A slowly inactivating calcium current works as a calcium sensor in calcitonin-secreting cells

AbstractCalcitonin (CT)-secreting cells (C-cells) are remarkably sensitive to changes in the extracellular Ca2+ concentration. In order to detect the mechanism by which C-cells monitor Ca2+, we compared a C-cell line responding to Ca2+ (rMTC cells) with another one known to have a defect in this Ca2+ signal transduction (TT cells). Rises of the Ca2+ concentration caused rMTC cells to depolarize and/or elicited spontaneous action potentials. Under voltage-clamp conditions, rMTC cells showed a slowly decaying Ca2+ inward current which was sensitive to dihydropyridines but not to Ni2+ at a low concentration. In contrast, the ‘defective’ TT cells neither depolarized nor fired action potentials with high Ca2+; they only exhibited an Ni2+-sensitive, transient Ca2+ current. The data strongly suggest that the slowly inactivating Ca2+ current is a prerequisite for Ca2+-sensitivity of C-cells and that fast inactivating channels are not sufficient to act as sensors of the extracellular Ca2+ concentration

Involvement of A pertussis Toxin Sensitive G-Protein in the Inhibition of Inwardly Rectifying K+ Currents by Platelet-Activating Factor in Guinea-Pig Atrial Cardiomyocytes

Platelet-activating factor (PAF) inhibits single inwardly rectifying K+ channels in guinea-pig ventricular cells. There is currently little information as to the mechanism by which these channels are modulated. The effect of PAF on quasi steady-state inwardly rectifying K+ currents (presumably of the IK1 type) of auricular, atrial and ventricular cardiomyocytes from guinea-pig were studied. Applying the patch-clamp technique in the whole-cell configuration, PAF (10 nM) reduced the K+ currents in all three cell types. The inhibitory effect of PAF occurred within seconds and was reversible upon wash-out. It was almost completely abolished by the PAF receptor antagonist BN 50730. Intracellular infusion of atrial cells with guanine 5′-(β-thio)diphosphate (GDPS) or pretreatment of cells with pertussis toxin abolished the PAF dependent reduction of the currents. Neither extracellularly applied isoproterenol nor intracellularly applied adenosine 3′,5′-cyclic monophosphate (cyclic AMP) attenuated the PAF effect. In multicellular preparations of auricles, PAF (10 nM) induced arrhythmias. The arrhythmogenic activity was also reduced by BN 50730. The data indicate that activated PAF receptors inhibit inwardly rectifying K+ currents via a pertussis toxin sensitive G-protein without involvement of a cyclic AMP-dependent step. Since IK1 is a major component in stabilizing the resting membrane potential, the observed inhibition of this type of channel could play an important role in PAF dependent arrhythmogenesis in guinea-pig heart

Gene expression signatures defining fundamental biological processes in pluripotent, early, and late differentiated embryonic stem cells

Investigating the molecular mechanisms controlling the in vivo developmental program postembryogenesis is challenging and time consuming. However, the developmental program can be partly recapitulated in vitro by the use of cultured embryonic stem cells (ESCs). Similar to the totipotent cells of the inner cell mass, gene expression and morphological changes in cultured ESCs occur hierarchically during their differentiation, with epiblast cells developing first, followed by germ layers and finally somatic cells. Combination of high throughput -omics technologies with murine ESCs offers an alternative approach for studying developmental processes toward organ-specific cell phenotypes. We have made an attempt to understand differentiation networks controlling embryogenesis in vivo using a time kinetic, by identifying molecules defining fundamental biological processes in the pluripotent state as well as in early and the late differentiation stages of ESCs. Our microarray data of the differentiation of the ESCs clearly demonstrate that the most critical early differentiation processes occur at days 2 and 3 of differentiation. Besides monitoring well-annotated markers pertinent to both self-renewal and potency (capacity to differentiate to different cell lineage), we have identified candidate molecules for relevant signaling pathways. These molecules can be further investigated in gain and loss-of-function studies to elucidate their role for pluripotency and differentiation. As an example, siRNA knockdown of MageB16, a gene highly expressed in the pluripotent state, has proven its influence in inducing differentiation when its function is repressed

Reactive oxygen species-linked regulation of the multidrug resistance transporter P-glycoprotein in Nox-1 overexpressing prostate tumor spheroids

AbstractExpression of the multidrug resistance (MDR) transporter P-glycoprotein (P-gp) has been demonstrated to be regulated by hypoxia-inducible factor-1α (HIF-1α) and inhibited by intracellular reactive oxygen species (ROS). Herein, P-gp and HIF-1α expression were investigated in multicellular prostate tumor spheroids overexpressing the ROS-generating enzyme Nox-1 in comparison to the mother cell line DU-145. In Nox-1-overexpressing tumor spheroids (DU-145Nox1) generation of ROS as well as expression of Nox-1 was significantly increased as compared to DU-145 tumor spheroids. ROS generation was significantly inhibited in the presence of the NADPH-oxidase antagonists diphenylen-iodonium chloride (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). Albeit growth kinetic of DU-145Nox1 tumor spheroids was decreased as compared to DU-145 spheroids, elevated expression of Ki-67 was observed indicating increased cell cycle activity. In DU-145Nox1 tumor spheroids, expression of HIF-1α as well as P-gp was significantly decreased as compared to DU-145 spheroids, which resulted in an increased retention of the anticancer agent doxorubicin. Pretreatment with the free radical scavengers vitamin E and vitamin C increased the expression of P-gp as well as HIF-1α in Nox-1-overexpressing cells, whereas no effect of free radical scavengers was observed on mdr-1 mRNA expression. In summary, the data of the present study demonstrate that the development of P-gp-mediated MDR is abolished under conditions of elevated ROS levels, suggesting that the MDR phenotype can be circumvented by modest increase of intracellular ROS generation

Identification of genomic biomarkers for anthracycline-induced cardiotoxicity in human iPSC-derived cardiomyocytes: an in vitro repeated exposure toxicity approach for safety assessment.

The currently available techniques for the safety evaluation of candidate drugs are usually cost-intensive and time-consuming and are often insufficient to predict human relevant cardiotoxicity. The purpose of this study was to develop an in vitro repeated exposure toxicity methodology allowing the identification of predictive genomics biomarkers of functional relevance for drug-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The hiPSC-CMs were incubated with 156 nM doxorubicin, which is a well-characterized cardiotoxicant, for 2 or 6 days followed by washout of the test compound and further incubation in compound-free culture medium until day 14 after the onset of exposure. An xCELLigence Real-Time Cell Analyser was used to monitor doxorubicin-induced cytotoxicity while also monitoring functional alterations of cardiomyocytes by counting of the beating frequency of cardiomyocytes. Unlike single exposure, repeated doxorubicin exposure resulted in long-term arrhythmic beating in hiPSC-CMs accompanied by significant cytotoxicity. Global gene expression changes were studied using microarrays and bioinformatics tools. Analysis of the transcriptomic data revealed early expression signatures of genes involved in formation of sarcomeric structures, regulation of ion homeostasis and induction of apoptosis. Eighty-four significantly deregulated genes related to cardiac functions, stress and apoptosis were validated using real-time PCR. The expression of the 84 genes was further studied by real-time PCR in hiPSC-CMs incubated with daunorubicin and mitoxantrone, further anthracycline family members that are also known to induce cardiotoxicity. A panel of 35 genes was deregulated by all three anthracycline family members and can therefore be expected to predict the cardiotoxicity of compounds acting by similar mechanisms as doxorubicin, daunorubicin or mitoxantrone. The identified gene panel can be applied in the safety assessment of novel drug candidates as well as available therapeutics to identify compounds that may cause cardiotoxicity

Combining Hypoxia and Bioreactor Hydrodynamics Boosts Induced Pluripotent Stem Cell Differentiation Towards Cardiomyocytes

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O(2) tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O(2) tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12015-014-9533-0) contains supplementary material, which is available to authorized users

Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

<p>Abstract</p> <p>Background</p> <p>Functional and molecular integrity of cardiomyocytes (CMs) derived from induced pluripotent stem (iPS) cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs) microdissected from differentiating human iPS cells and embryonic stem (ES) cells.</p> <p>Results</p> <p>Hierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes.</p> <p>Conclusions</p> <p>These data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both.</p