Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

• MeSH
• biologické markery MeSH
• buněčná diferenciace * MeSH
• buněčné kultury MeSH
• buněčné sféroidy MeSH
• exprese genu MeSH
• kardiomyocyty cytologie   metabolismus   MeSH
• kyslík metabolismus   MeSH
• lidé MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• sarkoplazmatické retikulum metabolismus   MeSH
• srdeční mitochondrie metabolismus   MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

It has become evident that epitranscriptome events, mediated by specific enzymes, regulate gene expression and, subsequently, cell differentiation processes. We show that methyltransferase-like proteins METTL3/METTL14 and N6-adenosine methylation (m6A) in RNAs are homogeneously distributed in embryonic hearts, and histone deacetylase (HDAC) inhibitors valproic acid and Trichostatin A (TSA) up-regulate METTL3/METTL14 proteins. The levels of METTL3 in mouse adult hearts, isolated from male and female animals, were lower in the aorta and pulmonary trunks when compared with atria, but METT14 was up-regulated in the aorta and pulmonary trunk, in comparison with ventriculi. Aging caused METTL3 down-regulation in aorta and atria in male animals. Western blot analysis in differentiated mouse embryonic stem cells (mESCs), containing 10-30 percent of cardiomyocytes, showed METTL3/METTL14 down-regulation, while the differentiation-induced increased level of METTL16 was observed in both wild type (wt) and HDAC1 depleted (dn) cells. In parallel, experimental differentiation in especially HDAC1 wild type cells was accompanied by depletion of m6A in RNA. Immunofluorescence analysis of individual cells revealed the highest density of METTL3/METTL14 in α-actinin positive cardiomyocytes when compared with the other cells in the culture undergoing differentiation. In both wt and HDAC1 dn cells, the amount of METTL16 was also up-regulated in cardiomyocytes when compared to co-cultivated cells. Together, we showed that distinct anatomical regions of the mouse adult hearts are characterized by different levels of METTL3 and METTL14 proteins, which are changed during aging. Experimental cell differentiation was also accompanied by changes in METTL-like proteins and m6A in RNA; in particular, levels and distribution patterns of METTL3/METTL14 proteins were different from the same parameters studied in the case of the METTL16 protein.

• MeSH
• adenosin analogy a deriváty   genetika   metabolismus   MeSH
• buněčná diferenciace MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• kardiomyocyty cytologie   metabolismus   MeSH
• lidé MeSH
• methyltransferasy metabolismus   MeSH
• myší embryonální kmenové buňky cytologie   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• stárnutí metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

12-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in biomedical studies. TPA has been proposed as a pluripotent cell differentiation factor, but results obtained have been inconsistent. In the present study we show that TPA can be applied as a cardiomyogenesis-promoting factor for the differentiation of mouse embryonic stem (mES) cells in vitro. The mechanism of TPA action is mediated by the induction of extracellular signal-regulated kinase (ERK) activity and the subsequent phosphorylation of GATA4 transcription factor. Interestingly, general mitogens (FGF, EGF, VEGF and serum) or canonical WNT signalling did not mimic the effect of TPA. Moreover, on the basis of our results, we postulate that a TPA-sensitive population of cardiac progenitor cells exists at a certain time point (after days 6-8 of the differentiation protocol) and that the proposed treatment can be used to increase the multiplication of ES cell-derived cardiomyocytes.

• MeSH
• buněčná diferenciace účinky léků   MeSH
• embryonální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosforylace MeSH
• kardiomyocyty cytologie   metabolismus   MeSH
• myši MeSH
• proteinkinasa C metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The stimulation of myocardium repair is restricted due to the limited understanding of heart regeneration. Interestingly, endogenous opioid peptides such as dynorphins and enkephalins are suggested to support this process. However, the mechanism-whether through the stimulation of the regenerative capacity of cardiac stem cells or through effects on other cell types in the heart-is still not completely understood. Thus, a model of the spontaneous cardiomyogenic differentiation of mouse embryonic stem (mES) cells via the formation of embryoid bodies was used to describe changes in the expression and localization of opioid receptors within cells during the differentiation process and the potential of the selected opioid peptides, dynorphin A and B, and methionin-enkephalins and leucin-enkephalins, to modulate cardiomyogenic differentiation in vitro. The expressions of both κ- and δ-opioid receptors significantly increased during mES cell differentiation. Moreover, their primary colocalization with the nucleus was followed by their growing presence on the cytoplasmic membrane with increasing mES cell differentiation status. Interestingly, dynorphin B enhanced the downregulation gene expression of Oct4 characteristic of the pluripotent phenotype. Further, dynorphin B also increased cardiomyocyte-specific Nkx2.5 gene expression. However, neither dynorphin A nor methionin-enkephalins and leucin-enkephalins exhibited any significant effects on the course of mES cell differentiation. In conclusion, despite the increased expression of opioid receptors and some enhancement of mES cell differentiation by dynorphin B, the overall data do not support the notion that opioid peptides have a significant potential to promote the spontaneous cardiomyogenesis of mES cells in vitro.

• MeSH
• buněčná diferenciace fyziologie   MeSH
• kardiomyocyty cytologie   fyziologie   MeSH
• myokard cytologie   MeSH
• myší embryonální kmenové buňky cytologie   metabolismus   MeSH
• myši MeSH
• opioidní peptidy metabolismus   MeSH
• receptory opiátové metabolismus   MeSH
• regenerace fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cardiomyocytes (CM) placed on microelectrode array (MEA) were simultaneously probed with cantilever from atomic force microscope (AFM) system. This electric / nanomechanical combination in real time recorded beating force of the CMs cluster and the triggering electric events. Such "organ-on-a-chip" represents a tool for drug development and disease modeling. The human pluripotent stem cells included the WT embryonic line CCTL14 and the induced dystrophin deficient line reprogrammed from fibroblasts of a patient affected by Duchenne Muscular Dystrophy (DMD, complete loss of dystrophin expression). Both were differentiated to CMs and employed with the AFM/MEA platform for diseased CMs' drug response testing and DMD characterization. The dependence of cardiac parameters on extracellular Ca2+ was studied. The differential evaluation explained the observed effects despite variability of biological samples. The β-adrenergic stimulation (isoproterenol) and antagonist trials (verapamil) addressed ionotropic and chronotropic cell line-dependent features. For the first time, a distinctive beating-force relation for DMD CMs was measured on the 3D cardiac in vitro model.

• MeSH
• biosenzitivní techniky * MeSH
• buněčná diferenciace genetika   MeSH
• Duchennova muskulární dystrofie patofyziologie   MeSH
• dystrofin genetika   MeSH
• fibroblasty účinky léků   ultrastruktura   MeSH
• indukované pluripotentní kmenové buňky metabolismus   ultrastruktura   MeSH
• isoprenalin farmakologie   MeSH
• kardiomyocyty cytologie   MeSH
• kontrakce myokardu genetika   fyziologie   MeSH
• lidé MeSH
• mikroelektrody MeSH
• mikroskopie atomárních sil MeSH
• verapamil farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.

• MeSH
• analýza dat MeSH
• elektrofyziologické jevy * MeSH
• kardiomyocyty cytologie   fyziologie   MeSH
• mechanické jevy * MeSH
• mikroskopie atomárních sil * přístrojové vybavení   metody   MeSH
• molekulární zobrazování MeSH
• spřažení excitace a kontrakce * MeSH
• vápníková signalizace MeSH
• Publikační typ
• časopisecké články MeSH

Atomic force microscopy (AFM) is not only a high-resolution imaging technique but also a sensitive tool able to study biomechanical properties of bio-samples (biomolecules, cells) in native conditions-i.e., in buffered solutions (culturing media) and stable temperature (mostly 37 °C). Micromechanical transducers (cantilevers) are often used to map surface stiffness distribution, adhesion forces, and viscoelastic parameters of living cells; however, they can also be used to monitor time course of cardiomyocytes contraction dynamics (e.g. beating rate, relaxation time), together with other biomechanical properties. Here we describe the construction of an AFM-based biosensor setup designed to study the biomechanical properties of cardiomyocyte clusters, through the use of standard uncoated silicon nitride cantilevers. Force-time curves (mechanocardiograms, MCG) are recorded continuously in real time and in the presence of cardiomyocyte-contraction affecting drugs (e.g., isoproterenol, metoprolol) in the medium, under physiological conditions. The average value of contraction force and the beat rate, as basic biomechanical parameters, represent pharmacological indicators of different phenotype features. Robustness, low computational requirements, and optimal spatial sensitivity (detection limit 200 pN, respectively 20 nm displacement) are the main advantages of the presented method.

• MeSH
• biomechanika * MeSH
• biosenzitivní techniky MeSH
• kardiomyocyty cytologie   MeSH
• lidé MeSH
• mikroskopie atomárních sil * přístrojové vybavení   metody   MeSH
• pluripotentní kmenové buňky cytologie   MeSH
• preklinické hodnocení léčiv MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The cytocompatibility of cardiomyocytes derived from embryonic stem cells and neural progenitors, which were seeded on the surface of composite films made of graphene oxide (GO) and polypyrrole (PPy-GO) or poly(3,4-ethylenedioxythiophene) (PEDOT-GO) are reported. The GO incorporated in the composite matrix contributes to the patterning of the composite surface, while the electrically conducting PPy and PEDOT serve as ion-to-electron transducers facilitating electrical stimulation/sensing. The films were fabricated by a simple one-step electropolymerization procedure on electrically conducting indium tin oxide (ITO) and graphene paper (GP) substrates. Factors affecting the cell behaviour, i.e. the surface topography, wettability, and electrical surface conductivity, were studied. The PPy-GO and PEDOT-GO prepared on ITO exhibited high surface conductivity, especially in the case of the ITO/PPy-GO composite. We found that for cardiomyocytes, the PPy-GO and PEDOT-GO composites counteracted the negative effect of the GP substrate that inhibited their growth. Both the PPy-GO and PEDOT-GO composites prepared on ITO and GP significantly decreased the cytocompatibility of neural progenitors. The presented results enhance the knowledge about the biological properties of electroactive materials, which are critical for tissue engineering, especially in context stimuli-responsive scaffolds.

• MeSH
• bicyklické sloučeniny heterocyklické chemie   MeSH
• elektrická vodivost * MeSH
• elektrochemie * MeSH
• grafit farmakologie   MeSH
• kardiomyocyty cytologie   účinky léků   MeSH
• myši MeSH
• nervové kmenové buňky cytologie   účinky léků   MeSH
• neurogeneze účinky léků   MeSH
• polymery chemie   farmakologie   MeSH
• pyrroly chemie   MeSH
• voda chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Computer assisted image acquisition techniques, including confocal microscopy, require efficient tools for an automatic sorting of vast amount of generated image data. The complexity of the classification process, absence of adequate tools, and insufficient amount of reference data has made the automated processing of images challenging. Mastering of this issue would allow implementation of statistical analysis in research areas such as in research on formation of t-tubules in cardiac myocytes. We developed a system aimed at automatic assessment of cardiomyocyte development stages (SAACS). The system classifies confocal images of cardiomyocytes with fluorescent dye stained sarcolemma. We based SAACS on a densely connected convolutional network (DenseNet) topology. We created a set of labelled source images, proposed an appropriate data augmentation technique and designed a class probability graph. We showed that the DenseNet topology, in combination with the augmentation technique is suitable for the given task, and that high-resolution images are instrumental for image categorization. SAACS, in combination with the automatic high-throughput confocal imaging, will allow application of statistical analysis in the research of the tubular system development or remodelling and loss.

• MeSH
• buněčná diferenciace MeSH
• fluorescenční barviva MeSH
• kardiomyocyty cytologie   ultrastruktura   MeSH
• konfokální mikroskopie metody   MeSH
• krysa rodu rattus MeSH
• modely kardiovaskulární MeSH
• neuronové sítě (počítačové) MeSH
• počítačové zpracování obrazu metody   MeSH
• sarkolema ultrastruktura   MeSH
• strojové učení MeSH
• umělá inteligence MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Modern diagnostic strategies for early recognition of cancer therapeutics-related cardiac dysfunction involve cardiac troponins measurement. Still, the role of other markers of cardiotoxicity is still unclear. The present study was designed to investigate dynamics of response of human cardiomyocytes derived from induced pluripotent stem cells (hiPCS-CMs) to doxorubicin with the special emphasis on their morphological changes in relation to expression and organization of troponins. The hiPCS-CMs were treated with doxorubicin concentrations (1 and 0.3 µM) for 48 h and followed for next up to 6 days. Exposure of hiPCS-CMs to 1 µM doxorubicininduced suppression of both cardiac troponin T (cTnT) and cardiac troponin I (cTnI) gene expression. Conversely, lower 0.3 µM doxorubicin concentration produced no significant changes in the expression of aforementioned genes. However, the intracellular topography, arrangement, and abundance of cardiac troponin proteins markedly changed after both doxorubicin concentrations. In particular, at 48 h of treatment, both cTnT and cTnI bundles started to reorganize, with some of them forming compacted shapes extending outwards and protruding outside the cells. At later intervals (72 h and onwards), the whole troponin network collapsed and became highly disorganized following, to some degree, overall changes in the cellular shape. Moreover, membrane permeability of cardiomyocytes was increased, and intracellular mitochondrial network rearranged and hypofunctional. Together, our results demonstrate complex effects of clinically relevant doxorubicin concentrations on hiPCS-CM cells including changes in cTnT and cTnI, but also in other cellular compartments contributing to the overall cytotoxicity of this class of cytostatics.

• MeSH
• antitumorózní látky farmakologie   toxicita   MeSH
• buněčné linie MeSH
• doxorubicin farmakologie   toxicita   MeSH
• indukované pluripotentní kmenové buňky cytologie   účinky léků   MeSH
• kardiomyocyty cytologie   účinky léků   metabolismus   MeSH
• kardiotoxicita MeSH
• lidé MeSH
• troponin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH