Human-based approaches to pharmacology and cardiology: an interdisciplinary and intersectorial workshop

Both biomedical research and clinical practice rely on complex datasets for the physiological and genetic characterization of human hearts in health and disease. Given the complexity and variety of approaches and recordings, there is now growing recognition of the need to embed computational methods in cardiovascular medicine and science for analysis, integration and prediction. This paper describes a Workshop on Computational Cardiovascular Science that created an international, interdisciplinary and inter-sectorial forum to define the next steps for a human-based approach to disease supported by computational methodologies. The main ideas highlighted were (i) a shift towards human-based methodologies, spurred by advances in new in silico, in vivo, in vitro, and ex vivo techniques and the increasing acknowledgement of the limitations of animal models. (ii) Computational approaches complement, expand, bridge, and integrate in vitro, in vivo, and ex vivo experimental and clinical data and methods, and as such they are an integral part of human-based methodologies in pharmacology and medicine. (iii) The effective implementation of multi- and interdisciplinary approaches, teams, and training combining and integrating computational methods with experimental and clinical approaches across academia, industry, and healthcare settings is a priority. (iv) The human-based cross-disciplinary approach requires experts in specific methodologies and domains, who also have the capacity to communicate and collaborate across disciplines and cross-sector environments. (v) This new translational domain for human-based cardiology and pharmacology requires new partnerships supported financially and institutionally across sectors. Institutional, organizational, and social barriers must be identified, understood and overcome in each specific setting

Human-based approaches to pharmacology and cardiology: an interdisciplinary and intersectorial workshop.

Both biomedical research and clinical practice rely on complex datasets for the physiological and genetic characterization of human hearts in health and disease. Given the complexity and variety of approaches and recordings, there is now growing recognition of the need to embed computational methods in cardiovascular medicine and science for analysis, integration and prediction. This paper describes a Workshop on Computational Cardiovascular Science that created an international, interdisciplinary and inter-sectorial forum to define the next steps for a human-based approach to disease supported by computational methodologies. The main ideas highlighted were (i) a shift towards human-based methodologies, spurred by advances in new in silico, in vivo, in vitro, and ex vivo techniques and the increasing acknowledgement of the limitations of animal models. (ii) Computational approaches complement, expand, bridge, and integrate in vitro, in vivo, and ex vivo experimental and clinical data and methods, and as such they are an integral part of human-based methodologies in pharmacology and medicine. (iii) The effective implementation of multi- and interdisciplinary approaches, teams, and training combining and integrating computational methods with experimental and clinical approaches across academia, industry, and healthcare settings is a priority. (iv) The human-based cross-disciplinary approach requires experts in specific methodologies and domains, who also have the capacity to communicate and collaborate across disciplines and cross-sector environments. (v) This new translational domain for human-based cardiology and pharmacology requires new partnerships supported financially and institutionally across sectors. Institutional, organizational, and social barriers must be identified, understood and overcome in each specific setting

In silico assessment of the effects of various compounds in MEA/hiPSC-CM assays: Modelling and numerical simulations

International audienceWe propose a mathematical approach for the analysis of drugs effects on the electrical activity of human induced pluripotent stem cell-derived car-diomyocytes (hiPSC-CMs) based on multi-electrode array (MEA) experiments. Our goal is to produce an in silico tool able to simulate drugs action in MEA/hiPSC-CM assays. The mathematical model takes into account the geometry of the MEA and the electrodes' properties. The electrical activity of the stem cells at the ion-channel level is governed by a system of ordinary differential equations (ODEs). The ODEs are coupled to the bidomain equations, describing the propagation of the electrical wave in the stem cells preparation. The field potential (FP) measured by the MEA is modeled by the extra-cellular potential of the bidomain equations. First, we propose a strategy allowing us to generate a field potential in good agreement with the experimental data. We show that we are able to reproduce realistic field potentials by introducing different scenarios of heterogeneity in the action potential. This heterogeneity reflects the differentiation atria/ventricles and the age of the cells. Second, we introduce a drug/ion channels interaction based on a pore block model. We conduct different simulations for five drugs (mexiletine, dofetilide, bepridil, ivabradine and BayK). We compare the simulation results with the field potential collected from experimental measurements. Different biomarkers computed on the FP are considered, including depolarization amplitude, repolarization delay, repolarization amplitude and depolarization-repolarization segment. The simulation results show that the model reflect properly the main effects of these drugs on the FP

Modelling the interaction between induced pluripotent stem cells derived cardiomyocytes patches and the recipient hearts

Cardiovascular diseases are the main cause of death worldwide. The single biggest killer is represented by ischemic heart disease. Myocardial infarction causes the formation of non-conductive and non-contractile, scar-like tissue in the heart, which can hamper the heart's physiological function and cause pathologies ranging from arrhythmias to heart failure. The heart can not recover the tissue lost due to myocardial infarction due to the myocardium's limited ability to regenerate. The only available treatment is heart transpalant, which is limited by the number of donors and can elicit an adverse response from the recipients immune system. Recently, regenerative medicine has been proposed as an alternative approach to help post-myocardial infarction hearts recover their functionality. Among the various techniques, the application of cardiac patches of engineered heart tissue in combination with electroactive materials constitutes a promising technology. However, many challenges need to be faced in the development of this treatment. One of the main concerns is represented by the immature phenotype of the stem cells-derived cardiomyocytes used to fabricate the engineered heart tissue. Their electrophysiological differences with respect to the host myocardium may contribute to an increased arrhythmia risk. A large number of animal experiments are needed to optimize the patches' characteristics and to better understand the implications of the electrical interaction between patches and host myocardium. In this Thesis we leveraged cardiac computational modelling to simulate \emph{in silico} electrical propagation in scarred heart tissue in the presence of a patch of engineered heart tissue and conductive polymer engrafted at the epicardium. This work is composed by two studies. In the first study we designed a tissue model with simplified geometry and used machine learning and global sensitivity analysis techniques to identify engineered heart tissue patch design variables that are important for restoring physiological electrophysiology in the host myocardium. Additionally, we showed how engineered heart tissue properties could be tuned to restore physiological activation while reducing arrhythmic risk. In the second study we moved to more realistic geometries and we devised a way to manipulate ventricle meshes obtained from magnetic resonance images to apply \emph{in silico} engineered heart tissue epicardial patches. We then investigated how patches with different conduction velocity and action potential duration influence the host ventricle electrophysiology. Specifically, we showed that appropriately located patches can reduce the predisposition to anatomical isthmus mediated re-entry and that patches with a physiological action potential duration and higher conduction velocity were most effective in reducing this risk. We also demonstrated that patches with conduction velocity and action potential duration typical of immature stem cells-derived cardiomyocytes were associated with the onset of sustained functional re-entry in an ischemic cardiomyopathy model with a large transmural scar. Finally, we demonstrated that patches electrically coupled to host myocardium reduce the likelihood of propagation of focal ectopic impulses. This Thesis demonstrates how computational modelling can be successfully applied to the field of regenerative medicine and constitutes the first step towards the creation of patient-specific models for developing and testing patches for cardiac regeneration.Open Acces

Stem Cell Research on Cardiology

Even today, cardiovascular diseases are the main cause of death worldwide, and therapeutic approaches are very restricted. Due to the limited regenerative capabilities of terminally differentiated cardiomyocytes post injury, new strategies to treat cardiac patients are urgently needed. Post myocardial injury, resident fibroblasts begin to generate the extracellular matrix, resulting in fibrosis, and finally, cardiac failure. Recently, preclinical investigations and clinical trials raised hope in stem cell-based approaches, to be an effective therapy option for these diseases. So far, several types of stem cells have been identified to be promising candidates to be applied for treatment: cardiac progenitor cells, bone marrow derived stem cells, embryonic and induced pluripotent stem cells, as well as their descendants. Furthermore, the innovative techniques of direct cardiac reprogramming of cells offered promising options for cardiovascular research, in vitro and in vivo. Hereby, the investigation of underlying and associated mechanisms triggering the therapeutic effects of stem cell application is of particular importance to improve approaches for heart patients. This Special Issue of Cells provides the latest update in the rapidly developing field of regenerative medicine in cardiology

Induced Pluripotent Stem Cell-Derived Disease Model for Catecholaminergic Polymorphic Ventricular Tachycardia

Human induced pluripotent stem cells (hiPSCs) offer significant opportunities for cardiac research. With this technology, it is possible to create patient-specific stem cell lines and differentiate them into cardiomyocytes for cardiac research. hiPSC technology has created many expectations for new therapeutic possibilities, and it holds promise for use in drug-testing platforms and in patient-specific drug therapy optimization, as well as later in regenerative medicine.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited, highly lethal arrhythmogenic cardiac disorder. It is primarily caused by cardiac ryanodine receptor gene (RyR2) mutations that result in abnormal calcium release from the sarcoplasmic reticulum to the cytosol, leading to the generation of afterdepolarizations and triggered activity. The estimated clinical prevalence of CPVT is 1:10000. Intracellular calcium ions are crucial to the function of the heart muscle, and disturbances in this process can have fatal consequences, as observed in CPVT. Understanding the mechanisms of arrhythmia and the role of intracellular calcium in CPVT pathophysiology is important for improving disease prevention, diagnosis, and treatment.The main objective of this work was to develop and characterize models of cardiac cells and to develop and improve techniques for studying electrical field stimulation and calcium cycling of cardiomyocytes. Utilizing electrical field stimulation, the orientation and maturation of neonatal rat cardiomyocytes and the increase in the beating rate of an in vitro disease model for CPVT were studied. For the cell model of CPVT, human iPSC-derived cardiomyocytes were obtained from CPVT patients carrying RyR2 mutations. These iPSCs disease models were used to study the disease mechanisms of CPVT, mutation-specific differences in intracellular calcium cycling and the effect of antiarrhythmic treatment of the cells. Mechanistic insights regarding CPVT arrhythmias and drug responses were also validated in the index patients. Additionally, a new calcium cycling analysis software tool was developed for characterizing abnormal intracellular calcium transients of disease-specific cardiomyocytes.The results of this work demonstrate that patient-specific iPSC-derived cardiomyocytes corresponded to the clinical phenotype in both the pathophysiology and drug responses of CPVT and encourages the continuation of disease modeling utilizing iPSCs. These studies also presented a new mechanism for arrhythmias in CPVT. These findings encourage the translation of findings in basic research to benefit patients in clinical practice, e.g., in the form of potentially new medications

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

Human pluripotent stem cell-based microtechnologies for in vitro modeling of cardiac diseases

Human pluripotent stem cells are quickly emerging as a fundamental tool for in vitro studies. In particular, the advent of “induced pluripotency” opened completely new horizons for in vitro disease modeling and patient-specific disease-on-a-dish therapeutic approach screening. The easy access to cell types of human origin hardly available otherwise, with virtually infinite amounts in a donor-unrestricted manner, unlocked in vitro studies for human tissues such as brain, pancreas and the heart. In the latter case, the need for new models of human cardiac physiology and physiopathology is highlighted by the severe fallouts of heart conditions on worldwide health and economy. The main focus of this thesis are human cardiomyocytes derived through differentiation of pluripotent stem cells, and their application as an in vitro model of the human cardiac tissue. In particular, the stress point of the work is their early and immature phenotype, that often limits their application and frustrates the potential of a human heart model in a Petri dish. After introducing the current scenario of study models for heart diseases and describing the main features of human pluripotent stem cells (hPSCs) and their cardiac derivatives (hPSC-CMs), this thesis will separately focus on the two main aspects of the cardiomyocyte physiology: structural and functional features and metabolic profile. From these perspectives, human cardiomyocytes derived from hPSCs display in vitro an early and immature phenotype, closely resembling cardiomyocytes at early stage of the development, such as fetal cardiomyocytes. Cell ultrastructural organization and functional performance are two strictly related features that find in adult cardiomyocytes perfect synthesis, with a very specialized function performed through a finely orchestrated sequence of events hugely relying on the right spatial distribution of key molecular components. In Chapter 2, biomaterials and microengineered substrates are employed to address the molecular mechanisms triggering cardiac maturation in vitro, in order to provide insight in the process and drive hPSC-CMs towards more adult-like phenotypes, better suiting disease modeling and drug screening. Cardiac metabolism is likewise a characterizing feature of the tissue supporting in a unique fashion the impressive workload of the heart. In Chapter 3, hPSC-CM metabolism is described and a novel microfluidic technology is developed for metabolic maturation screening of cardiac cultures. With this approach, hPSC-CMs are shown to positively respond to an optimized metabolic maturation protocol, similar to the very rapid fetal-to-adult metabolism switch in hCMs after birth in response to changing metabolite availability. Finally, in perspective of the maturation approaches previously described and their feasible application to human cardiac cultures, in Chapter 4 are discussed two human genetic diseases affecting the heart muscle. For both Duchenne’s muscular dystrophy and arrhythmogenic right ventricular cardiomyopathy/dysplasia, cardiac cellular models are set up and proven to display in vitro the molecular hallmarks of the disease, thus providing the biological substrate for further studies on human cardiomyocyte cultures

Ionic parameters identification of an inverse problem of strongly coupled PDE's system in cardiac electrophysiology using Carleman estimates

International audienceIn this paper, we consider an inverse problem of determining multiple ionic parameters of a 2 × 2 strongly coupled parabolic-elliptic reaction-diffusion system arising in cardiac electrophysiology modelling. We use the bidomain model coupled to an ODE system and we consider a general formalism of physiologicaly-detailed cellular membrane models to describe the ionic exchanges at the microscopic level. Our main result is the uniqueness and a Lipschitz stability estimate of the ion channels con-ductance parameters of the model using subboundary observations over an interval of time. The key ingredients are a global Carleman-type estimate with a suitable observations acting on a part of the boundary

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