QuinoMit Q10-Fluid attenuates hydrogen peroxide-induced irregular beating in mouse pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Coenzyme Q10 (CoQ10) is a crucial component of the mitochondrial structure which is involved in producing more than 90% of cellular ATP. This study aimed to investigate the protective effects and underlying mechanisms of QuinoMit Q10-Fluid against hydrogen peroxide (H2O2)-induced arrhythmias on cardiomyocytes (CMs). METHODS: Undifferentiated stem cell-derived CMs were cultured in the presence of different concentrations of QuinoMit Q10-Fluid. To investigate if CoQ10 has anti-apoptotic activity, CMs were exposed to H2O2 for up to 100 h with or without CoQ10. The expression levels of cardiac reference genes were determined by RT-PCR. The structural and functional properties of CMs were examined by immunofluorescence and the xCELLigence system. Caspase 3/7 assay was also performed for cell apoptosis study. RESULTS: The study showed that QuinoMit Q10-Fluid inhibits the proliferation of pluripotent stem cells at high concentrations and had less effect on cardiomyogenesis. However, the beating rate of clusters containing CMs generated under QuinoMit Q10-Fluid (1:100) was significantly increased. This increase was accompanied by the up-regulated expression level of some important cardiac markers during differentiation. Treatment of CMs with H2O2 notably induced irregular beating and decreased the amplitude of the beating signal of CMs, concomitantly with increased caspase-3/7 activity. However, CMs pretreated with QuinoMit exhibited a protective effect against H2O2-induced arrhythmia. CONCLUSION: Our results reveal that QuinoMit Q10-Fluid attenuates H2O2-induced irregular beating in mouse pluripotent stem cell-derived CMs, at least partly by reducing the generation of ROS, suggesting a protective effect against CM dysfunctions.

Stem cells in natural product and medicinal plant drug discovery-An overview of new screening approaches.

Natural products remain a rich source of new drugs, and the search for bioactive molecules from nature continues to play an important role in the development of new medicines. Also, there is increasing use of herbal medicines for the treatment of a plethora of diseases, and demands for more scientific evidence for their efficacy and safety remains a huge challenge. The propensity of stem cells to differentiate into almost every cell type not only holds promise for the delivery of cell-based therapies for currently incurable diseases or a useful tool in studying cell physiology and pathophysiology. Increasingly, stem cells are becoming an important tool in preclinical drug screening and toxicity testing. In this review, we examine the scientific advances made towards the use of pluripotent stem cells as a model for the screening of plant-based medicines. The combination of well-established in vitro electrophysiological and a plethora of toxicogenomic technologies, together with the optimisation of culture methods of herbal plants and pluripotent stem cells can be explored to establish the basis for efficacy, and tissue/organ-based toxicities of many currently used medicinal plants whose efficacies and toxicities remain unknown.

Correction: Nemade, H.; et al. Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells. Cells 2020, 9, 554.

The authors wish to make the following corrections to this paper [...].

Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells.

Application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited by the challenges in their efficient differentiation. Recently, the Wingless (Wnt) signaling pathway has emerged as the key regulator of cardiomyogenesis. In this study, we evaluated the effects of cyclooxygenase inhibitors on cardiac differentiation of hPSCs. Cardiac differentiation was performed by adherent monolayer based method using 4 hPSC lines (HES3, H9, IMR90, and ES4SKIN). The efficiency of cardiac differentiation was evaluated by flow cytometry and RT-qPCR. Generated hPSC-CMs were characterised using immunocytochemistry, electrophysiology, electron microscopy, and calcium transient measurements. Our data show that the COX inhibitors Sulindac and Diclofenac in combination with CHIR99021 (GSK-3 inhibitor) efficiently induce cardiac differentiation of hPSCs. In addition, inhibition of COX using siRNAs targeted towards COX-1 and/or COX-2 showed that inhibition of COX-2 alone or COX-1 and COX-2 in combination induce cardiomyogenesis in hPSCs within 12 days. Using IMR90-Wnt reporter line, we showed that inhibition of COX-2 led to downregulation of Wnt signalling activity in hPSCs. In conclusion, this study demonstrates that COX inhibition efficiently induced cardiogenesis via modulation of COX and Wnt pathway and the generated cardiomyocytes express cardiac-specific structural markers as well as exhibit typical calcium transients and action potentials. These cardiomyocytes also responded to cardiotoxicants and can be relevant as an in vitro cardiotoxicity screening model.

Altered Functional Expression of ß-Adrenergic Receptors in Rhesus Monkey Embryonic Stem Cell-Derived Cardiomyocytes.

Pluripotent stem cells have demonstrated the potential to generate large numbers of functional cardiomyocytes (CMs) from different cell sources. Besides Wnt signaling, additional pathways are involved in early cardiac development and function. To date however, no study exists showing the effects of perturbing the canonical Wnt pathway using nonhuman primate embryonic stem (ES) cells. In this study, we investigated the effect of canonical Wnt inhibition during differentiation of nonhuman primate ES cell-derived CMs under defined, growth factor conditions. Rhesus monkey ES (rES) cells were differentiated into spontaneously beating CMs in the absence (control) or presence (treated) of Wnt inhibitor Dickkopf1 (DKK1), vascular endothelial growth factor, and basic fibroblast growth factor combined or added in a sequential manner during differentiation. Quantification and functional characterization of CMs were assessed by molecular and electrophysiological techniques. Analysis revealed no difference in average ratio of spontaneously beating clusters in both control and treated groups. However, the percentage of CMs was significantly reduced and the expressions of specific cardiac markers tested were also decreased in the treated group. Interestingly, we found that in CMs obtained from treated group, ß-adrenergic receptors (ß-ARs) were less expressed, their function was altered and electrophysiological studies revealed differences in action potential responsiveness to ß-AR stimulation. We demonstrated that the Wnt/ß-catenin pathway inhibitor, DKK1 associated with other growth factors repressed functional expression of ß-ARs in rES cell-derived CMs. Thus, control of this pathway in each cell line and source is important for proper basic research and further cell therapy applications.

Multiple nickel-sensitive targets elicit cardiac arrhythmia in isolated mouse hearts after pituitary adenylate cyclase-activating polypeptide-mediated chronotropy.

The pituitary adenylate cyclase-activating polypeptide (PACAP)-27 modulates various biological processes, from the cellular level to function specification. However, the cardiac actions of this neuropeptide are still under intense studies. Using control (+|+) and mice lacking (-|-) either R-type (Cav2.3) or T-type (Cav3.2) Ca2+ channels, we investigated the effects of PACAP-27 on cardiac activity of spontaneously beating isolated perfused hearts. Superfusion of PACAP-27 (20nM) caused a significant increase of baseline heart frequency in Cav2.3(+|+) (156.9±10.8 to 239.4±23.4 bpm; p<0.01) and Cav2.3(-|-) (190.3±26.4 to 270.5±25.8 bpm; p<0.05) hearts. For Cav3.2, the heart rate was significantly increased in Cav3.2(-|-) (133.1±8.5 bpm to 204.6±27.9 bpm; p<0.05) compared to Cav3.2(+|+) hearts (185.7±11.2 bpm to 209.3±22.7 bpm). While the P wave duration and QTc interval were significantly increased in Cav2.3(+|+) and Cav2.3(-|-) hearts following PACAP-27 superfusion, there was no effect in Cav3.2(+|+) and Cav3.2(-|-) hearts. The positive chronotropic effects observed in the four study groups, as well as the effect on P wave duration and QTc interval were abolished in the presence of Ni2+ (50µM) and PACAP-27 (20nM) in hearts from Cav2.3(+|+) and Cav2.3(-|-) mice. In addition to suppressing PACAP's response, Ni2+ also induced conduction disturbances in investigated hearts. In conclusion, the most Ni2+-sensitive Ca2+ channels (R- and T-type) may modulate the PACAP signaling cascade during cardiac excitation in isolated mouse hearts, albeit to a lesser extent than other Ni2+-sensitive targets.

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities.

Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

Dynamic Support Culture of Murine Skeletal Muscle-Derived Stem Cells Improves Their Cardiogenic Potential In Vitro.

Ischemic heart disease is the main cause of death in western countries and its burden is increasing worldwide. It typically involves irreversible degeneration and loss of myocardial tissue leading to poor prognosis and fatal outcome. Autologous cells with the potential to regenerate damaged heart tissue would be an ideal source for cell therapeutic approaches. Here, we compared different methods of conditional culture for increasing the yield and cardiogenic potential of murine skeletal muscle-derived stem cells. A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation. In contrast to static culture conditions, dynamic culture with or without previous hanging drop preculture led to significantly increased cluster diameters and the expression of cardiac specific markers on the protein and mRNA level. Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli. This data indicates that skeletal muscle-derived stem cells are capable of adopting enhanced cardiac muscle cell-like properties by applying specific culture conditions. Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells.

Effects of synthetic neural adhesion molecule mimetic peptides and related proteins on the cardiomyogenic differentiation of mouse embryonic stem cells.

BACKGROUND/AIMS: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. METHODS: In the present study, using a transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain (α-MHC) promoter (pαMHC-EGFP), we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGL(L), hNgf_C2, EnkaminE, Plannexin and C3) on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. RESULTS: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF) peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. CONCLUSION: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to cardiac pathologies where BDNF levels are impaired.

In vitro chronotropic effects of Erythrina senegalensis DC (Fabaceae) aqueous extract on mouse heart slice and pluripotent stem cell-derived cardiomyocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Erythrina senegalensis DC (Fabaceae) bark is commonly used in sub-Saharan traditional medicine for the treatment of many diseases including gastrointestinal disorders and cardiovascular diseases. In this study, we investigated the effect of the aqueous extract of the stem bark of Erythrina senegalensis on the contractile properties of mouse ventricular slices and human induced pluripotent stem (hiPS) cell-derived cardiomyocytes. We also investigated the cytotoxic effect of the extract on mouse embryonic stem (ES) cells differentiating into cardiomyocytes (CMs). MATERIALS AND METHODS: We used well-established electrophysiological technologies to assess the effect of Erythrina senegalensis aqueous extract (ESAE) on the beating activity of mouse ventricular slices, mouse ES and hiPS cell-derived CMs. To study the cytotoxic effect of our extract, differentiating mouse ES cells were exposed to different concentrations of ESAE. EB morphology was assessed by microscopy at different stages of differentiation whereas cell viability was measured by flow cytometry, fluorometry and immunocytochemistry. The electrical activity of CMs and heart slices were respectively captured by the patch clamp technique and microelectrode array (MEA) method following ESAE acute exposure. RESULTS: Our findings revealed that ESAE exhibits a biphasic chronotropic activity on mouse ventricular slices with an initial low dose (0.001 and 0.01 µg/mL) decrease in beating activity followed by a corresponding significant increase in chronotropic activity at higher doses above 10 µg/mL. The muscarinic receptor blocker, atropine abolished the negative chronotropic activity of ESAE, while propranolol successfully blocked its positive chronotropic activity. ESAE showed a significant dose-dependent positive chronotropic activity on hiPS cell-derived CMs. Also, though not significantly, ESAE decreased cell viability and increased total caspase-3/7 activity of mouse ES cells in a concentration-dependent manner. CONCLUSION: Erythrina senegalensis aqueous extract exhibits a biphasic chronotropic effect on mouse heart and a positive chronotropic activity on hiPS cell-derived CMs, suggesting a possible mechanism through muscarinic and ß-adrenergic receptor pathways. Also, ESAE is not cytotoxic on mouse ES cells at concentrations up to 100 µg/mL.

Cardiac functions of voltage-gated Ca(2+) channels: role of the pharmacoresistant type (E-/R-Type) in cardiac modulation and putative implication in sudden unexpected death in epilepsy (SUDEP).

Voltage-gated Ca(2+) channels (VGCCs) are ubiquitous in excitable cells. These channels play key roles in many physiological events like cardiac regulation/pacemaker activity due to intracellular Ca(2+) transients. In the myocardium, the Cav1 subfamily (L-type: Cav1.2 and Cav1.3) is the main contributor to excitation-contraction coupling and/or pacemaking, whereas the Cav3 subfamily (T-type: Cav3.1 and Cav3.2) is important in rhythmically firing of the cardiac nodal cells. No established cardiac function has been attributed to the Cav2 family (E-/R-type: Cav2.3) despite accumulating evidence of cardiac dysregulation observed upon deletion of the Cav2.3 gene, the only member of this family so far detected in cardiomyocytes. In this review, we summarize the pathophysiological changes observed after ablation of the E-/R-type VGCC and propose a cardiac mechanism of action for this channel. Also, considering the role played by this channel in epilepsy and its reported sensitivity to antiepileptic drugs, a putative involvement of this channel in the cardiac mechanism of sudden unexpected death in epilepsy is also discussed.

The proliferative and chronotropic effects of Brillantaisia nitens Lindau (Acanthaceae) extracts on pluripotent stem cells and their cardiomyocytes derivatives.

ETHNOPHARMACOLOGICAL RELEVANCE: Brillantaisia nitens Lindau (Acanthaceae) leaves are commonly used in traditional medicine in Africa for the treatment of many disorders including heart diseases and malaria. In this study, we therefore evaluated the effect of the methylene chloride/methanol leaf extract of Brillantaisia nitens on the proliferation of mouse pluripotent stem cells and their cardiomyocyte derivatives. MATERIALS AND METHODS: In this study, we combined two emerging technologies, pluripotent stem cell-derived cardiomyocytes and modern electrophysiology systems (impedance-based real-time) to assess the cytotoxicity of Brillantaisia nitens extract (BNE). Undifferentiated pluripotent cells and cardiomyocytes were exposed to different concentrations of BNE. Cell viability and contraction were monitored by impedance using the xCELLigence system for short- and long-term treatment whereas the excitability of single cardiomyocytes was captured by patch clamp technique after BNE acute exposure. RESULTS: Brillantaisia nitens extract inhibited the proliferation and increased cytotoxicity of embryonic stem cells in a concentration-dependent manner. With the increase in concentration of BNE, beating rate and the contractile amplitude of cardiomyocytes changed significantly. Spontaneous rhythmic activity of cardiomyocytes was completely suppressed after 48 and 24h exposures to relatively low (4.16 mg/ml) and high (8.32 mg/ml) concentrations of BNE, respectively. Moreover, acute application of 4.16 mg/ml of BNE led to a significant alteration of action potential (AP) parameters such as beating frequency, amplitude and AP duration at 90% of repolarization. CONCLUSION: Brillantaisia nitens extract inhibits the proliferative capacity of pluripotent stem cells and reduces electrical activity of cardiomyocytes, confirming its depressant action on the heart.