A novel statistical signal processing method to estimate effects of compounds on contractility of cardiomyocytes using impedance assays

International audienceLabel free methods such as cell impedance assays are in vitro tests increasingly used in drug development and producing large and high-content data files. Since the current commercial software are not suited for fully automated analysis , there is a need to develop validated and rapid solutions to extract relevant information for biologists. This need is particularly obvious in the case of impedance signals analysis from cardiomyocytes. The proposed solution is based on three main steps. The first one consists in calculating five indices informing about the time variations of frequency (F), amplitude (A), shape (S) of beatings, trends (T) of the cardiomyocyte dependent on spreading, viability and attachment as well as irregularity (I) of the contractility. In a second phase, two summary statistics are proposed to test the concentration effect of drugs on the five FASTI indices. Results of the statistical tests are finally aggregated in a cardio-effect grade to compare the tested molecules in a cardio-impact scale graduated from 0 (no influence) to 10 (highly disturbed effects in cardiomy-ocytes). This innovative approach was tested using in vitro data obtained from cell impedance analysis of three known molecules (2 cardiotoxic and 1 non-cardiotoxic compounds). Results have clearly shown the ability of the proposed approach to identify significant effects on the contractility of cardiomyocytes. This solution speeds up the analysis of cardiomyocyte impedance data, takes into account all the kinetic data generated and is now available for biologists on a web-platform: i-Cardio TM developed by CYBERnano TM

Functional Human Cell-Based Cardiac Tissue Model with Contraction Force Measurements

Sydämeen kohdistuvat lääkkeiden haittavaikutukset ovat yksi suurimpia syitä lääkeainekandidaattien hylkäämiselle sekä jo markkinoille vietyjen lääkkeiden poisvedoille. Nykyiset menetelmät lääkkeiden turvallisuuden ja tehokkuuden testaamiseen eivät ennusta lääkkeiden vaikutuksia ihmiselle riittävän tarkasti. Koska sydänten toiminnassa on lajikohtaisia eroja, eläinkokeissa ei välttämättä tunnisteta kaikkia ihmisen sydämelle haitallisia aineita. Tätä varten tarvitaan toiminnaltaan mahdollisimman hyvin ihmisen sydänkudosta vastaavia ihmissolupohjaisia sydänkudosmalleja. Tämän väitöskirjan tavoitteena oli kehittää toiminnallinen sydänkudosmalli, joka mallintaa aikuisen ihmisen sydäntä, sekä yhdistää tähän malliin sykintävoiman mittaus. Tässä työssä kehitetty sydänkudosmalli koostuu ihmisen rasvan kantasoluista (hASC) ja ihmisen napanuoran laskimon endoteelisoluista (HUVEC) muodostuvasta verisuoniverkostosta, jonka päälle kasvatetaan ihmisen indusoiduista kantasoluist (hiPSC) erilaistetut sydänlihassolut. Tämä sydänkudosmalli karakterisoitiin rakenteellisesti, geenien ilmentymisen tasolla ja toiminnallisesti. Sydänkudosmallien sykintävoimanmittaukseen kehitettiin yksi- ja kaksisuuntaisia pietsosähköisiä sensoreita. Tulosten perusteella yhteisviljelmä verisuonipohjan kanssa parantaa sydänlihassolujen kypsymistä edistämällä niiden järjestäytymistä ja morfologiaa, sarkomeerirakennetta ja solu-solu-liitoksia. Myös sydänlihassolujen geenien ilmentymisessä oli vastaavuuksia aikuisen sydämeen. Toiminnallinen karakterisointi tunnetuilla sydämeen vaikuttavilla lääkeaineilla osoitti mallin tunnistavan tarkasti aineiden vaikutuksia. Kehitetyt pietsosähköiset voima-anturit soveltuivat sydänmallien sykintävoiman mittaamiseen. Antureiden todettiin pystyvän mittaamaan sekä voimaa eri mekanismein lisäävien että sitä vähentävien aineiden vaikutuksia. Yhteenvetona voidaan todeta, että työssä kehitetty sydänkudosmalli jäljittelee sydänlihaskudoksen rakennetta ja toimintaa. Malli sopii testaamaan ihmisen sydämeen kohdistuvia akuutteja lääkeaineiden haittavaikutuksia ja sydänlääkkeiden tehoa. Kehitettyyn sydänkudosmalliin liitetyllä pietsosähköisellä voima-anturilla on potentiaalia voimaankohdistuvien lääkeainevaikutusten testaamiseen.Adverse cardiac effects are a major reason for drug attrition during drug development and for post-approval market withdrawals. Therefore, drug development would greatly benefit from tests that better predict human cardiac function. Due to intrinsic species-to-species differences in the functionality of the heart, nonclinical animal testing does not fully represent the effects of the drugs on human. Thus, there is a need for reliable, human cell -based standardised in vitro models for cardiotoxicity and drug efficacy testing. The aim of this thesis was to develop a functional human cell -based cardiac tissue model that mimics the adult human heart and to develop a contraction force measurement system for measuring the cardiac contractility of the cardiac tissue model. The cardiac tissue model that was optimised in this thesis consisted of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes that are cocultured with preformed human adipose stromal cells (hASC) and human umbilical vein endothelial cells (HUVEC) vascular-like networks. The model was characterized structurally, in gene expression levels, and functionally. For measuring the cardiac contraction force, piezoelectric cantilever sensors with single axis and dual axis sensor designs were developed. The cell culture method was adjusted according to the sensor designs. The functionality of the cardiac tissue model and contraction force measurement technology were confirmed by known inotropic drug exposures. The results show that the coculture with the vascular-like networks improved the cardiomyocyte maturity and the tissue like structure of the model. The cardiomyocytes in the model showed improved organization and morphology, well- developed sarcomeres, and cell-cell connections. The gene expression of the cardiac tissue model also showed characteristics of the adult human heart. The functional characterization with known reference compounds showed that the model had good predictivity with high correlation to human data. The developed piezoelectric contraction force sensors were suitable for measuring the contraction force of cardiac tissue constructs. Both positive and negative inotropic effects including different mechanisms were measurable in the model with the system. In conclusion, the developed cardiac tissue model mimics the myocardium structure and functionality. The model is suitable for testing cardiotoxicity and efficacy of acute drug-induced effects on human heart. Moreover, the functionality of the cardiac tissue model with the developed contraction force measurement system was shown on a proof-of-concept level

Experimental and computational biomedicine : Russian Conference with International Participation in memory of Professor Vladimir S. Markhasin : abstract book

Toward 100 Anniversary of I. P. Pavlov's Physiological Society.The volume contains the presentations that were made during Russian conference with international participation "Experimental and Computational Biomedicine" dedicated to corresponding member of RAS V.S. Markhasin (Ekaterinburg, April 10‒12, 2016). The main purpose of the conference is the discussion of the current state of experimental and theoretical research in biomedicine. For a wide range of scientists, as well as for lecturers, students of the biological and medical high schools.Сборник содержит тезисы докладов, представленных на российской конференции с международным участием «Экспериментальная и компьютерная биомедицина», посвященной памяти члена‐корреспондента РАН В. С. Мархасина (г. Екатеринбург, 10‒12 апреля 2016 г.). Основной целью конференции является обсуждение современного состояния экспериментальных и теоретических исследований в области биомедицины. Сборник предназначен для ученых, преподавателей, студентов и аспирантов биологического и медицинского профиля.МАРХАСИН ВЛАДИМИР СЕМЕНОВИЧ (1941-2015)/ MARKHASIN VLADIMIR SEMENOVICH (1941-2015). [3] PROGRAMM COMMITTEE. [5] ORGANIZING COMMITTEE. [6] KEYNOTE SPEAKERS. [7] CONTENTS. [9] PLENARY LECTURES. [10] Fedotov S. Non-Markovian random walks and anomalous transport in biology. [10] Hoekstra A. Multiscale modelling in vascular disease. [10] Kohl P. Systems biology of the heart: why bother? [10] Meyerhans A. On the regulation of virus infection fates. [11] Panfilov A.V., Dierckx H., Kazbanov I., Vandersickel N. Systems approach to studying mechanisms of ventricular fibrillationusing anatomically accurate modeling. [11] Revishvili A.S. Atrial fibrillation. Noninvasive diagnostic and treatment:from fundamental studies to clinical practice. [12] Rice J. Life sciences research at IBM. [12] Roshchevskaya I.M., Smirnova S., Roshchevsky M.P. Regularities of the depolarization of an atria:an experimental comparative-physiological study. [12] Rusinov V.L., Chupahin O.N., Charushin V.N Scientific basis for development of antiviral drugs. [13] Solovyova O.E. Tribute Lecture. Mechano-electric heterogeneity of the myocardiumas a paradigm of its function. [13] Veksler V. Myocardial energy starvation in chronic heart failure:perspectives for metabolic therapy. [13] Wladimiroff J.W. Fetal cardiac assessment using new methodsof ultrasound examination. [14] Yushkov B.G., Chereshnev V.A. The important questions of regeneration theory. [14] EXPERIMENTAL AND COMPUTATIONAL MODELS IN CARDIOVASCULARPHYSIOLOGY AND CARDIOLOGY. [15] EXPERIMENTAL AND COMPUTATIONAL MODELS IN CARDIOVASCULARPHYSIOLOGY AND CARDIOLOGY. [15] Arteyeva N. T-wave area along with Tpeak-Tend interval is the most accurateindex of the dispersion of repolarization. [15] Borodin N., Iaparov B.Y., Moskvin A. Mathematical modeling of the calmodulin effect on the RyR2 gating. [15] Dokuchaev A., Katsnelson L.B., Sulman T.B., Shikhaleva E.V., Vikulova N.A. Contribution of cooperativity to the mechano-calcium feedbacksin myocardium. Experimental discrepancy and mathematicalapproach to overcome it. [16] Elman K.A., Filatova D.Y., Bashkatova Y.V., Beloschenko D.V. The stochastic and chaotic estimation of parametersof cardiorespiratory system of students of Ugra. [16] Erkudov V.O., Pugovkin A.P., Verlov N.A., Sergeev I.V., Ievkov S.A., Mashood S., Bagrina J.V. Characteristics of the accuracy of calculation of values of systemic blood pressure using transfer functions in experimental blood loss and its compensation. [16] Ermolaev P., Khramykh T.Mechanisms of cardiodepression after 80% liver resection in rats. [17] Filatova O.E., Rusak S.N., Maystrenko E.V., Dobrynina I.Y. Aging dynamics of cardio-vascular parameters аboriginal systemand alien population of the Russian North. [17] Frolova S., Agladze K.I., Tsvelaya V., Gaiko O. Photocontrol of voltage-gated ion channel activity by azobenzenetrimethylammonium bromide in neonatal rat cardiomyocytes. [18] Gorbunov V.S., Agladze K.I., Erofeev I.S. The application of C-TAB for excitation propagation photocontrolin cardiac tissue. [18] Iribe G. Localization of TRPC3 channels estimated by in-silicoand cellular functional experiments. [19] Kachalov V.N., Tsvelaya V., Agladze K.I. Conditions of the spiral wave unpinning from the heterogeneitywith different boundary conditions in a model of cardiac tissue. [19] Kalita I., Nizamieva A.A., Tsvelaya V., Kudryashova N., Agladze K.I. The influence of anisotropy on excitation wave propagationin neonatal rat cardiomyocytes monolayer. [19] Kamalova Y. The designing of vectorcardiograph prototype. [20] Kapelko V., Shirinsky V.P., Lakomkin V., Lukoshkova E., Gramovich V.,Vyborov O., Abramov A., Undrovinas N., Ermishkin V. Models of chronic heart failure with acute and gradual onset. 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Quantification of Stem Cell Derived Cardiomyocyte Beating Mechanics using Video Microscopy Image Analysis

Until recently, the studying of human cardiac cells had been a difficult and to some extent dangerous task due to the risks involved in cardiac biopsies. Induced pluripotent stem cell technology enables the conversion of human adult cells to stem cells, which can be further differentiated to cardiac cells. These cells have the same genotype as the patient from whom they were derived, allowing the studying of genetic cardiac diseases, as well as the cardiac safety and efficacy screening of pharmaceutical agents using human cardiac cells instead of animal cell models. Using the stem cell derived cardiac cells in these studies, however, requires novel and specialized measurement methods for understanding the functioning of these cells.Long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT) are genetic cardiac diseases, which can induce deadly arrhythmias. The induced pluripotent stem cell derived cardiac cells allow the studying of these diseases in laboratory conditions. A greater understanding of these diseases is important for prevention of sudden cardiac death, more accurate diagnosis, and development of possible treatment options. In order to understand the functioning of these cells, new methods are sought after. Traditionally, the electrical function of these cells are measured. However, the primary function of the cardiac cells is to beat in order to pump blood for circulation. The methods to quantify this mechanical function, the contraction and relaxation movement of cells, has been in lesser focus.The main objective of this work is to develop a measurement method, which allows the in vitro quantification of biomechanics of single human cardiac cells using video microscopy. The method uses digital image correlation to determine movement occurring in cardiac cells during contractile movement. The method is implemented in a software tool, which enables the characterization and parametrization of the cardiomyocyte beating function. The beating function itself can be affected by environmental factors, pharmacological agents and cardiac disease.Here, the quantification of mechanical function is performed using digital image correlation to estimate displacement between subsequent video frames. Velocity vector fields can then be used to calculate signals that characterize the contraction and relaxation movement. We estimate its accuracy in cardiac cell studies using artificial data sets and its feasibility with concurrent electrical measurements. Cardiac diseases are studied by quantifying beating mechanics from Long QT and CPVT specific cell lines. Traditional electrophysiological measurements are used for validation and comparison. The interaction between calcium and contraction is studied with a simultaneous measurement of biomechanics and calcium imaging.This thesis resulted a new and accessible analysis method capable of measuring cardiomyocyte biomechanics. This method was determined to be non-toxic and minimally invasive, and found capable to be automated for high-throughput analysis. Due to not harming the cells, repeated measurements are enabled. Using the method, we observed for the first time abnormal beating phenotypes in two long QT associated mutations in single cardiomyocytes. Further, we demonstrated a concurrent calcium and motion measurement without background corrections. This provided also evidence that this combined analysis could be particularly useful in some cardiac disease cases. The methods and results shown in the thesis represent key early advances in the field.The method was implemented in a software tool, which enabled cell biologists to use it different stages of cardiomyocyte studies. Overall, the results of the thesis represent an accessible method of studying cardiomyocyte biomechanics, which improves the understanding of contraction-calcium coupling and paves way for high-throughput analysis of cardiomyocytes in genetic cardiac disease and pharmacological research.Viime aikoihin asti ihmisen sydämen solujen tutkiminen on ollut vaikeaa ja vaarallista, sillä näytepalojen ottamiseen sydämestä liittyy paljon riskejä. Menetelmä indusoitujen pluripotenttien kantasolujen tuottamiseen sallii aikuisten solujen muuntamisen takaisin kantasolumuotoon, josta ne voidaan vielä erilaistaa sydänsoluiksi. Näillä soluilla on sama geeniperimä kuin potilaalla, josta ne on johdettu. Tämä luo mahdollisuuden tutkia geneettisiä sydänsairauksia, ja sallii lääkeaineiden sydänturvallisuuden ja tehokkuuden tutkimisen käyttäen ihmisen sydänsoluja eläinkokeiden sijaan. Kantasolupohjaisten sydänsolujen käyttäminen näissä tutkimuksissa kuitenkin vaatii uusia ja erityisiä mittausmenetelmiä solujen toiminnan ymmärtämiseksi.Pitkä QT-syndrooma (LQTS) ja katekolamiiniherkkä polymorfinen kammiotakykardia (CPVT) ovat perinnöllisiä sydänsairauksia, jotka voivat aiheuttaa kuolemaan johtavia rytmihäiriöitä. Indusoiduista pluripotenteista kantasoluista johdettujen sydänsolujen avulla voidaan tutkia näitä sairauksia laboratorio-oloissa. Ymmärtämällä paremmin näitä sairauksia voidaan saavuttaa tarkempia diagnooseja ja kehittää mahdollisia uusia hoitomuotoja sydänperäisten äkkikuolemien estämiseksi. Uusia mittausmenetelmiä tarvitaan, jotta näiden solujen toimintaa voidaan tutkia. Näiden solujen toiminnallisuutta on perinteisesti tutkittu mittaamalla niiden sähköistä toimintaa. Sydänsolujen pääasiallinen tehtävä on kuitenkin mekaaninen: pumpata verta sydämestä verenkiertoon. Tätä solujen supistumista ja rentoutumista mittaavia menetelmiä on tutkittu vähemmän.Tämän väitöskirjan päämäärä on kehittää mittausmenetelmä, jolla voidaan määrittää yksittäisten ihmisen sydänsolujen biomekaniikkaa in vitro videomikroskopiaa käyttäen. Menetelmä käyttää digitaalista kuvien korrelaatiota määrittämään sydänsoluissa supistusliikkeen aikana tapahtuvan liikkeen. Menetelmää käytetään ohjelmistotyökalussa, jolla voidaan karakterisoida ja parametrisoida sydänsolun syketoimintaa. Syketoimintaan voi vaikuttaa niin ympäristötekijät, lääkeaineet kuin sydänsairaudetkin.Tässä väitöskirjassa sydänsolujen mekaanista toimintaa mitataan videomikroskopian avulla määrittämällä liikettä videon peräkkäisistä kuvista digitaalista kuvakorrelaatiota käyttäen. Saaduista nopeusvektorikentistä lasketaan supistumista ja rentoutumista kuvaavia signaaleja. Arvoimme sen tarkkuutta sydänsolututkimuksissa käyttäen keinotekoisia tietoaineistoja ja sen soveltuvuutta yhtäaikaisilla sähköisillä mittauksilla. Tutkimme perinnöllisiä sydänsairauksia (LQTS ja CPVT) mittaamalla sykinnän mekaniikkaa yksittäisistä sydänsoluista, jotka ovat johdettu näitä sairauksia kantavien potilaiden kantasolulinjoista. Perinteisiä sähköfysiologisia mittauksia käytetään menetelmän validointiin ja vertailuun. Kalsiumin ja sykinnän vuorovaikutusta tutkitaan yhtäaikaisella biomekaniikan ja kalsiumaineenvaihdunnan mittauksella.Tämän väitöskirjan tuloksena saatiin aikaan uusi ja helposti lähestyttävä menetelmä sydänlihassolujen biomekaniikan tutkimiseen. Menetelmä ei ole soluille haitallinen ja se vaikuttaa solujen toimintaan perinteisiin menetelmiin verrattuna vain vähän. Se on automatisoitavissa suuria näytemääriä varten. Koska se ei vahingoita soluja, mittaukset voidaan myös toistaa samoilla soluilla. Tätä menetelmää käyttäen havaitsimme ensimmäisinä kahdesta eri LQT1-mutaatiota kantavista potilaista johdetuissa yksittäisissä sydänsoluissa poikkeavia sykintätyyppejä. Lisäksi, osoitimme yhtäaikaisen kalsiumin ja liikkeen mittauksen olevan mahdollinen ilman laskennallisia taustan korjauksia ja havaitsimme, että näin yhdistetystä analyysista voi olla erityistä hyötyä sydänsairauksien tutkimisessa. Väitöskirjassa esitetyt menetelmät ja tulokset edustavat alan tärkeitä ensimmäisiä edistysaskelia.Tätä menetelmää käytettiin väitöskirjan ohella tehdyssä ohjelmistotyökalussa, jota voidaan käyttää sydänlihassolujen tutkimuksen eri vaiheissa. Väitöskirjan tuloksena syntynyt helposti lähestyttävä menetelmä sallii sydänlihassolujen biomekaniikan analyysin. Sen avulla voidaan myös ymmärtää paremmin supistusliikkeen ja kalsiumin kytkentää. Kokonaisuutena, väitöskirja luo pohjaa sydänlihassolujen suurten näytemäärien analyysille sydänsairauksien ja lääkeaineiden tutkimuksessa

Cardiac organoid technology and computational processing of cardiac physiology for advanced drug screening applications

Stem cell technology has gained considerable recognition since its inception to advance disease modeling and drug screening. This is especially true for tissues that are difficult to study due to tissue sensitivity and limited regenerative capacity, such as the heart. Previous work in stem cell-derived cardiac tissue has exploited how we can engineer biologically functional heart tissue by providing the appropriate external stimuli to facilitate tissue development. The goal of this dissertation is to explore the potentials of stem cell cardiac organoid models to recapitulate heart development and implement analytical computational tools to study cardiac physiology. These new tools were implemented as potential advancements in drug screening applications for better predictions of drug-related cardiotoxicity. Cardiac organoids, generated via micropatterning techniques, were explored to determine how controlling engineering parameters, specifically the geometry, direct tissue fate and organoid function. The advantage of cardiac organoid models is the ability to recapitulate and study human tissue morphogenesis and development, which has currently been restricted through animal models. The cardiac organoids demonstrated responsiveness manifested as impairments to tissue formation and contractile functions as a result of developmental drug toxicity. Single-cell genomic characterization of cardiac organoids unveiled a co-emergence of cardiac and endoderm tissue, which is seen in vivo through paracrine signaling between the liver and heart. We then implemented computational tools based on nonlinear mathematical analysis to evaluate the cardiac physiological drug response of stem cell-derived cardiomyocytes. This dissertation discusses in vitro tissue platforms as well as computational tools to study drug-induced cardiotoxicity. Using these tools, we can extend current toolboxes of understanding cardiac physiology for advanced investigations of stem-cell based cardiac tissue engineering

Application of Parylene C thin films in cardiac cell culturing

There are two main challenges when producing in vitro cell systems: first, to reconstitute the in situ cellular microenvironment, thus delivering more representative and reliable cell models for drug screening and disease modelling studies. Second, to record and quantify the electrical and chemical gradients across the culture. Ideally, both challenges should be accomplished within a single platform towards a lab-on-chip implementation. This research work investigates the application of Parylene C in cardiac cell scaffolding and its integrability with electrochemical monitoring technologies for measuring extracellular action potentials and pH. The surface properties of Parylene C in terms of water affinity, chemical composition and nanotopography were characterised before and after modifying the material's inherent hydrophobicity through oxygen plasma. A technology was developed to selectively alter the surface hydrophobicity of Parylene C through standard lithography and oxygen plasma, which is characterised by μm-resolution and long-term pattern stability, and can accurately control the extent of induced hydrophilicity, the pattern layout and 3-D geometry. The micro-engineered Parylene C films were employed as scaffolds for cardiac cells with immature physiological properties, such as neonatal rat ventricular myocytes (NRVM). The scaffolds promoted a more in situ cellular structure and organisation, while they improved important calcium (Ca2+) cycling parameters such as fluorescent amplitude, time to peak (Tp), time to 50% (T50) and 90% (T90) decay at 0.5-2 Hz field stimulation. The thickness of the patterned Parylene C films was found to regulate the shape of the cells by controlling their adhesion area on the Parylene substrate through a thickness-dependent hydrophobicity. NRVM on thin (2 μm) membranes tended to bridge across the hydrophobic areas and adopt a spread-out shape (average contact angle at the level of the nucleus was 64.51o). On the other hand, cells on thick (10 μm) films were mostly constrained on the hydrophilic areas and demonstrated a more elongated, cylindrical (in vivo-like) shape (average contact angle was 84.73o). The cylindrical shape and a significantly (p <0.05) denser microtubule structure in cells on thick films possibly suggest a more mature cardiomyocyte. However, there was no significant effect on the Ca2+ physiology between the two groups. The micro-patterning technology was able to deliver free-standing Parylene C thin films (2-10 μm) to study the effect of substrate elasticity and flexibility on the Ca2+ physiology of NRVM. Preliminary results showed that fluorescent amplitude and time to peak were improved in structured NRVM cultures on stand-alone Parylene films compared to rigid Parylene-coated glass surfaces. However, no such trend was present in Ca2+ release parameters (T50, T90). The flexibility of the culture substrate was also manipulated by employing free-standing micro-patterned Parylene C films of distinct thicknesses (2-10 μm), but did not affect the cellular Ca2+ physiology. Further biological validation is needed with a larger sample size to draw a certain conclusion. The cell patterning technology was transferred to commercially available planar Multi-Electrode arrays (MEAs) to demonstrate integrability of this method with existing monitoring tools. The micro-patterned MEAs induced anisotropic cardiomyocyte cultures, as they substantially increased the longitudinal-to-transverse velocity anisotropy ratios (1.09, n=4 to 1.69, n=2), promoting action potential propagation profiles that closer resembled native cardiac tissue. Furthermore, the micro-engineered MEAs were proven to be reusable, yielding a versatile and low-cost approach that is compatible with state-of-art recording equipment and can be employed as a more reliable, off-the-shelf tool for drug screening studies. Selective hydrophilic modification of Parylene C was also employed to activate locally the H+ sensing capacity of such films, implementing extended-gate pH sensors. The ability of Parylene C to act in a dual way - as an encapsulation material and as an active pH sensing membrane - was demonstrated. The material exhibited a distinguishable sensitivity dependent on the oxygen plasma recipe, relatively low drift rates and excellent encapsulation quality. Based on these principles, flexible Parylene-based high-density miniaturised electrode arrays were fabricated, employing Parylene as a flexible structure material and as a H+ sensing membrane for local detection of pH. The presented Parylene-based technology has the potential to deliver integrated lab-on-chip implementations for growing cells in vitro with controlled microtopography while monitoring the extracellular electrical and pH gradients across the culture in a non-invasive way, with application in drug screening and disease modelling.Open Acces

Metabolic control of human cardiomyocyte function and maturation

Cardiovascular diseases remain a leading cause of morbidity and mortality in the world, despite advances in drug and therapeutic developments. The complex nature of cardiac diseases such as myocardial infarction and heart failure require innovative approaches to elucidate disease mechanisms, identify molecular targets and develop novel therapies. The advent of human pluripotent stem cell (hPSC) technologies allowed for robust and reliable generations of contracting human cardiomyocytes (CMs) in vitro. hPSC-CMs hold great promise for a broad range of research and clinical applications including studying myocardial physiology, modeling cardiac diseases, and transplanting healthy cells to repair the damaged heart. However, one major limitation of hPSC-CMs differentiated in vitro is that they are relatively immature and resemble embryonic CMs. These cells lack well defined cellular edges and mature sarcomeres, which makes it difficult to quantitatively assess contractile functions using traditional edge detection technologies. In addition, hPSC-CMs cultured in traditional glucose rich media lack metabolic and functional maturity, utilizing mainly glycolysis for energy production, similar to the embryonic heart. To address these limitations, we first devised a novel technology to simultaneously quantify hPSC-CMs’ contractile kinetics, force generation and electrical activities at the single cell resolution. This methodology allowed us to examine the impact of energy substrates and metabolic pathway utilization on CM physiology and function. We identified that Hypoxia Inducible Factor 1 alpha (HIF1α) and its transcriptional target Lactate Dehydrogenase A (LDHA) are aberrantly upregulated in hPSC-CMs cultured in traditional glucose rich media. By using small molecules and siRNA, we demonstrated that inhibition of HIF1α/LDHA shifts hPSC-CMs’ metabolism from glycolysis to oxidative phosphorylation, which resulted in improved CM structural and functional maturation. Furthermore, we investigated the energy substrate dependency of hPSC-CMs in response to in vitro hypoxic and ischemia-reperfusion injuries. We observed that hPSC-CMs cultured in glucose rich media lack physiological responses to hypoxic insults. On the other hand, in vitro coverslip ischemia-reperfusion resulted in CM death and apoptosis, independent of glucose cultures. These findings highlighted the importance of bioenergetics in modeling cardiac diseases in vitro and provided us with the basis for a potential drug screening platform using hPSC-CMs.2020-07-02T00:00:00

Mechanisms of receptor tyrosine kinase signaling diversity: a focus in cardiac growth

To understand organism function and disease and to target perturbed processes for therapy, comprehensive knowledge of the underlying cell signaling networks is required. However, mapping the interplay of the vast number of biomolecules involved in these networks remains challenging. As a result, efforts have focused on identifying the structural elements within biomolecules that facilitate signal transmission. Receptor tyrosine kinases (RTKs) regulate the function of several important organs and are most recognized as oncogenes in cancer. Research into the structural determinants of RTKs that govern their signaling has led to clinically approved therapies. However, some structural regions of these kinases remain poorly understood. In this thesis, the diversity of cell signaling arising from variation in an overlooked region in RTKs known as the extracellular juxtamembrane region was explored. A sequence motif that controls the cell surface location and the signaling of RTKs was identified, presenting a potential novel way to target RTKs for therapy. The cell signaling pathways that regulate myocardial growth could be putatively re-activated to treat heart failure or inhibited to treat pathological hypertrophy. Additionally, these pathways may hold the key to regenerating the myocardium post-injury. A pathway promoting myocardial growth involving STAT5b and the RTK ErbB4 was uncovered in this thesis. VEGFB, traditionally associated with endothelial cells, was additionally observed to elicit myocardial growth through paracrine signaling involving ErbB RTKs. Activation of ErbB4 pathways in the heart with NRG-1 has improved the cardiac function of heart failure patients implying that the discoveries made in this thesis may aid in heart failure therapy development. Finally, recent developments in omics technologies have facilitated the detection and quantification of the different layers of cell signaling networks. Consequently, a growing need for computational analyses capable of reverse-engineering cell signaling pathways from multi-omics data has emerged. In this thesis, a new computational approach specifically designed to discover cell signaling pathways from multi-omics data without the use of prior information was developed. These types of de novo methods remain essential for uncovering new cell signaling connections, which, in turn, can unveil potential new drug targets to treat disease. Reseptorityrosiinikinaasien viestinnän monimuotoisuuden mekanismit: painotus sydänlihaksen kasvussa Elimistön toiminnan ja sairauksien ymmärtäminen sekä lääkekehitys edellyttää kattavaa tietoa solujen soluviestintäverkostoista. Koska soluviestintämolekyylejä on lukuisia, soluviestinnän tutkimus on keskittynyt löytämään toistuvia rakenteellisia soluviestintää välittäviä alueita soluviestintämolekyyleistä. Reseptorityrosiinikinaasit (RTK:t) ovat solun pinnan soluviestintämolekyylejä, jotka säätelevät useita elimistön tärkeitä toimintoja ja joiden rakenteen tutkimus on johtanut useisiin käytössä oleviin lääkkeisiin. RTK:iden rakenteessa sijaitsee alue solun ulkopuolella, jonka merkitystä ei ole aikaisemmin juurikaan selvitetty. Tämän alueen vaikutusta RTK:iden viestinnän monimuotoisuudelle tutkittiin tässä väitöskirjassa. Alueelta löydettiin sekvenssimotiivi, joka säätelee RTK:iden sijaintia solun pinnalla sekä niiden viestintää. Alueelle voidaan tulevaisuudessa mahdollisesti kohdentaa RTK:iden viestintää muuttavia lääkkeitä. Soluviestintäreittejä, jotka säätelevät sydänlihaksen kasvua, voidaan mahdollisesti aktivoida sydämen vajaatoiminnan hoitamiseksi tai estää vahingollisen sydämen liikakasvun lieventämiseksi. Lisäksi näitä soluviestintäreittejä voidaan hyödyntää vaurion jälkeiseen sydänlihassolujen regeneraatioon. Sydänlihaksen kasvun soluviestintäreitteihin liittyviä havaintoja tehtiin tässä väitöskirjassa. RTK ErbB4:n todettiin aiheuttavan sydänlihaksen kasvua STAT5b viestinnän kautta. RTK ligandi VEGF-B:n puolestaan todettiin vaikuttavan sydänlihaksen kasvuun ErbB RTK:iden viestinnän avulla. Koska ErbB4 viestinnän aktivointi on parantanut sydämen vajaatoimintapotilaiden sydämen toimintaa, nämä havainnot saattavat edesauttaa sydämen vajaatoiminnan hoitojen kehitystä. Omiikka-teknologioilla voidaan mitata soluviestintäverkostojen eri tasoja lähes kattavasti. Laskennallisia työkaluja kuitenkin tarvitaan, jotta omiikka-teknologioilla tuotettu tieto voidaan mallintaa soluviestintäreiteiksi. Uusi soluviestintäreittien mallinnusohjelma kehitettiin tässä väitöskirjassa. Mallinnusohjelma käyttää ainoastaan omiikka-teknologioilla saatua tietoa soluviestintäreittien mallinnukseen. Tämän kaltaisia vain mitattuun tietoon perustuvia menetelmiä tarvitaan uusien soluviestintäreittien löytämiseksi. Uudet soluviestintäreittien yhteydet puolestaan voivat paljastaa uusia tautimekanismeja ja toimia uusina lääkekohteina