denota la importància del seu estudi i prevenció. La investigació sobre electrofisiologia cardíaca ha demostrat que les anomalies elèctriques que afecten a canals cardiacs poden desencadenar arrítmies. Sorprenentment, s'ha descobert una gran varietat de fàrmacs proarrítmics, inclosos aquells que utilitzem per a previndre-les. Les indicacions d'ús de fàrmacs actuals van intentar solucionar aquest problema dissenyant una prova per a identificar aquells fàrmacs que podien ser perillosos basada en el bloqueig d'un sol canal iònic. L'estudi de les interaccions fàrmac-canal ha revelat l'existència no sols de compostos que bloquegen múltiples canals, sinó també una gran complexitat en aquestes interaccions. Això podria explicar per què alguns medicaments poden mostrar efectes molt diferents en la mateixa malaltia. Existeixen dos desafiaments importants respecte als efectes dels fàrmacs en la electrofisiologia cardíaca. D'una banda, les empreses i entitats reguladores estan buscant una eina d'alt rendiment que millore la detecció del potencial proarrítmic durant el desenvolupament de fàrmacs. D'altra banda, els pacients amb anomalies elèctriques sovint requereixen tractaments personalitzats més segurs. Les simulacions computacionals contenen un poder sense precedents per a abordar fenòmens biofísics complexos. Haurien de ser d'utilitat a l'hora de determinar les característiques que defineixen tant els efectes beneficiosos com no desitjats dels fàrmacs mitjançant la reproducció de dades experimentals i clíniques. En aquesta tesi doctoral, s'han utilitzat models computacionals i simulacions per a donar resposta a aquests dos desafiaments. L'estudi dels efectes dels fàrmacs sobre l'activitat cardíaca es va dividir en l'estudi de la seva seguretat i la seva eficacia. Per a donar resposta al primer desafiament, es va adoptar un enfocament més ampli i es va generar un nou biomarcador fàcil d'usar per a la classificació del potencial proarrítmic dels fàrmacs utilitzant models del potencial d'acció de cèl·lules i teixits cardíacs humans. Es va integrar el bloqueig de múltiples canals a través d'IC50 i l'ús de concentracions terapèutiques amb la finalitat de millorar el poder predictiu. Després, es va entrenar el biomarcador quantificant el potencial proarrítmic de 84 fàrmacs. Els resultats obtinguts suggereixen que el biomarcador podria usar-se per a provar el potencial proarrítmic de nous fàrmacs. Respecte al segon desafiament, es va adoptar un enfocament més específic i es va buscar millorar la teràpia de pacients amb anomalies elèctriques cardíaques. Per tant, es va crear un model detallat de la mutació V411M del canal de sodi, causant de la síndrome de QT llarg, reproduint dades clíniques i experimentals. Es van avaluar els possibles efectes beneficiosos de ranolazina, a l'una que es va aportar informació sobre els mecanismes que impulsen l'efectivitat de la flecainida. Els resultats obtinguts suggereixen que, si bé tots dos fàrmacs van mostrar diferents mecanismes de bloqueig dels canals de sodi, un tractament amb ranolazina podria ser beneficiós en aquests pacients.
[EN] Cardiovascular disease remains the main cause of death in Europe. Cardiac arrhythmias are an important cause of sudden death, but their mechanisms are complex. This denotes the importance of their study and prevention. Research on cardiac electrophysiology has shown that electrical abnormalities caused by mutations in cardiac channels can trigger arrhythmias. Surprisingly, a wide variety of drugs have also shown proarrhythmic potential, including those that we use to prevent arrhythmia. Current guidelines designed a test to identify dangerous drugs by assessing their blocking power on a single ion channel to address this situation. Study of drug-channel interactions has revealed not only compounds that block multiple channels but also a great complexity in those interactions. This could explain why similar drugs can show vastly different effects in some diseases. There are two important challenges regarding the effects of drugs on cardiac electrophysiology. On the one hand, companies and regulators are in search of a high throughput tool that improves proarrhythmic potential detection during drug development. On the other hand, patients with electrical abnormalities often require safer personalized treatments owing to their condition. Computer simulations provide an unprecedented power to tackle complex biophysical phenomena. They should prove useful determining the characteristics that define the drugs' beneficial and unwanted effects by reproducing experimental and clinical observations. In this PhD thesis, we used computational models and simulations to address the two abovementioned challenges. We split the study of drug effects on the cardiac activity into the study of their safety and efficacy, respectively. For the former, we took a wider approach and generated a new easy-to-use biomarker for proarrhythmic potential classification using cardiac cell and tissue human action potential models. We integrated multiple channel block through IC50s and therapeutic concentrations to improve its predictive power. Then, we quantified the proarrhythmic potential of 84 drugs to train the biomarker. Our results suggest that it could be used to test the proarrhythmic potential of new drugs. For the second challenge, we took a more specific approach and sought to improve the therapy of patients with cardiac electrical abnormalities. Therefore, we created a detailed model for the long QT syndrome-causing V411M mutation of the sodium channel reproducing clinical and experimental data. We tested the potential benefits of ranolazine, while giving insights into the mechanisms that drive flecainide's effectiveness. Our results suggest that while both drugs showed different mechanisms of sodium channel block, ranolazine could prove beneficial in these patients.
This PhD thesis was developed within the following projects: Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional (FEDER) DPI2015-69125-R (MINECO/FEDER, UE): Simulación computacional para la predicción personalizada de los efectos de los fármacos sobre la actividad cardiaca. Dirección General de Política Científica de la Generalitat Valenciana (PROMETEU2016/088): “Modelos computacionales personalizados multiescala para la optimización del diagnóstico y tratamiento de arritmias cardiacas (personalised digital heart). Vicerrectorado de Investigación, Innovación y Transferencia de la Universitat Politècnica de València, Ayuda a Primeros Proyectos de Investigación (PAID-06-18), and by Memorial Nacho Barberá. Instituto de Salud Carlos III (La Fe Biobank PT17/0015/0043).
Cano García, J. (2021). Prediction of the effects of drugs on cardiac activity using computer simulations [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/164094
TESIS

Les Síndromes de QT Llarg (SQTL) i de Brugada (SBr) són malalties hereditàries amb un alt risc de mort sobtada cardíaca degut a alteracions elèctriques cardíaques sense cap defecte estructural del cor. Estan relacionades amb mutacions a gens que codifiquen pels canals iònics cardíacs o proteïnes reguladores. No obstant això, resten sense diagnòstic genètic un percentatge elevat de pacients amb SQTL (20-35%) i SBr (60-75%) després de la sequenciació dels gens relacionats. En aquesta tesi es planteja si mutacions a les subunitats β del canal de sodi no associades prèviament a SQTL i SBr poden ser responsables d’aquestes malalties en els pacients sense mutació. Els resultats obtinguts relacionen per primera vegada mutacions als gens SCN1Bb i SCN2B a la SQTL i SBr, respectivament; i aporten noves evidències de les bases genètiques d’aquestes malalties i del paper fonamental de les subunitats β en la funció del canal de sodi cardíac.
Long QT Syndrome (LQTS) and Brugada Syndrome (BrS) are inheritable cardiac diseases with a high risk of sudden cardiac death due to electric alterations without structural defects of the heart. These alterations are determined by mutations in cardiac ion channels or regulatory proteins. However, near 20-35% of LQTS patients and 60-75% of BrS patients remain without a genetic diagnosis after the screening of LQTS and BrS-previously related genes. Our assumption was that mutations in the sodium channel β subunits, still not related to the respective disease, could explain the phenotype in patients without a mutation after genetic screening. We identify for the first time mutations in two β subunits genes (SCN1Bb and SCN2B) responsible for LQTS and BrS, respectively; and our studies provide new evidences of the genetic basis of LQTS and BrS, and support the growing evidence of the important role of β subunits on cardiac sodium channel function.

碩士
國立中央大學
生命科學研究所
98
Long QT syndrome (LQTS) is referred to as a clinical constellation in which patients manifest episodic syncope, cardiac arrest or sudden death due to torsades de pointes associated with electrocardiographic QT prolongation and otherwise normal hearts. Of the ten types of LQTS, LQT1 is caused by mutations in the gene encoding KCNQ1 leading to “loss of function” of the IKs channel. Of note, clinically LQT1 patients tend to develop cardiac events during physical exertion (esp. swimming)[1] and beta-adrenergic receptor blocking agents provide only incomplete protection. In the proposed study, we sought to define mechanisms by which beta-adrenergic stimulation (BAS) provoke cardiac arrhythmias and more importantly to search for an alternative or adjunctive regimen for more effectively to prevent cardiac arrhythmias from occurrence. We plan to apply the Luo-Rudy ventricular myocyte model which has incorporated a relatively detailed dynamics of intracellular Ca2+ cycling to simulate LQT1. Mechanisms to be defined include abnormal automaticity, reentry, and triggered activity whereas potential targeted sites of therapy will be focused on components enhanced by BAS, ICa,L, IKs, IK1, INaK and Iup. We anticipate that LQT1 will have inducible delayed afterdepolarizations that are aggravated by BAS-induced enhancement of ICa,L and Iup, and an inhibitor capable of blocking both ICa,L and Iup should be an useful adjunctive to beta-adrenergic receptor blocking agents. Keywords：Long QT syndrome; Torsades de pointes; Beta-adrenergic stimulation (BAS); Intracellular Ca2+ cycling

碩士
國立臺灣大學
生醫電子與資訊學研究所
102
SCN5A encodes a cardiac sodium channel. Its mutations are associated with Brugada Syndrome (BrS) and Long QT Syndrome Type 3 (LQT3). Both diseases are often neglected by the clinicians because they are difficult to diagnose. Hundreds of non-synonymous variants have been identified in SCN5A; however, the underlying mechanism and the relationship between the genotype and phenotype remain unclear. A new approach that helps to screen and prioritize identified mutations is beneficial for researchers to identify a novel pathogenic mutation in this high-throughput sequencing era. Therefore, we aim to study and analyze the characteristics of SCN5A variants in order to evaluate the possibility of these mutations developing into BrS or LQT3. In this study, 4 prediction algorithms were used to predict whether a variant is pathogenic or benign. The algorithms includes: Sorts Intolerant From Tolerant (SIFT), Protein Variation Effect Analyzer (PROVEAN), Polymorphism Phenotyping v2 (PolyPhen2) and Genomic Evolutionary Rate Profiling++ (GERP++). Several variants (BrS N=425, LQT3 N=136) were collected from literatures and published reports. Furthermore, Estimated Predictive Values (EPV) is used to evaluate the frequency of one variant in a rare disease, such as BrS or LQT3. Therefore, for each variant, EPV was calculated and all variants were classified into different groups based on the protein structures and exon information. The results demonstrated that higher prediction performances can be obtained when at least 3 prediction algorithms agreed on pathogenicity. For example, the EPVs increased from 56% to 75% and 60% to 83% in the domains of Pfam-B3701 and Na transmembrane in BrS, respectively. In general, the results showed that the proposed approach was able to discriminate case-derived variants and general-population-derived variants. Based on the filtered variants, a prediction model was developed to evaluate potential risk for each variant. In addition, the associations between the SCN5A domains and the two diseases, BrS and LQT3, were evaluated. Intriguingly, the results showed that a variant in domain II (DII) transmembrane has a higher possibility that can develop into BrS. Similarly, a variant in the C-terminal may have a higher chance turning into LQT3. In conclusion, a probability model that integrates EPV and 4 prediction algorithms was developed in this study in order to classify variants identified in SCN5A and evaluate the chance that such variants may lead to BrS or LQT3.

碩士
國立成功大學
生理學研究所
96
Timothy syndrome (TS) is a malignant form of congenital long QT syndrome caused by genetic mutations that result in “nearly complete failure” of voltage-dependent inactivation (VDI) of the Cav1.2 channel leading to "gain of function”. We set out to explore electrophysiological mechanisms underlying the propensity to develop lethal ventricular arrhythmias associated with TS. We adopted a modified Luo-Rudy cell model of ventricular myocyte along with 1-dimensional multicellular strand model. To simulate mutant-induced reduction of VDI of the Cav1.2 channel, the time constant of VDI was multiplied by 2-fold to 20-fold. This resulted in progressive prolongation of action potential duration (APD) and QT interval without eliciting abnormal automaticity. Delayed afterdepolarizations (DADs) and DAD-mediated trigger activity first appeared in the mid-myocardial cell, and intermittently, and we also observed APD alternans with early afterdepolarizations (EADs). Additionally, there was a progressive increase in transmural dispersion of repolarization (TDR) alongside steepening of APD restitution. Beta-adrenergic stimulation further amplified TDR and steepened APD restitution, and importantly, facilitated induction of DAD-mediated triggered activity culminating in its transformation into a “flutter- or fibrillatory-like” rhythm driven by a transient inward current (ITi) generated by forward mode Na+-Ca2+ (INCX) and nonspecific Ca2+-activated currents (INS(Ca)) evoked by spontaneous Ca2+ release from the sarcoplasmic reticulum (Irel,overload) related to “Ca2+ overload.” Of note, bradycardia further prolonged APD and exerted similar proarrhythmic effects. In patients with TS, significant reduction of VDI of the Cav1.2 channel may provide not only a trigger but also a substrate for the propensity to develop lethal ventricular arrhythmia aggravated by enhanced sympathetic tone and bradycardia.

The human ether-à-go-go-related gene (hERG) encodes the pore-forming α-subunits of the Kv11.1 channel that is responsible for the cardiac rapidly activating delayed rectifier K+ current (IKr), which plays a critical role in cardiac repolarization. Dysfunction of hERG causes long QT syndrome (LQTS), a cardiac electrical disorder that can lead to severe cardiac arrhythmias and sudden death (Mitcheson et al., 2000a; Roden, 2004; Maier et al., 2006; Misner et al., 2012). The overall function of hERG channels is dependent on the channel density at the plasma membrane as well as proper channel gating. Previous work from our lab demonstrated that degradation of hERG protein in the lysosome is regulated by ubiquitin ligase Nedd4-2-mediated monoubiquitination (Sun et al., 2011; Guo et al., 2012). However, whether the internalized hERG proteins can be recycled back to the plasma membrane remains to be determined. In the present study, we investigated the regulatory effects of various Rabs on hERG channels using Western blot analysis, co-immunoprecipitation (Co-IP), whole-cell patch clamp and immunofluorescence microscopy. The data revealed that, among hERG, human Kv1.5 (cardiac ultra-rapidly activating delayed rectifier K+ channel), and human EAG (ether-à-go-go gene) potassium channels, Rab4 selectively decreased the mature hERG protein expression on the plasma membrane. Mechanistically, Rab4 did not directly target the internalized hERG protein for recycling. Instead, Rab4 increased the expression level of the E3 ubiquitin ligase Nedd4-2 (Neural Precursor Cell-expressed Developmentally Downregulated Protein 4-2), which has been shown to mediate hERG ubiquitination and degradation (Guo et al., 2012). Nedd4-2 binding site mutations ∆1073 (binding site is removed) and Y1078A (binding site is modified) in hERG completely abolished the effect of Rab4. It has been shown that Nedd4-2 undergoes self-ubiquitination after targeting substrates (Bruce et al., 2008). My data further demonstrated that Rab4 decreased the degradation rate of Nedd4-2 and increased the rate of recycling. The increased Nedd4-2 then decreases hERG expression at the plasma membrane by targeting the PY-motif in the C-terminus of hERG channels. In summary, the present study showed that Rab4 decreases the expression and function of hERG potassium channels on the plasma membrane through enhancing the recycling of the ubiquitin ligase Nedd4-2.
Thesis (Master, Physiology) -- Queen's University, 2013-08-09 12:11:27.938

Background: Long QT syndrome (LQTS) is a cardiac condition which predisposes individuals to syncope, seizures, and sudden cardiac death. There is a high prevalence of congenital LQTS in a First Nations community in Northern British Columbia due to the founder variant p.V205M in the KCNQ1 gene. Additionally, two other variants of interest are present in this population: the KCNQ1 p.L353L variant, previously noted to modify the phenotype of LQTS in adults, and the CPT1A p.P479L variant, a metabolic variant common in Northern Indigenous populations associated with hypoglycemia and sudden unexpected infant death. Methods: We performed a mixed methods study to better understand the impact of LQTS in children and youth in this First Nations community. To learn about the clinical impact of LQTS, and better understand the effects of the KCNQ1 and CPT1A variants in children, we used statistical analysis to compare the cardiac phenotypes of 211 First Nations children with and without the p.V205M, p.L353L and p.P479L variants, alone and in combination. Ordinary Least Squares linear regression was used to compare the highest peak corrected QT interval (QTc). The peak QTc is an electrocardiogram measurement used in risk stratification of LQTS patients. Logistic regression was used to compare the rates of syncope and seizures experienced in childhood. Additionally, to learn about the lived-experience of LQTS, we interviewed one young First Nations adult about her experiences growing up with LQTS as a teenager. From this interview, we conducted a qualitative case study analysis using Interpretative Phenomenological Analysis. All research was done in partnership with the First Nations community using community-based participatory methods. Results: We found that the p.V205M variant conferred a 22.4ms increase in peak QTc (p<0.001). No other variants or variant interaction effects were observed to have a significant impact on peak QTc. No association between the p.V205M variant and loss of consciousness (LOC) events (syncope and seizures) was observed (OR(95%CI)=1.3(0.6-2.8); p=0.531). However, children homozygous for p.P479L were found to experience 3.3 times more LOC events compared to non-carriers (OR=3.3(1.3-8.3); p=0.011). With regard to the qualitative portion of the thesis, four superordinate (main) themes emerged from the case study: Daily life with Long QT Syndrome, Interactions with Medical Professionals, Finding Reassurance, and The In-Between Age. We found that even though our participant was asymptomatic and felt that she was not impacted by LQTS in her daily life, she considered certain elements of the condition to be stressful, such as taking a daily beta-blocker. Conclusion: These results suggest that while the KCNQ1 p.V205M variant is observed to significantly prolong the peak QTc, the CPT1A p.P479L variant is more strongly associated with LOC events in children from this community. More research is needed to further determine the effect of these variants; however, our preliminary findings suggest management strategies, such as whether beta-blockers are indicated for p.V205M carriers, may need to be reassessed. The importance of developing a holistic, well-balanced approach to medical care, taking into consideration the personal perspectives and unique medical circumstances of each child is exemplified in this study.
Graduate

yes
Type II congenitalLong QT syndrome (LQT2) is due to genetic mutations in hERG channel. Genetic or pharmacological factors could potentially affect hERG channel biogenesis and contributes to LQTS, for example, disease mutations G601S and T473P result in hERG trafficking deficiency [1,2]. Various rescue strategies for hERG dysfuction are being developed. Some correctors for CFTR channel have been reported to act indirectly on proteostasis pathways to promote folding and correction on hERG trafficking deficiency [3]. In this study, we tested the hypothesis that the CFTR corrector KM-11060 and the potentiator PG-01 may correct hERG mutation trafficking diseases. We use HEK293 cell line expressing a well-studied trafficking disease mutation G601S-hERG channel [4]. We treated cells with CFTR potentiator PG-01and corrector KM-11060, which function through different cellular mechanisms, and assessed whether correction occurred via immunoblotting. Whole cell proteins from HEK 293 cells expressing hERG channels were used for analysis [5]. Proteins were separated on 8% SDS-polyacrylamide electrophoresis gels for 1 hour, transferred onto PVDF membrane, and blocked for 1 h with 5% nonfat milk. The blots were incubated with the primary antibody (Santa Cruz Biotechnology) for 12-16 h at 4C temperature and then incubated with a donkey antigoat horseradish peroxidase-conjugated secondary antibody( Santa Cruz Biotechnology). Actin expression was used for loading controls. The blots were visualized using the ECL detection kit (Genshare).Results were deemed significantly different from controls by a one-way ANOVA (p < 0.05). Our results show that both KM-11060 (5, 10, 20uM) and PG-01(5, 15 uM) can correct G601S mutant alleles of hERG protein trafficking (Fig 1, 2). KM-11060 (20uM) but not PG-01(15 uM) enhance protein expression of wild type hERG channel (Fig 2). Further treatment on cells at low temperature with different drug concentration will be tested. Functional studies are also needed to test whether the drugs can correct the function of hERG mutation channel. These results could potentially provide novel insight into the correction mechanism of CFTR potentiator and also help to develop new treatment for LQT2.