48 research outputs found
HiPSC-derived cardiac tissue for disease modeling and drug discovery
Li, J.; Hua, Y.; Miyagawa, S.; Zhang, J.; Li, L.; Liu, L.; Sawa, Y. hiPSC-Derived Cardiac Tissue for Disease Modeling and Drug Discovery. Int. J. Mol. Sci. 2020, 21, 8893
Syncytial Model of Human Pluripotent Stem Cell-Derived Cardiomyocytes for Electrophysiology Studies
Human pluripotent stem cells (hPSCs) are a valuable resource for generating human cardiomyocytes and modeling human cardiac physiology in vitro. In this thesis, the electrophysiology of hPSC derived cardiomyocytes was studied in large populations of cells in syncytial models. It was found that heterogeneity in electrophysiology, as represented by action potential variability, is common in small clusters of hPSC-derived cardiomyocytes. A waveform-based automated algorithm was used to identify groups of cardiomyocytes based on similarity of their action potentials. It was found that, unlike in small cell clusters, action potential variability in monolayer culture was relatively low, resembling mainly a single electrophysiological phenotype. The utility of a monolayer hPSC-CM model was explored in two applications: 1) modeling the monogenic disease, type 2 Long QT syndrome (LQT2), using a human induced pluripotent stem cell (hiPSC) line carrying a hERG-A422T mutation, and 2) studying responses to cardioactive drugs using hiPSC-derived cardiomyocytes. The monolayer model with LQT2 hiPSC-derived cardiomyocytes had prolonged action potentials and an increased sensitivity to IKr block compared to that of non-disease cardiomyocytes, consistent with the expected LQT2 phenotype. The prolonged action potentials could be normalized by activation of IKr with ML-T531, a compound that delays the inactivation of the hERG channel. However, ectopic activity, such as early-afterdepolarizations (EADs), were mostly absent in LQT2 monolayers, in contrast to the frequent occurrence reported in smaller cell cultures or single cells. For drug testing, monolayers of hiPSC-derived cardiomyocytes responded to a panel of eight cardioactive drugs in a manner consistent with the mechanism of the drugs: blockers of repolarizing currents prolonged the action potentials, while blockers of depolarizing currents shortened them. At a tissue level, blockers of excitatory INa slowed propagation of action potentials. Computational modeling showed that a drug can alter the repolarization gradient, a proarrhythmia biomarker, in monolayers having a defined electrophysiological gradient
The hERG1 (KV11.1) potassium channel : its modulation and the functional characterisation of genetic variants
The human ether ĂĄ-go-go related gene (hERG1 or KCNH2) encodes the pore forming subunit of the cardiac delayed rectifier potassium (IKr) channel. Its unique kinetics result in a resurgent current crucial for the repolarisation of the cardiac action potential and a capability to suppress premature excitation. hERG1 is widely expressed with roles e.g. in neuronal firing, intestinal and uterine contractility, and insulin secretion. Furthermore overexpression and ectopic expression of hERG1 occurs in cancer where it is involved in proliferation, migration, chemotherapy resistance etc.
The long QT syndrome (LQTS) often presents as sudden cardiac death in children and young adults. LQTS is characterised by electrocardiogram abnormalities with arrhythmia that can lead to palpitations, syncope, seizure, cardiac arrest and death. Underlying the congenital form of LQTS are mutations in ion channel proteins (including hERG1, the loss-of-function of which gives rise to LQT2) and their interacting proteins. Carriers of a particular mutation may be symptomatic (to varying extents) or asymptomatic, with the deleterious effects only emerging due to the presence of other factors. This is analogous to drug-induced LQTS where arrhythmia may occur in 1 of 120,000 users of certain non-cardiac drugs. Virtually all drug-induced LQTS is caused by inhibition of hERG1. Consequently in the field of safety pharmacology the hERG1 channel has for the last 20 years and continues to have a huge impact as the primary in vitro predictor of the proarrhythmic risk for a drug.
Various aspects of the hERG1 channel are investigated in the studies presented in this thesis. The effect of prucalopride, a gastrointestinal prokinetic drug, on hERG1 was examined. Prucalopride exhibited rapid state and concentration dependent inhibition of hERG1 however, at therapeutic concentrations block is insignificant (hERG safety margin of â„300). This in vitro prediction has translated to the clinical studies of this drug and the market.
The heterogeneous phenotype associated with LQTS may arise from genetic modifiers such polymorphisms and mutations. Heterologous expression of the prevalent hERG1 K897T polymorphism identified a reduced hERG1 current density as the primary difference from wild-type, a result of decreased protein expression. Additionally a slowing of deactivation and alteration of inactivation was evident. Also studied but using induced pluripotent stem cell (iPSC) derived cardiomyocytes was hERG1 R176W. Unlike previous LQT2 iPSC models the origin here was a relatively asymptomatic individual. The phenotypic characteristics of LQT2 were however still reproduced in vitro (i.e. a decrease in IKr and action potential prolongation) though as a milder version.
Finally the effect of ceramide, a sphingolipid which accumulates in heart failure and is involved in lipotoxicity, on hERG1 was investigated. Ceramide was found to reduce hERG1 current in a time dependent manner through tagging (ubiquitination) of the cell surface protein for internalisation and targeting to lysosomes.HERG (human ether-a-go-go related gene, hERG1 tai KCNH2) geenin ilmentÀmÀ proteiini tuottaa kaliumkanavan, joka vastaa sydÀnsolujen sÀhköfysiologisissa mittauksissa kuvattua IKr virtaa. HERG kaliumkanava aukeaa sydÀmen aktiopotentiaalin repolarisaatiovaiheessa ja estÀÀ nÀin ennenaikaisen aktiopotentiaalin laukeamisen. hERG1 proteiinia ilmentyy sydÀmen lisÀksi useissa muissa kudoksissa mm. hermosoluissa, suolessa, kohdussa ja haiman insuliinia tuottavissa soluissa. hERG1 ilmentyy lisÀksi useissa eri syöpÀsoluissa, joissa se osallistuu syöpÀsolujen jakautumisen sÀÀtelyyn, etÀpesÀkkeiden syntymiseen ja kemoterapiaresistenssin muodostumiseen.
PitkÀ QT-oireyhtymÀ on harvinainen perinnöllinen sairaus, joka ilmentyy lasten ja nuorten aikuisten sydÀnperÀisenÀ Àkkikuolemana. PitkÀ QT-oireyhtymÀ voidaan tunnistaa sydÀnsÀhkökÀyrÀstÀ kÀÀntyvien kÀrkien kammiotakykardiana. PitkÀ QT-oireyhtymÀn oireita ovat tajuttomuus, epilepsia, sydÀnpysÀhdys ja Àkkikuolema. Geneettisten tutkimusten ansiosta tiedetÀÀn ettÀ perinnöllisen pitkÀ-QT oireyhtymÀn alaluokka LQT2 johtuu joko HERG geenin mutaatioista tai sen kanssa vuorovaikuttavien proteiinien mutaatioista. HERG mutaatioiden kantajat voivat olla tÀysin oireettomia mutta haitalliset henkeÀ uhkaavat vaikutukset saattavat tulla esille muiden tekijöiden vaikutuksesta. Joidenkin lÀÀkkeiden erittÀin harvinaisena haittavaikutuksena ilmentyvÀ rytmihÀiriö on analoginen pitkÀ QT-oireyhtymÀn kanssa. LÀÀkeen aiheuttaman rytmihÀiriön esiintyvyys on 1:120 000 tiettyjen muiden kuin sydÀnlÀÀkkeiden lÀÀkkeiden kÀyttÀjistÀ. KÀytÀnnössÀ lÀhes kaikki lÀÀkkeiden aiheuttamat sydÀmen rytmihÀiriöt aiheutuvat HERG kaliumkanavan salpauksesta. Sen takia ei ole sattumaa ettÀ lÀÀketeollisuuden varhainen lÀÀketurvallisuutta ennustava tutkimus on siirtynyt laaja-alaisesti kÀyttÀmÀÀn HERG kaliumkanavan sÀhköfysiologisia mittauksia entistÀkin turvallisempien lÀÀkemolekyylien seulomiseksi ja kehittÀmiseksi lÀÀkkeeksi asti.
TÀssÀ vÀitöskirjassa on tutkittu hERG1 kaliumkanavaa useasta eri lÀhtökohdasta kÀsin. EnsimÀisessÀ julkaisussa kyettiin osoittamaan sÀhköfysiologisin in vitro mittauksin ettÀ ummetuksen hoitoon kehitetty lÀÀke prucalopride salpasi hERG1 kaliumkanavaa vasta yli 300-kertaa suuremmissa pitoisuuksissa kuin mitÀ tarvitaan lÀÀkkeen kliinisen tehon esille saamiseksi. NÀin ollen in vitro mittauksin kyettiin ennustamaan ettÀ lÀÀkkeellÀ on ÀÀrimmÀisen pieni riski aihuttaa sydÀnperÀisiÀ oireita, joka on sittemmin nÀytetty toteen kliinisissÀ tutkimuksissa.
Toisessa julkaisussa tutkittiin pitkÀ-QT oireyhtymÀÀn liittyvÀÀ vaihtelevaa sÀhköfysiologista ilmiasua, joka voi aiheutua joko HERG geenin mutaatioista tai monimuotoisuudesta. HERG geenin K897T monimuotoinen versio vÀhensi solumallissa HERG proteiinin mÀÀrÀÀ verrattuna valtamutaatioon. LisÀksi monimuotoisen ionikanavan aukeamisen ja sulkeutumisen nopeus erosi valtamutaatiosta. Kolmannessa julkaisussa tutkittiin uudelleenohjelmoidussa sydÀnkantasolumallissa (iPS kardiomyosyytti) HERG proteiinin R176W monimuotoista versiota, joka oli perÀisin melko oireettomalta henkilöltÀ. PitkÀ QT-oireyhtymÀn ilmiasu (sydÀnsolun IKr virran vÀheneminen ja aktiopotentiaalin keston piteneminen) tuli esille sykkivien uudelleenohjelmoitujen sydÀnkantasolujen sÀhköfysiologisissa mittauksissa.
ViimeisessÀ julkaisussa tutkittiin miten sydÀmen vajaatoiminnan aikana sydÀnlihakseen kertyvÀ sfingolipidi keramidi vaikuttaa HERG kaliumkanavan toimintaan. Osoittautui ettÀ keramidi vÀhensi HERG ionivirtaa ajasta riippuvasti, jolloin solukalvolla oleva HERG proteiini leimattiin ubikitiinillÀ solun sisÀlle lysosomiin hajotettavaksi
Allosteric modulation and ligand binding kinetics at the Kv11.1 channel
Kv11.1-induced cardiotoxicity has emerged as an unanticipated adverse effect of many pharmacological agents and has become a major obstacle in drug development over the past decades. In this thesis, allosteric modulation of the Kv11.1 channel has been extensively explored, and negative allosteric modulators were shown to relieve the proarrhythmic effects of structurally and therapeutically diverse Kv11.1 blockers. The most potent modulators may be developed as a new class of antiarrhythmic medications in the future. On the other hand, kinetic binding parameters of a wide range of Kv11.1 blockers at the channel have been thoroughly investigated in this thesis. Association and dissociation rates or residence times are strongly suggested to be integrated with equilibrium affinity values into the future paradigms for a better and more comprehensive evaluation of Kv11.1 liability of drug candidates. The __kon-koff-KD__ kinetic map provides a first and promising classification of Kv11.1 blockers, which could be beneficial and indicative for drug researchers to design compounds with less Kv11.1-mediated cardiac side effects in the early stage of drug development. Hopefully, all findings in this thesis have brought new insights into Kv11.1-induced cardiac arrhythmias, and will offer opportunities for restoring or preventing this kind of arrhythmias in the near future.Chinese Scholarship CouncilUBL - phd migration 201
Cardiac Arrhythmias
The most intimate mechanisms of cardiac arrhythmias are still quite unknown to scientists. Genetic studies on ionic alterations, the electrocardiographic features of cardiac rhythm and an arsenal of diagnostic tests have done more in the last five years than in all the history of cardiology. Similarly, therapy to prevent or cure such diseases is growing rapidly day by day. In this book the reader will be able to see with brighter light some of these intimate mechanisms of production, as well as cutting-edge therapies to date. Genetic studies, electrophysiological and electrocardiographyc features, ion channel alterations, heart diseases still unknown , and even the relationship between the psychic sphere and the heart have been exposed in this book. It deserves to be read
Cellular models for fundamental and applied biomedical research
Cell culture models play an important role in biomedical
research and will continue to do so given the growing opposition to
vivisection and the limited predictive value of animal models for human
disease. Moreover, cell culture models can be easily established to mimic
physiological or pathological processes, which is difficult to accomplish
using in silico models. While non-cellular in vitro models are highly
suitable for studying simple biochemical processes, cell culture models
recapitulate many of the complex regulatory circuits governing protein
activity in vivo and hence allow investigation of diverse physiological
processes. Also, cell culture models offer the possibility to address
fundamental research questions in a much more simplified, specific and
controllable manner than can be achieved using in vivo models.
CSCLUMC / Geneeskund
Modifications post-traductionnelles des canaux calciques cardiaques de type L : identification des résidus asparagine qui participent à la glycosylation de la sous-unité auxiliaire CaVα2Ύ1
Les canaux calciques de type L CaV1.2 sont principalement responsables de lâentreÌe des ions calcium pendant la phase plateau du potentiel dâaction des cardiomyocytes ventriculaires. Cet influx calcique est requis pour initier la contraction du muscle cardiaque. Le canal CaV1.2 est un complexe oligomeÌrique qui est composeÌ de la sous-uniteÌ principale CaVα1 et des sous-uniteÌs auxiliaires CaVÎČ et CaVα2ÎŽ1. CaVÎČ joue un roÌle deÌterminant dans lâadressage membranaire de la sous-uniteÌ CaVα1. CaVα2ÎŽ1 stabilise lâeÌtat ouvert du canal mais le meÌcanisme moleÌculaire responsable de cette modulation nâa pas eÌteÌ encore identifieÌ. Nous avons reÌcemment montreÌ que cette modulation requiert une expression membranaire significative de CaVα2ÎŽ1 (Bourdin et al. 2015). CaVα2ÎŽ1 est une glycoproteÌine qui posseÌde 16 sites potentiels de glycosylation de type N. Nous avons donc eÌvalueÌ le roÌle de la glycosylation de type-N dans lâadressage membranaire et la stabiliteÌ de CaVα2ÎŽ1. Nous avons dâabord confirmeÌ que la proteÌine CaVα2ÎŽ1 recombinante, telle la proteÌine endogeÌne, est significativement glycosyleÌe puisque le traitement aÌ la PNGase F se traduit par une diminution de 50 kDa de sa masse moleÌculaire, ce qui est compatible avec la preÌsence de 16 sites Asn. Il sâest aveÌreÌ par ailleurs que la mutation simultaneÌe de 6/16 sites (6xNQ) est suffisante pour 1) reÌduire significativement la densiteÌ de surface de! CaVα2ÎŽ1 telle que mesureÌe par cytomeÌtrie en flux et par imagerie confocale 2) acceÌleÌrer les cineÌtiques de deÌgradation telle quâestimeÌe apreÌs arreÌt de la syntheÌse proteÌique et 3) diminuer la modulation fonctionnelle des courants geÌneÌreÌs par CaV1.2 telle quâeÌvalueÌe par la meÌthode du « patch-clamp ». Les effets les plus importants ont toutefois eÌteÌ obtenus avec les mutants N663Q, et les doubles mutants N348Q/N468Q, N348Q/N812Q, N468Q/N812Q. Ensemble, ces reÌsultats montrent que Asn663 et aÌ un moindre degreÌ Asn348, Asn468 et Asn812 contribuent aÌ la biogeneÌse et la stabiliteÌ de CaVα2ÎŽ1 et confirment que la glycosylation de type N de CaVα2ÎŽ1 est neÌcessaire aÌ la fonction du canal calcique cardiaque de type L.L-type CaV1.2 channels play a key role in the excitation-contraction coupling in the heart. They are formed of a pore-forming CaVα1 subunit in complex with the intracellular CaVÎČ and the disulfur-linked CaVα2ÎŽ accessory subunits. CaVα2ÎŽ significantly increases peak current densities of CaV1.2. The mechanism underlying this effect is still under study but requires that CaVα2ÎŽ be trafficked at the cell surface. CaVα2ÎŽ contains 16 putative N-glycosylation sites. A study was carried out to identify the role of N-glycosylation in the trafficking and protein stability of the subunit CaVα2ÎŽ. Herein we show that enzymatic removal of N-glycans produced a 50 kDa shift in the mobility of cardiac and recombinant CaVα2ÎŽ1 proteins. Simultaneous mutation of the 16 Asn sites was required to fully account for this change in protein mobility. Nonetheless, the mutation of only 6/16 sites was sufficient to 1) significantly reduce the steady-state cell surface fluorescence of CaVα2ÎŽ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) accelerate the degradation kinetics estimated from cycloheximide chase assays; and 3) prevent the CaVα2ÎŽ1-mediated increase in peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational 6 Asn mutant functionally rescued CaVα2ÎŽ1. Single mutation N663Q and double mutations N348Q/ N468Q, N348Q/ N812Q, N468Q/N812Q decreased protein stability/synthesis and abolished steady-state cell surface density as well as upregulation of L-type currents. These results demonstrate that Asn663, and to a lesser extent Asn348, Asn468, and Asn812 contribute to the stability of CaVα2ÎŽ1 function and furthermore that N- glycosylation of CaVα2ÎŽ1 is essential to produce functional L-type Ca2+ channels
Marine Toxins Targeting Kv1 Channels: Pharmacological Tools and Therapeutic Scaffolds
Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential for the treatment of ion channel-related diseases or channelopathies. Here, we review specific peptide and small-molecule marine toxins modulating Kv1 channels and thus cover recent findings of bioactives found in the venoms of marine Gastropod (cone snails), Cnidarian (sea anemones), and small compounds from cyanobacteria. Furthermore, we discuss pivotal advancements at exploiting the interaction of ÎșM-conotoxin RIIIJ and heteromeric Kv1.1/1.2 channels as prevalent neuronal Kv complex. RIIIJâs exquisite Kv1 subtype selectivity underpins a novel and facile functional classification of large-diameter dorsal root ganglion neurons. The vast potential of marine toxins warrants further collaborative efforts and high-throughput approaches aimed at the discovery and profiling of Kv1-targeted bioactives, which will greatly accelerate the development of a thorough molecular toolbox and much-needed therapeutics
Optogenetics in striated muscle: defibrillation of the heart and direct stimulation of skeletal muscles with light
Optogenetic depolarization of cells using the non-selective cation channel Channelrhodopsin-2 (ChR2) enables precise control over the membrane potential of cells within a specific area of intact organs. Furthermore, the selective overexpression of light-gated proteins allows cell type-specific and pain-free stimulation which could be of great benefit for future scientific and therapeutic approaches. In my thesis, I explored two potential applications of optogenetic methods in striated muscle: optogenetic defibrillation to terminate ventricular arrhythmia in intact mouse hearts and direct optogenetic stimulation of skeletal muscles. These new approaches could lead in the future to the development of optogenetic defibrillators and laryngeal pacemakers. Most experiments were performed with explanted hearts, isolated skeletal fibers and muscles or larynges from transgenic ChR2 expressing mice. To add translational perspectives, we also explored optogenetic defibrillation and intralaryngeal muscles stimulation after ChR2 gene transfer to wild type mice using adeno-associated virus (AAV). Optogenetic defibrillation by epicardial illumination was highly efficient in terminating ventricular arrhythmia in transgenic hearts and the success rate of optogenetic defibrillation was depending on the pulse duration, the size of illumination and the light intensity. Importantly, we were also able to terminate ventricular arrhythmia in non-transgenic hearts even one year after AAV mediated gene transfer. The potential applicability of optogenetic defibrillation in the human heart was assessed in experimentally-calibrated computer simulations of a patientâs heart with infarct-related ventricular tachycardia. Because optogenetic stimulation would be in principle pain-free in patients, the proof for its feasibility could lay the foundation for the development of a new treatment option for patients at high risk for ventricular arrhythmia. Direct optogenetic stimulation of skeletal muscle was first proven in isolated Flexor digitorum brevis fibers and in intact soleus muscles, which could both be stimulated using brief light pulses. The force of light-induced single twitches could be precisely controlled by varying the pulse duration and light intensity. Optogenetic stimulation was most efficient with 10 ms long pulses at a repetition rate of 40 Hz reaching ~84% of the maximum force generated by electrical stimulation with 100 Hz. Recurrent nerve paralysis is a severe complication of neck surgery, malignant processes or central neurological diseases and results in a fixed paramedian position of the vocal cords as well as life-threatening dyspnea in the case of bilateral paralysis. Current treatment options consist only of destructive surgery. Unfortunately the use of local electrical stimulation to restore laryngeal function faces severe technical limitations. Therefore I sought to explore direct optogenetic stimulation of intralaryngeal muscles in explanted larynges from ChR2 transgenic mice. Specific illumination of the individual intralaryngeal muscle groups led to an opening or closing of the vocal cords depending on the site of illumination. This proves the sufficient spatial resolution of light for selective stimulation of the intralaryngeal muscles groups. In addition, we were able to induce opening of the vocal cords in wild type mice after AAV-based gene transfer of ChR2 with light. Thus optogenetic stimulation could become a new treatment option for patients suffering from bilateral laryngeal paralysis. In conclusion, optogenetic stimulation can overcome the severe limitations of electrical stimulation of the heart and skeletal muscles. The new technologies, I have developed and characterized in this thesis, allow for the design of completely new stimulation patterns to address open questions in muscle physiology. Furthermore, optogenetic stimulation of striated muscles could become a new treatment option for patients enabling selective and pain-free stimulation with few side effects