Effects of uniaxial strain in hypertrophic cardiomyopathy -patient specific human induced pluripotent stem cell -derived cardiomyocytes

Background and aims: Hypertrophic cardiomyopathy (HCM) is genetically inherited autosomal-dominant disease with significant genotypic and phenotypic heterogeneity. HCM is one of the most common inherited cardiovascular disorders and the leading cause of sudden cardiac death (SCD) in young adults. Typically hypertrophy affects the left ventricle and interventricular septum and may eventually lead to various cardiac complications. No specific preventive therapy is available for HCM. The aim of this study was to identify and compare the effects of uniaxial mechanical strain on HCM –patient specific and control human induced pluripotent stem cell -derived cardiomyocytes. Methods: Four induced pluripotent stem cell (hiPSC) lines were used in this study. Three cell lines were HCM –patient specific carrying the Q1061X- Finnish founder mutation in MYBPC3 gene and one wild type cell line was generated from a healthy individual. Cardiomyocytes (CMs) were differentiated from the hiPSC lines by END-2 method, stretched uniaxially by Flexcell®-apparatus, immunostained, imaged and analyzed. From immunostained images, hiPSC –derived CMs’ proliferation, alignment, multinucleation, area and sarcomere protein organization were analyzed. Results: Stretched and control hiPSC –derived CMs were compared. Stretching decreased proliferation and multinuclearism. Stretching induced growth of area in wild type CMs, formation of atrial CMs and alignment against the axis of the strain. Stretching had diverse effects on sarcomere protein organization. When HCM and wild type cells were compared, proliferation and alignment were similar, there were more multinuclear and atrial HCM CMs and wildtype CMs were smaller in area. Conclusions: Uniaxial mechanical stretch has diverse effects on hiPSC –derived CMs. By mechanical stimulation, hiPSC –derived CMs could be made more mature to better resemble native adult CMs. Yhdensuuntaisen mekaanisen venytyksen vaikutukset hypertrofista kardiomyopatiaa sairastavista potilaista tuotettuihin indusoiduista pluripotenteista kantasoluista erilaistettuihin sydänlihassoluihin Tutkimuksen tausta ja tavoitteet: Hypertrofinen kardiomyopatia (HCM) on autosomaali-dominantisti periytyvä sairaus joka on sekä geno- että fenotyypiltään hyvin heterogeeninen. HCM on yksi yleisimmistä periytyvistä sydänsairauksista sekä erittäin merkittävä tekijä sydämen komplikaatioista johtuvissa äkkikuolemissa nuorilla aikuisilla. Taudille tyypillinen vasemman kammion- ja kammioiden välisen seinämän sydänlihaksen paksuuntuminen voi johtaa useisiin erimuotoisiin sydämen toiminnallisiin komplikaatioihin. Hoitoa HCM:aa vastaan ei ole vielä löydetty. Tämän tutkimuksen tavoitteena oli identifioida ja verrata yhdensuuntaisen mekaanisen venytyksen vaikutuksia HCM –potilaiden ja kontrollihenkilöiden ihmisen indusoiduista pluripotenteista kantasoluista erilaistettuihin sydänlihassoluihin. Tutkimusmenetelmät: Tutkimuksessa käytettiin neljää eri ihmisen fibroblasteista uudelleenohjelmoitua indusoitua pluripotenttia kantasolulinjaa (hiPSC) joista kolme oli HCM-potilasspesifisiä kantaen suomalaista MYBPC3-valtamutaatiota (Q1061X) ja yksi kontrollilinja terveestä henkilöstä. Näistä linjoista erilaistettuja sydänlihassoluja venytettiin yhdensuuntaisesti Flexcell®-laitteella, immunovärjättiin, kuvattiin ja analysoitiin. Immunovärjätyistä kuvista analysoitiin hiPS-soluista erilaistettujen sydänlihassolujen jakautumista, järjestäytymistä, monitumaisuutta, pinta-alaa, sarkomeeriproteiinien organisoitumista sekä kammio- ja eteisspesifisten sydänlihassolujen muodostumista. Tutkimustulokset: Venytettyjä ja venyttämättömiä iPS-soluista erilaistettuja sydänlihassoluja verrattiin keskenään. Venytys vähensi jakaantumista ja monitumaisuutta. Venytys indusoi pinta-alan kasvua villityypin soluissa, eteisspesifisten sydänlihassolujen muodostumista sekä järjestäytymistä venytyksen suuntaa vasten. Venytyksellä oli erilaisia vaikutuksia eri sarkomeeriproteiinien järjestäytymiseen. Verrattaessa HCM- ja kontrolli-sydänlihassoluja keskenään, jakautuminen ja järjestäytyminen olivat samaa luokkaa, monitumaisia sekä eteisspesifisiä HCM -sydänlihassoluja oli enemmän ja kontrollisolut olivat pienempiä pinta-alaltaan. Johtopäätökset: Yhdensuuntaisella mekaanisella venytyksellä on useita eri vaikutuksia hiPS-soluista erilaistettuihin sydänlihassoluihin. Mekaanisesti stimuloimalla hiPSC sydänlihassoluja voidaan saada kypsymään ja paremmin vastaamaan aikuisen kudoksen kypsiä sydänlihassoluja

Machine learning of drug influence based on iPSC cardiomyocyte calcium transient signals

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CytoSpectre: a tool for spectral analysis of oriented structures on cellular and subcellular levels

CytoSpectre user guide. This file contains the user guide of the software. (PDF 464 kb

Data analytics for cardiac diseases

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Pneumatic unidirectional cell stretching device for mechanobiological studies of cardiomyocytes

In this paper, we present a transparent mechanical stimulation device capable of uniaxial stimulation, which is compatible with standard bioanalytical methods used in cellular mechanobiology. We validate the functionality of the uniaxial stimulation system using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The pneumatically controlled device is fabricated from polydimethylsiloxane (PDMS) and provides uniaxial strain and superior optical performance compatible with standard inverted microscopy techniques used for bioanalytics (e.g., fluorescence microscopy and calcium imaging). Therefore, it allows for a continuous investigation of the cell state during stretching experiments. The paper introduces design and fabrication of the device, characterizes the mechanical performance of the device and demonstrates the compatibility with standard bioanalytical analysis tools. Imaging modalities, such as high-resolution live cell phase contrast imaging and video recordings, fluorescent imaging and calcium imaging are possible to perform in the device. Utilizing the different imaging modalities and proposed stretching device, we demonstrate the capability of the device for extensive further studies of hiPSC-CMs. We also demonstrate that sarcomere structures of hiPSC-CMs organize and orient perpendicular to uniaxial strain axis and thus express more maturated nature of cardiomyocytes

Automatic Optimization of an in Silico Model of Human iPSC Derived Cardiomyocytes Recapitulating Calcium Handling Abnormalities

The growing importance of human induced pluripotent stem cell-derived cardiomyoyctes (hiPSC-CMs), as patient-specific and disease-specific models for studying cellular cardiac electrophysiology or for preliminary cardiotoxicity tests, generated better understanding of hiPSC-CM biophysical mechanisms and great amount of action potential and calcium transient data. In this paper, we propose a new hiPSC-CM in silico model, with particular attention to Ca2+ handling. We used (i) the hiPSC-CM Paci2013 model as starting point, (ii) a new dataset of Ca2+ transient measurements to tune the parameters of the inward and outward Ca2+ fluxes of sarcoplasmic reticulum, and (iii) an automatic parameter optimization to fit action potentials and Ca2+ transients. The Paci2018 model simulates, together with the typical hiPSC-CM spontaneous action potentials, more refined Ca2+ transients and delayed afterdepolarizations-like abnormalities, which the old Paci2013 was not able to predict due to its mathematical formulation. The Paci2018 model was validated against (i) the same current blocking experiments used to validate the Paci2013 model, and (ii) recently published data about effects of different extracellular ionic concentrations. In conclusion, we present a new and more versatile in silico model, which will provide a platform for modeling the effects of drugs or mutations that affect Ca2+ handling in hiPSC-CMs

Simultaneous recordings of action potentials and calcium transients from human induced pluripotent stem cell derived cardiomyocytes

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer a unique in vitro platform to study cardiac diseases, as they recapitulate many disease phenotypes. The membrane potential (Vm) and intracellular calcium (Ca2+) transient (CaT) are usually investigated separately, because incorporating different techniques to acquire both aspects concurrently is challenging. In this study, we recorded Vm and CaT simultaneously to understand the interrelation between these parameters in hiPSC-CMs. For this, we used a conventional patch clamp technique to record Vm, and synchronized this with a Ca2+ imaging system to acquire CaT from same hiPSC-CMs. Our results revealed that the CaT at 90% decay (CaT90) was longer than action potential (AP) duration at 90% repolarization (APD90). In addition, there was also a strong positive correlation between the different parameters of CaT and AP. The majority of delayed after depolarizations (DADs) observed in the Vm recording were also characterized by elevations in the intracellular Ca2+ level, but in some cases no abnormalities were observed in CaT. However, simultaneous fluctuations in CaT were always observed during early after depolarizations (EADs) in Vm. In summary, simultaneous recording of Vm and CaT broadens the understanding of the interrelation between Vm and CaT and could be used to elucidate the mechanisms underlying arrhythmia in cardiac disease condition

Simultaneous Measurement of Contraction and Calcium Transients in Stem Cell Derived Cardiomyocytes

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) provide a powerful platform for disease modeling and drug development in vitro. Traditionally, electrophysiological methods or fluorescent dyes (e.g. calcium) have been used in their functional characterization. Recently, video microscopy has enabled non-invasive analysis of CM contractile motion. Simultaneous assessments of motion and calcium transients have not been generally conducted, as motion detection methods are affected by changing pixel intensities in calcium imaging. Here, we present for the first time a protocol for simultaneous video-based measurement of contraction and calcium with fluorescent dye Fluo-4 videos without corrections, providing data on both ionic and mechanic activity. The method and its accuracy are assessed by measuring the effect of fluorescence and background light on transient widths and contraction velocity amplitudes. We demonstrate the method by showing the contraction-calcium relation and measuring the transient time intervals in catecholaminergic polymorphic ventricular tachycardia patient specific iPSC-CMs and healthy controls. Our validation shows that the simultaneous method provides comparable data to combined individual measurements, providing a new tool for measuring CM biomechanics and calcium simultaneously. Our results with calcium sensitive dyes suggest the method could be expanded to use with other fluorescent reporters as well.publishedVersionPeer reviewe

Pneumatic unidirectional cell stretching device for mechanobiological studies of cardiomyocytes

In this paper, we present a transparent mechanical stimulation device capable of uniaxial stimulation, which is compatible with standard bioanalytical methods used in cellular mechanobiology. We validate the functionality of the uniaxial stimulation system using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The pneumatically controlled device is fabricated from polydimethylsiloxane (PDMS) and provides uniaxial strain and superior optical performance compatible with standard inverted microscopy techniques used for bioanalytics (e.g., fluorescence microscopy and calcium imaging). Therefore, it allows for a continuous investigation of the cell state during stretching experiments. The paper introduces design and fabrication of the device, characterizes the mechanical performance of the device and demonstrates the compatibility with standard bioanalytical analysis tools. Imaging modalities, such as high-resolution live cell phase contrast imaging and video recordings, fluorescent imaging and calcium imaging are possible to perform in the device. Utilizing the different imaging modalities and proposed stretching device, we demonstrate the capability of the device for extensive further studies of hiPSC-CMs. We also demonstrate that sarcomere structures of hiPSC-CMs organize and orient perpendicular to uniaxial strain axis and thus express more maturated nature of cardiomyocytes

Mutation-Specific Phenotypes in hiPSC-Derived Cardiomyocytes Carrying Either Myosin-Binding Protein C Or α-Tropomyosin Mutation for Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disease, which affects the structure of heart muscle tissue. The clinical symptoms include arrhythmias, progressive heart failure, and even sudden cardiac death but the mutation carrier can also be totally asymptomatic. To date, over 1400 mutations have been linked to HCM, mostly in genes encoding for sarcomeric proteins. However, the pathophysiological mechanisms of the disease are still largely unknown. Two founder mutations for HCM in Finland are located in myosin-binding protein C (MYBPC3-Gln1061X) and α-tropomyosin (TPM1-Asp175Asn) genes. We studied the properties of HCM cardiomyocytes (CMs) derived from patient-specific human induced pluripotent stem cells (hiPSCs) carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. Both types of HCM-CMs displayed pathological phenotype of HCM but, more importantly, we found differences between CMs carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn gene mutation in their cellular size, Ca2+ handling, and electrophysiological properties, as well as their gene expression profiles. These findings suggest that even though the clinical phenotypes of the patients carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn gene mutation are similar, the genetic background as well as the functional properties on the cellular level might be different, indicating that the pathophysiological mechanisms behind the two mutations would be divergent as well