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Case study PDF
Diabetic cardiomyopathy – “In vitro cardiomyopathy model to evaluate ischemic sensitivity”
Publication link
2024 – Wnt dose escalation during the exit from pluripotency identifies tranilast as a regulator of cardiac mesoderm
CardioExcyte 96 Publication in Developmental Cell (2024) Authors: Wu Z., Shen S., Mizikovsky D., Cao Y., Naval-Sanchez M., Tan S.Z., Alvarez Y., Sun Y., Chen X., Zhao Q., Kim D., Yang P., Hill T., Jones A., Fairlie D., Pébay A., Hewitt A., Tam P., White M., Nefzger C., Palpant N.

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.

Publication link
2024 – Reprogramming of DNA methylation patterns mediates perfluorooctane sulfonate-induced fetal cardiac dysplasia
CardioExcyte 96 Publication in Science of the Total Environment (2024) Authors: Qiu M., Chen J., Liu M., Shi Y., Nie Z., Dong G., Li X., Chen J., Ou Y., Zhuang J.

Prenatal exposure to perfluorooctane sulfonate (PFOS) is associated with adverse health effects, including congenital heart disease, yet the underlying mechanisms remain elusive. Herein, we aimed to evaluate the embryotoxicity of PFOS using C57BL/6 J mice to characterize fetal heart defects after PFOS exposure, with the induction of human embryonic stem cells (hESC) into cardiomyocytes (CMs) as a model of early-stage heart development. We also performed DNA methylation analysis to clarify potential underlying mechanisms and identify targets of PFOS. Our results revealed that PFOS caused septal defects and excessive ventricular trabeculation cardiomyopathy at 5 mg/kg/day in embryonic mice and inhibited the proliferation and pluripotency of ESCs at concentrations >20 μM. Moreover, it decreased the beating rate and the population of CMs during cardiac differentiation. Decreases were observed in the abundances of NPPA+ trabecular and HEY2+ compact CMs. Additionally, DNA methyl transferases and ten-eleven translocation (TET) dioxygenases were regulated dynamically by PFOS, with TETs inhibitor treatment inducing significant decreases similar as PFOS. 850 K DNA methylation analysis combined with expression analysis revealed several potential targets of PFOS, including SORBS2FHOD1SLIT2SLIT3ADCY9, and HDAC9. In conclusion, PFOS may reprogram DNA methylation, especially demethylation, to induce cardiac toxicity, causing ventricular defects in vivo and abnormal cardiac differentiation in vitro.

 

Application Note PDF
Hepatocytes – “Comprehensive impedance-based hepatotoxicity assay for metabolically active iPSC-derived hepatocytes”
CardioExcyte 96, AtlaZ Application Note: Cells were kindly provided by FUJIFILM CellularDynamics, FCDI

Hepatotoxicity and drug-induced liver injury (DILI) are leading reasons for drug failure to market, leading to approximately 18% of market withdrawals of drugs in the last decade.
Additionally, only half of drugs with a potential to induce hepatotoxicity are actually identified during preclinical animal studies. This highlights the importance of generating more
advanced cell-based models and experimental strategies to enhance the predictivity of these assays. Current existing in vitro models employed to predict DILI mostly focus on
hepatocytes, though primary hepatocytes do not maintain their phenotype. iCell® Hepatocytes 2.0 (FUJIFILM Cellular Dynamics, FCDI) are human iPSC-derived cells with a
wide variety of basic and functional characteristics which make them amenable to applications such as compound mediated ADME-T and DILI toxicity. In addition to displaying
characteristic hepatocyte morphology, (i.e., polygonal shape, polynucleation and formation of bile canalicular channels), these cells also express liver cell markers, including albumin,
A1AT, and HNF4a, and exhibit basic and induced P450 functions, as observed in primary human hepatocytes. iCell® Hepatocytes 2.0 maintain morphology, marker expression,
and metabolic function in culture over a longer time frame compared to primary human hepatocytes, rendering these cells useful for investigations of acute and chronic DILI responses in a 2D culture system using impedance. Combining these cells with the planar gold-film electrodes on the impedance systems reveal alterations in confluency, cell contact (morphological shape) and conductivity of adherent cells, thereby providing a measure of toxicity. Dose-dependent harmful effects of drugs could be evaluated over time in a functional 2D cell-based model without the need for 3D spheroid formation.

Publication link
2024 – Cellular-level analyses of SCN5A mutations in left ventricular noncompaction cardiomyopathy suggest electrophysiological mechanisms for ventricular tachycardia
CardioExcyte 96 Publication in Biochemistry and Biophysics Reports (2024) Authors: Li Y., Liu S., Huang J., Xie Y., Hou A., Wei Y.

Left ventricular noncompaction cardiomyopathy (LVNC) is a cardiovascular disease characterized by arrhythmia and heart failure. In this study, LVNC myocardial samples were collected from patients who underwent heart transplantation and were analyzed using exome sequencing. Approximately half of the LVNC patients carried SCN5A variants, which are associated with clinical symptoms of ventricular tachycardia. To investigate the electrophysiological functions of these SCN5A variants and the underlying mechanism by which they increase arrhythmia susceptibility in LVNC patients, functional evaluations were conducted in CHO–K1 cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using patch-clamp or microelectrode array (MEA) techniques. These findings demonstrated that these SCN5A mutants exhibited gain-of-function properties, leading to increased channel activation and enhanced fast inactivation in CHO–K1 cells. Additionally, these mutants enhanced the excitability and contractility of the cardiomyocyte population in hESC-CMs models. All SCN5A variants induced fibrillation-like arrhythmia and increased the heart rate in cardiomyocytes. However, the administration of Lidocaine, an antiarrhythmic drug that acts on sodium ion channels, was able to rescue or alleviate fibrillation-like arrhythmias and secondary beat phenomenon. Based on these findings, it is speculated that SCN5A variants may contribute to susceptibility to arrhythmia in LVNC patients. Furthermore, the construction of cardiomyocyte models with SCN5A variants and their application in drug screening may facilitate the development of precise therapies for arrhythmia in the future.

Application Note PDF
Cardiomyocytes – Monitoring the effect of Triiodothyronine (T3) and Dexmethasone (Dex) on the calcium handling property of hiPSC-Cardiomyocytes
CardioExcyte 96 Application Note - Cells kindly provided by Bayer AG, and analysis software kindly provided by Hamamatsu

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) provide new avenues for disease modelling, drug discovery and cell therapy. However, structural and functional immaturity of these cells, poses a great challenge. Therefore, there is a highly unmet need for hiPSC-CMs with a mature phenotype. Recently, it has been reported that triiodothyronine (T3) and dexamethasone (Dex) play significant roles in hiPSCCMs maturation, by promoting the structural T-tubule development, enhancing the electrophysiological maturation and improving calcium handling ability.

Publication link
2024 – Revisiting the urease production of MICP-relevant bacterium Sporosarcina pasteurii during cultivation
CardioExcyte 96 Publication in Biocatalysis and Agricultural Biotechnology (2024) Authors: Lapierre FM. Huber R.

The ureolytic bacterium Sporosarcina pasteurii is commonly used for Microbial Induced Calcium Carbonate Precipitation (MICP). This process has a variety of applications in the fields of construction, geotechnical engineering, and environmental remediation. However, the factors influencing urease activity of S. pasteurii during cultivation are not yet fully understood. Even though there is debate over whether higher urease activity is in fact beneficial for MICP applications, knowledge about urease production can help provide ureolytic biomass more cost- effectively, reducing overall production costs. This study revisits the effect of various cultivation conditions on growth and urease activity by using an automated high-throughput microbioreactor system combined with a novel system for high-throughput urease activity quantification. This experimental set-up allows for unprecedented data density and enables new insights into urease production of S. pasteurii. Several factors were tested, including oxygen limitation, the feeding of urea, choice of ammonium salt, the proportion of ammonium salt in the medium, and lowering the pH during cultivation; none of these had a notable impact on urease production. Only the addition of a broad spectrum of nutrients through regular feeding with highly concentrated yeast extract and glucose led to a 70 % increased specific urease activity compared to a batch culture. These results can improve the understanding of the regulatory mechanisms governing urease expression in S. pasteurii during cultivation, and allow to adapt the cultivation process accordingly. Additionally, the new system for automated high-throughput enzyme activity determination presented in this study could be applied to optimize bioprocesses involving other ion producing enzymes.

 

Application protocol
Contractility Analysis of iCell Cardiomyocytes2 : Evaluating iCell Cardiomyocytes2 in co-culture with iCell Cardiac Fibroblasts on the FLEXcyte 96 System
CardioExcyte 96, FLEXcyte 96, collaboration with FUJIFILM Cellular Dynamics, Inc. and InnoVitro
Publication link
2023 – Berberine attenuates sunitinib-induced cardiac dysfunction by normalizing calcium regulation disorder via SGK1 activation
CardioExcyte 96 Publication in Food and Chemical Toxicology (2023) Authors: Li C., Wu W., Xing J., Yan W., Zhang J., Sun J., Zhang Z., Qiu Z., Xu Y., Wang X.

Sunitinib (SNT)-induced cardiotoxicity is associated with abnormal calcium regulation caused by phosphoinositide 3 kinase inhibition in the heart. Berberine (BBR) is a natural compound that exhibits cardioprotective effects and regulates calcium homeostasis. We hypothesized that BBR ameliorates SNT-induced cardiotoxicity by normalizing the calcium regulation disorder via serum and glucocorticoid-regulated kinase 1 (SGK1) activation. Mice, neonatal rat cardiomyocytes (NRVMs), and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to study the effects of BBR-mediated SGK1 activity on the calcium regulation disorder caused by SNT as well as the underlying mechanism. BBR offered prevention against SNT-induced cardiac systolic dysfunction, QT interval prolongation, and histopathological changes in mice. After the oral administration of SNT, the Ca2+ transient and contraction of cardiomyocytes was significantly inhibited, whereas BBR exhibited an antagonistic effect. In NRVMs, BBR was significantly preventive against the SNT-induced reduction of calcium transient amplitude, prolongation of calcium transient recovery, and decrease in SERCA2a protein expression; however, SGK1 inhibitors resisted the preventive effects of BBR. In hiPSC-CMs, BBR pretreatment significantly prevented SNT from inhibiting the contraction, whereas coincubation with SGK1 inhibitors antagonized the effects of BBR. These findings indicate that BBR attenuates SNT-induced cardiac dysfunction by normalizing the calcium regulation disorder via SGK1 activation.

 

Read more in the publication here.

Publication link
2023 – MFN2 deficiency promotes cardiac response to hypobaric hypoxia by reprogramming cardiomyocyte metabolism
CardioExcyte 96 Publication in Acta Physiologica (2023) Authors: Zhang R., Yang A., Zhang L., He L., Gu X., Yu C., Lu Z., Wang C., Zhou F., Li F., Ji L., Xing J., Guo H.

Aim

Under hypobaric hypoxia (HH), the heart triggers various defense mechanisms including metabolic remodeling against lack of oxygen. Mitofusin 2 (MFN2), located at the mitochondrial outer membrane, is closely involved in the regulation of mitochondrial fusion and cell metabolism. To date, however, the role of MFN2 in cardiac response to HH has not been explored.

Methods

Loss- and gain-of-function approaches were used to investigate the role of MFN2 in cardiac response to HH. In vitro, the function of MFN2 in the contraction of primary neonatal rat cardiomyocytes under hypoxia was examined. Non-targeted metabolomics and mitochondrial respiration analyses, as well as functional experiments were performed to explore underlying molecular mechanisms.

Results

Our data demonstrated that, following 4 weeks of HH, cardiac-specific MFN2 knockout (MFN2 cKO) mice exhibited significantly better cardiac function than control mice. Moreover, restoring the expression of MFN2 clearly inhibited the cardiac response to HH in MFN2 cKO mice. Importantly, MFN2 knockout significantly improved cardiac metabolic reprogramming during HH, resulting in reduced capacity for fatty acid oxidation (FAO) and oxidative phosphorylation, and increased glycolysis and ATP production. In vitro data showed that down-regulation of MFN2 promoted cardiomyocyte contractility under hypoxia. Interestingly, increased FAO through palmitate treatment decreased contractility of cardiomyocyte with MFN2 knockdown under hypoxia. Furthermore, treatment with mdivi-1, an inhibitor of mitochondrial fission, disrupted HH-induced metabolic reprogramming and subsequently promoted cardiac dysfunction in MFN2-knockout hearts.

Conclusion

Our findings provide the first evidence that down-regulation of MFN2 preserves cardiac function in chronic HH by promoting cardiac metabolic reprogramming.

 

Publication link
2023 – PO-01-173 Ibrutinib treatment results in increased phosphorylation of the ERK1/2 signaling pathway in atrial-specific human iPSC-derived cardiomyocytes
CardioExcyte 96 Publication in Heart Rhythm (2023) Authors: Fleming M., O'Neill M., Knollmann B., Moslehi J., Roden D.

Background

Cardiovascular sequelae of targeted cancer therapies may provide novel insights into cardiovascular biology and disease pathogenesis, including atrial fibrillation (AF). The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has revolutionized treatment for B-cell malignancies but increases the incidence of AF compared with conventional chemotherapy. Recent data in mouse models have shown that ibrutinib mediated-AF likely results not from inhibition of BTK (“on target” effect), but from off-target inhibition of a different kinase, C-terminal Src kinase (CSK). The signaling pathway by which CSK inhibition results in AF remains unknown and thus may represent a novel mechanism for AF molecular pathogenesis.

Objective

To test the hypothesis that ibrutinib generates atrial fibrillation in susceptible patients by targeting signaling pathways downstream from CSK.

Methods

We studied cellular monolayers of hiPSC-aCMs using extracellular field potential (EFP) recordings obtained by the Nanion CardioExcyte 96 system after 72-hour exposure to ibrutinib, the second-generation BTK inhibitor acalabrutinib, or vehicle. As an initial proteomic approach to determining which kinase signaling pathways are altered upon inhibition of CSK and hence may predispose to AF arrhythmogenesis, we used a human phospho-antibody array to study the relative phosphorylation state of 37 kinases in hiPSC-aCMs treated with ibrutinib or vehicle.

Results

EFP recordings from cellular monolayers of hiPSC-aCMs exposed to ibrutinib for 24 hours develop a striking increase in spontaneous beat-to-beat variability, an in vitro correlate of arrhythmogenic behavior. This effect was not seen with vehicle control or the second-generation BTK inhibitor acalabrutinib, which is less arrhythmogenic in clinical trials. Treatment with ibrutinib for 72 hours resulted in increased phosphorylation of multiple kinases in the Erk1/2 pathway, including Erk1 T202/Y204 and Erk2 T185/Y187, in human phospho-kinase array studies. Traditional Western Blotting confirmed these results and showed that ibrutinib, but not acalabrutinib, resulted in increased Erk1/2 phosphorylation.

Conclusion

Off-target inhibition of CSK by ibrutinib may result in increased AF through the ERK1/2 pathway, consistent with previous reports implicating increased phosphorylation in the ERK1/2 pathway in the pathogenesis of AF. The downstream mechanisms by which this occurs remain unknown and thus represents a novel potential target for AF therapeutics.
Read more in the publication here.
Publication link
2023 – Three tyrosine kinase inhibitors cause cardiotoxicity by inducing endoplasmic reticulum stress and inflammation in cardiomyocytes
CardioExcyte 96 Publication in BMC Medicine (2023) Authors: Wang H., Wang Y., Li J., He Z., Boswell S., Chung M., You F., Han S.

Background

Tyrosine kinase inhibitors (TKIs) are anti-cancer therapeutics often prescribed for long-term treatment. Many of these treatments cause cardiotoxicity with limited cure. We aim to clarify molecular mechanisms of TKI-induced cardiotoxicity so as to find potential targets for treating the adverse cardiac complications.

Methods

Eight TKIs with different levels of cardiotoxicity reported are selected. Phenotypic and transcriptomic responses of human cardiomyocytes to TKIs at varying doses and times are profiled and analyzed. Stress responses and signaling pathways that modulate cardiotoxicity induced by three TKIs are validated in cardiomyocytes and rat hearts.

Results

Toxicity rank of the eight TKIs determined by measuring their effects on cell viability, contractility, and respiration is largely consistent with that derived from database or literature, indicating that human cardiomyocytes are a good cellular model for studying cardiotoxicity. When transcriptomes are measured for selected TKI treatments with different levels of toxicity in human cardiomyocytes, the data are classified into 7 clusters with mainly single-drug clusters. Drug-specific effects on the transcriptome dominate over dose-, time- or toxicity-dependent effects. Two clusters with three TKIs (afatinib, ponatinib, and sorafenib) have the top enriched pathway as the endoplasmic reticulum stress (ERS). All three TKIs induce ERS in rat primary cardiomyocytes and ponatinib activates the IRE1α-XBP1s axis downstream of ERS in the hearts of rats underwent a 7-day course of drug treatment. To look for potential triggers of ERS, we find that the three TKIs induce transient reactive oxygen species followed by lipid peroxidation. Inhibiting either PERK or IRE1α downstream of ERS blocks TKI-induced cardiac damages, represented by the induction of cardiac fetal and pro-inflammatory genes without causing more cell death.

Conclusions

Our data contain rich information about phenotypic and transcriptional responses of human cardiomyocytes to eight TKIs, uncovering potential molecular mechanisms in modulating cardiotoxicity. ER stress is activated by multiple TKIs and leads to cardiotoxicity through promoting expression of pro-inflammatory factors and cardiac fetal genes. ER stress-induced inflammation is a promising therapeutic target to mitigate ponatinib- and sorafenib-induced cardiotoxicity.

Read more in the publication here.

Publication link
2023 – Omentin-1 drives cardiomyocyte cell cycle arrest and metabolic maturation by interacting with BMP7
CardioExcyte 96 Publication in Cell & Mol. Life Sci. (2023) Authors: Yang H., Song S., Li J., Li Y., Feng J., Sun Q., Qiu X., Chen Z., Bai X., Liu X., Lian H., Liu L., Bai Y., Zhang G., Nie Y.

Mammalian cardiomyocytes (CMs) undergo maturation during postnatal heart development to meet the increased demands of growth. Here, we found that omentin-1, an adipokine, facilitates CM cell cycle arrest and metabolic maturation. Deletion of omentin-1 causes mouse heart enlargement and dysfunction in adulthood and CM maturation retardation in juveniles, including delayed cell cycle arrest and reduced fatty acid oxidation. Through RNA sequencing, molecular docking analysis, and proximity ligation assays, we found that omentin-1 regulates CM maturation by interacting directly with bone morphogenetic protein 7 (BMP7). Omentin-1 prevents BMP7 from binding to activin type II receptor B (ActRIIB), subsequently decreasing the downstream pathways mothers against DPP homolog 1 (SMAD1)/Yes-associated protein (YAP) and p38 mitogen-activated protein kinase (p38 MAPK). In addition, omentin-1 is required and sufficient for the maturation of human embryonic stem cell-derived CMs. Together, our findings reveal that omentin-1 is a pro-maturation factor for CMs that is essential for postnatal heart development and cardiac function maintenance.

 

Publication link
2023 – D-galactose causes sinoatrial node dysfunction: from phenotype to mechanism
CardioExcyte 96 Publication in Aging (2023) Authors: Zhang H., Chen C., Liu Y., Chen W., Qi J., Xu Y., Ren Lu., Yang G., Min D., Liu Z., Cai X., Hao M., Xu G., Hou P.

With the population aging, age-related sinoatrial node dysfunction (SND) has been on the rise. Sinoatrial node (SAN) degeneration is an important factor for the age-related SND development. However, there is no suitable animal modeling method in this field. Here, we investigated whether D-galactose could induce SAN degeneration and explored the associated mechanism. In vivo, twelve C57BL/6 mice were divided into Control and D-galactose group to receive corresponding treatments. Senescence was confirmed by analyzing the hair and weight; cardiac function was evaluated through echocardiography, cerebral blood flux and serum-BNP; the SAN function was evaluated by electrocardiogram; fibrotic change was evaluated by Masson's trichrome staining and oxidative stress was assessed through DHE staining and serum indicators. Mechanism was verified through immunofluorescence-staining and Western blotting. In vitro, mouse-atrial-myocytes were treated with D-galactose, and edaravone was utilized as the ROS scavenger. Senescence, oxidative stress, proliferation ability and mechanism were verified through various methods, and intuitive evidence was obtained through electrophysiological assay. Finally, we concluded that D-galactose can be used to induce age-related SND, in which oxidative stress plays a key role, causing PITX2 ectopic expression and downregulates SHOX2 expression, then through the downstream GATA4/NKX2-5 axis, results in pacing-related ion channels dysfunction, and hence SND development.

 

Publication link
2023 – PTX3 from vascular endothelial cells contributes to trastuzumab-induced cardiac complications
CardioExcyte 96 Publication in Cardivasc. Res. (2023) Authors: Xu Z., Gao Z., Fu H., Zeng Y., Jin Y., Xu B., Zhang Y., Pan Z., Chen X., Zhang X., Wang X., Yan H., Yang X., Yang B., He Q., Luo P.
Aims

Trastuzumab, the first humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (ERBB2/HER2), is currently used as a first-line treatment for HER2 (+) tumours. However, trastuzumab increases the risk of cardiac complications without affecting myocardial structure, suggesting a distinct mechanism of cardiotoxicity.

Methods and results

We used medium from trastuzumab-treated human umbilical vein endothelial cells (HUVECs) to treat CCC-HEH-2 cells, the human embryonic cardiac tissue-derived cell lines, and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to assess the crosstalk between vascular endothelial cells (VECs) and cardiomyocytes. Protein mass spectrometry analysis was used to identify the key factors from VECs that regulate the function of cardiomyocytes. We applied RNA-sequencing to clarify the mechanism, by which PTX3 causes cardiac dysfunction. We used an anti-human/rat HER2 (neu) monoclonal antibody to generate a rat model that was used to evaluate the effects of trastuzumab on cardiac structure and function and the rescue effects of lapatinib on trastuzumab-induced cardiac side effects. Medium from trastuzumab-treated HUVECs apparently impaired the contractility of CCC-HEH-2 cells and iPSC-CMs. PTX3 from VECs caused defective cardiomyocyte contractility and cardiac dysfunction in mice, phenocopying trastuzumab treatment. PTX3 affected calcium homoeostasis in cardiomyocytes, which led to defective contractile properties. EGFR/STAT3 signalling in VECs contributed to the increased expression and release of PTX3. Notably, lapatinib, a dual inhibitor of EGFR/HER2, could rescue the cardiac complications caused by trastuzumab by blocking the release of PTX3.

Conclusion

We identified a distinct mode of cardiotoxicity, wherein the activation of EGFR/STAT3 signalling by trastuzumab in VECs promotes PTX3 excretion, which contributes to the impaired contractility of cardiomyocytes by inhibiting cellular calcium signalling. We confirmed that lapatinib could be a feasible preventive agent against trastuzumab-induced cardiac complications and provided the rationale for the combined application of lapatinib and trastuzumab in cancer therapy.

 

Case study PDF
Urease-positive bacteria – “Quantification of Urease Activity via High Throughput Conductivity Measurements”
2023 – Monitoring the Reversibility of GPCR Signaling by Combining Photochromic Ligands with Label-free Impedance Analysis
CardioExcyte 96 technology (custom-built system) Publication in Angewandte Chemie (2023) Authors: Wirth U., Erl J., Azzam S., Höring C., Skiba M., Singh R., Hochmuth K., Keller M., Wegener J., König B.

G protein-coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label-free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.

Webinar
12.09.2018 | Webinar: CiPA study: Bridging ion channel and myocyte data
Authors: Dr. Sonja Stölzle-Feix
CiPA myocyte phase II validation study results: cross-site comparison using the CardioExcyte 96;
HTS Phase I study: an update on progress of the CiPA Ion Channel Work Stream using the SyncroPatch 384PE and Patchliner

Get up-to-date with the CiPA progress of the Myocyte and Ion Channel Work Goups: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines.

Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed.Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values.

With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.

Webinar
12.09.2018 | Webinar: CiPA study: Bridging ion channel and myocyte data
Authors: Tim Strassmaier

Get up-to-date with the CiPA progress of the Myocyte and Ion Channel Work Goups: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines.

Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed.Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values.

With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.

Poster PDF
2015 – Complementary automated patch clamp, extracellular field potential and impedance recordings of iPSCs: safety screening tool box for the future
Webinar
2018 – HTS Phase I study: an update on progress of the CiPA Ion Channel Work Stream using the SyncroPatch 384PE and Patchliner
Presenter: Tim Strassmaier, Nanion Technologies Inc. USA, Source: Webinar: "CiPA study: Bridging ion channel and myocyte data", September 12, 2018
Title: CiPA myocyte phase II validation study results: cross-site comparison using the CardioExcyte 96

The CiPA HTS Ion Channel Working Group finalized its phase I study in 2017. Amongst other external sites, Nanion Technologies in Germany, USA and Japan participated with the Patchliner and the SyncroPatch 384PE in this study. A comparative view of the ion channel targets and a cross-platform and cross-site comparison will be presented. Furthermore, results from the myocyte Work Stream using arrhythmogenic compounds will be compared and confirmed with patch clamp data derived from the HTS Work Stream.

Please note: The original webinar presentation contained 8 slides with data of an upcoming publication. Due to confidentiality reasons, the relevant slides were cut out of the movie.

Poster PDF
2018 – Combining electrophysiology and contractility recordings for more complete assessment of hiPSC-CMs
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384), Patchliner and CardioExcyte 96 poster, Europhysiology Meeting 2018
Poster PDF
2016 – Next level toxicity screening: From single channel to overall cell behavior
Orbit mini, CardioExcyte 96 and SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, Meeting of the French Society of Toxinology (SFET) 2015 
Poster PDF
2018 – Optogenetic technologies enable high throughput ion channel drug discovery and toxicity screening
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) and CardioExcyte 96 poster, Biophysics Annual Meeting 2018
Presentation PDF
2018 – Innovations for cell monitoring in safety and toxicity assays
Presenter: Dr. Elena Dragicevic, Senior Scientist / Sales Manager, Nanion Technologies; Source: SOT, Ncardia & Nanion hosted session, March 12. 2018
Poster PDF
2018 – High throughput automatic patch clamp: Applications for Safety Pharmacology
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, JSPS Meeting 2018
Publication link
2015 – Novel screening techniques for ion channel targeting drugs
Patchliner, SyncroPatch 384PE (a predecessor model of SyncroPatch 384i) and CardioExcyte 96 Publication in Channels (2015) Authors: Obergrussberger A., Stölzle-Feix S., Becker N., Brüggemann A., Fertig N., Möller C.

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.

Publication link
2022 – Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation
FLEXcyte 96 and CardioExcyte 96 Publication in JoVe (2022) Authors: Lickiss B., Gossmann M., Linder P., Thomas U., Dragicevic E., Lemme M., George M., Fertig N., Stölzle-Feix S.

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform. The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling. The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

Application Note PDF
Cardiac Ion Channels – “High Throughput Screening of Cardiac Ion Channels”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384), Patchliner and CardioExcyte 96 application note   

In 2013 the Cardiac Safety Research Consortium (CSRC), the Health and Environmental Sciences Institute (HESI), and the US Food and Drug Administration (FDA) proposed a new paradigm to improve assessment of the proarrythmic risk of therapeutic compounds. Until now, drug safety testing has focussed on interaction with the hERG channel and QT prolongation which can lead to potentially fatal torsades de pointes (TdP). Although this approach has been largely successful in preventing new drugs reaching the market with unexpected potential to cause TdP, it is also possible that potentially valuable therapeutics have failed due to this early screening. The new paradigm, the Comprehensive In-vitro Proarrhythmia Assay (CiPA) was introduced to provide a more complete assessment of proarrythmic risk by evaluating and implementing currently available high throughput methods. An important part of this remains electrophysiological evaluation of not only hERG, but also other cardiac channels including NaV1.5, CaV1.2, KVLQT1 and Kir2.1. Additionally, new technologies, such as impedance measurements, and cells such as stem cell-derived cardiomyocytes, may provide useful tools for high throughput safety assessment. Here, we present high quality data with reliable pharmacology on hERG expressing CHO cells, NaV1.5, CaV1.2 or KVLQT1 expressed in HEK293 cells and Kir2.1 expressed in RBL cells on the SyncroPatch 384PE or Patchliner. Additionally, electrophysiological recordings on the Patchliner and Impedance measurements on the CardioExcyte 96 of stem cell-derived cardiomyocytes are shown

Application Note PDF
Cardiomyocytes – “Combining automated patch clamp, impedance and EFP of hiPSC-CMs”
CardioExcyte 96,  SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) and Patchliner Application Note    Cells kindly provided by Takara-Clonetech.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are gaining interest in cardiac safety screening. Given their recapitulation of native behavior, availability, ease of use and standardized production, they are likely to provide a viable alternative to acutely isolated cardiomyocytes to assess the pro-arrhythmic potentials of drug candidates. In 2013 the Comprehensive In-vitro Proarrhythmia Assay (CiPA) was introduced to provide a more complete assessment of pro- arrythmic risk by evaluating and implementing currently available high throughput methods and evaluating the potential use of hiPSC-CMs as a model  system for cardiac safety testing. Until now, drug safety testing has focussed on interaction with the hERG channel and QT prolongation which can lead to potentially fatal torsades de pointes (TdP). Although this approach has been largely successful in preventing new drugs reaching the market with unexpected potential to cause TdP, it is also possible that potentially valuable therapeutics have failed due to this early screening. The CiPA initiative has proposed an expansion of patch clamp assessment beyond hERG to include, e.g. NaV1.5 and CaV1.2. In addition, techniques such as multi-electrode array (MEA) and impedance are being thoroughly evaluated as complementary techniques to patch clamp. Here we present data recorded using the automated patch clamp platforms, the Patchliner, SyncroPatch 96 and SyncroPatch 384PE on Cellartis® Cardiomyocytes (Takara Bio Europe Cat nr. Y10075). Recordings of NaV1.5 and CaV1.2 are shown.  Impedance and EFP recordings were also performed using the CardioExcyte 96, and the effects of verapamil and sotalol are shown.

Poster PDF
2018 – A Sensor Based Technique for Pharmacological Safety Testing of Cardiac Transport Proteins NCX, NaKATPase and Respiratory Chain Complexes
CardioExcyte 96 and SUFE2R 96SE poster, SPS 2018
Webinar
17.03.2020 | Webinar: A new technology to evaluate mature cardiac contractility on high throughput level
The Webinar focuses on the FLEXcyte 96 technology, an Add-on of the CardioExcyte 96 instrument. The analysis of cardiomyocyte contractility as well as data of short-termed and long-termed compound applications is discussed.
Speakers:
Dr. Sonja Stölzle-Feix
Dr. Matthias Gossmann

Drug development is a costly and time-consuming process, with high drug failure rates both in early and late stages of the development process. Pre-clinical cardiac safety, toxicity and efficacy testing, usually performed using animal models with low predictive value or primary human cells, are one of the main reasons for high drug attrition rates.

To improve the drug development process, a suitable technology is required to acquire high quality data from physiologically relevant models on high throughput level. Standard cultivation methods for stem cell-derived cardiomyocytes are still based on stiff glass or plastic surfaces, creating an unphysiological environment to what cells would experience naturally and hinder them to further mature in vitro. In contrast, the FLEXcyte 96 plates mimic flexible mechanical conditions of real biological tissue and thereby enhancing the development of a mature cardiomyocyte phenotype which cannot be elicited with other assays commonly used. In combination with the FLEXcyte 96 platform, it is possible to analyze mature cardiac contractility on a 96 well high throughput level, both after acute and chronic compound treatment, ranging from 5 minutes to 5 days.

Hence, the FLEXcyte 96 system enables high throughput at lower costs and delivers highly predictive functional information on drug candidates early in the drug development process.

Product Sheet PDF
Cell Analytics – Interactive Brochure
Webinar
07.12.2021 | Webinar: Comprehensive Technologies for Quantitative Analysis of Cell Physiology
Speakers:
Ron Knox, PhD Scientist (Nanion Technologies) (University Hospital Regensburg; Germany)
Laurie A. Boyer, PhD Professor of Biology and Biological Engineering (Massachusetts Institute of Technology)

In this webinar we highlight impedance-based platforms (CardioExcyte 96 and FLEXcyte 96) and a high-throughput automated patch clamp (APC) instrument, the SyncroPatch 384.

The broad range and versatility of cell-based assays easily performed with these Automated Patch Clamp and cell monitoring systems, make them an excellent choice for integration into a core cell screening center/therapy/ biomanufacturing facility and traditional academic labs around the globe.

Presentation PDF
2018 – Innovations for cell monitoring in safety and toxicity assays
Presenter: Dr. Elena Dragicevic, Senior Scientist / Sales Manager, Nanion Technologies Source:
SOT, Ncardia & Nanion hosted session, March 12. 2018
Presentation PDF
2017 – Cross-site comparison of myocyte phase II compounds on different iPS cardiomyocytes based on CardioExcyte 96 recordings
Presenter: Dr. Corina Bot, Nanion Technologies, Inc. Source:
CiPA Cardiac stem cell working group webinar, September 20. 2017
Webinar
28.04.2015 | Webinar: Excited About Contraction – Combining Contractility and Excitability Measurements in Cardiotoxicity Screening
This Webinar covers the introduction of the CardioExcyte 96, a hybrid device for impedance- and MEA-type recordings from intact, beating networks of stem cell-derived cardiomyocytes.

Nanion invites you to a webinar on the CardioExcyte 96 - a hybrid device for impedance- and MEA-type recordings from intact, beating networks of stem cell-derived cardiomyocytes.
Combining impedance- and MEA-recordings increases the significance of toxicity screens as both the contractility and the electrical excitability of the cellular network are taken into account.

Presentation PDF
2018 – Optogenetic control of transiently transfected hiPSC-derived cardiomyocytes for the assessment of drug related cardiotoxicity
Presenter: Greg Luerman, PhD, Technical Director North America, Ncardia Source: SOT, Ncardia & Nanion hosted session, March 12. 2018
Webinar
13.10.2020 | Webinar: Profiling the pharmacology of G-Protein coupled receptors (GPCR) in cell-based assays using label-free impedance analysis
Speakers:
Michael Skiba (University Hospital Regensburg; Germany)
This is an on-demand webinar from Nan]i[on and Friends 2020.

G-protein coupled receptors (GPCRs) are among the most heavily addressed drug targets in medicinal chemistry and pharmacology. It has been estimated that about 40 % of all prescription pharmaceuticals on the market address GPCRs in different target tissues. The screening for new agonists or antagonists has been largely based on assays studying genetically engineered cells for the (i) potential binding of the ligand to their receptors or (ii) the production of second messengers upon receptor activation. Both approaches require invasive experimental procedures. Thus, they need to be performed as endpoint assays that do not reveal the time course of the cell response or details about intrinsic signal amplification. In contrast to that, non-invasive and label-free impedance monitoring has been developed over the last decades providing the response of target cells to receptor activation in real time. The technique is referred to as electric cell-substrate impedance sensing or short ECIS. In ECIS the cells are grown on planar gold-film electrodes that are integrated into regular cell culture dishes. Most recently, these electrode bearing dishes have been made commercially available in standard 96well format. The impedance of the cell-covered electrodes is measured with non-invasive electrical signals and reports on the cell response with a time resolution that is adjustable from minutes to milliseconds. This article will highlight several different approaches how non-invasive impedance measurements are used to characterize the pharmacology of GPCRs in cell-based assays comprising agonist assays, antagonist assays, dose-response relationships, signal transduction profiling and it will introduce a new dosing scheme that increases the experimental throughput significantly.

Webinar
2018 – CiPA myocyte phase II validation study results: cross-site comparison using the CardioExcyte 96
Presenter:
Dr. Sonja Stölzle-Feix, Nanion Technologies
Source:
Webinar: "CiPA study: Bridging ion channel and myocyte data", September 12, 2018

Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines.

Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed.Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values.

With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended

User meeting video
15.10.2020 | Webinar: Heart slice culture system reliably demonstrates clinical drug-related cardiotoxicity
Speakers: Dr. Tamer M. Mohamed (University of Louisville), This is an on-demand webinar from Nan]i[on and Friends 2020.

The limited availability of human heart tissue and its complex cell composition are major limiting factors for the reliable testing of drug efficacy and toxicity. Recently, we developed functional human and pig heart slice biomimetic culture systems that preserve the viability and functionality of 300 μm heart slices for up to 6 days. Here, we tested the reliability of this culture system for testing the cardiotoxicity of anti-cancer drugs. We tested three anti-cancer drugs (doxorubicin, trastuzumab, and sunitinib) with known different mechanisms of cardiotoxicity at three concentrations and assessed the effect of these drugs on heart slice viability, structure, function and gene expression. Slices incubated with any of these drugs for 48 h showed diminished in viability as well as loss of cardiomyocyte structure and function. Mechanistically, RNA sequencing of doxorubicin-treated tissues demonstrated a significant downregulation of cardiac genes and upregulation of oxidative stress responses. Trastuzumab treatment downregulated cardiac muscle contraction-related genes consistent with its clinically known effect on cardiomyocytes. Interestingly, sunitinib treatment resulted in significant downregulation of angiogenesis-related genes, in line with its mechanism of action. Similar to hiPS-derived-cardiomyocytes, heart slices recapitulated the expected toxicity of doxorubicin and trastuzumab, however, slices were superior in detecting sunitinib cardiotoxicity and mechanism in the clinically relevant concentration range of 0.1–1 μM. These results indicate that heart slice culture models have the potential to become a reliable platform for testing and elucidating mechanisms of drug cardiotoxicity.

Webinar
13.10.2020 | Webinar: Impedance measurements and Calcium imaging to investigate the role of store operated Ca2+ entry in GPCR-mediated endothelial barrier regulation
Speakers:
Dr. Judith Stolwijk (University Hospital Regensburg; Germany)
This is an on-demand webinar from Nan]i[on and Friends 2020.

The endothelium of our blood vessels is under the control of various G protein-coupled receptors (GPCRs), regulating endothelial barrier function, vascular tone, angiogenesis and inflammation. Some of these GPCRs signal via Gq,11 protein, which activates Ca2+-release from the IP3-sensitive internal stores of the endoplasmic reticulum (ER). Calcium store depletion can subsequently activate so-called store-operated Ca2+ entry (SOCE) via the ER-resident Ca2+-sensor stromal-interacting molecule 1 (STIM1) and Orai1 Ca2+ entry channels.

We used Impedance measurements and ratiometric Calcium imaging to investigate the role of calcium entry via Orai1 channels downstream of barrier-regulating GPCRs in response to their agonists Thrombin, Histamine and Sphingosine-1-Phosphate (S1P) in human endothelial cells.

Product Sheet PDF
CardioExcyte 96 – Product Sheet
Tutorial video
2020 – CardioExcyte 96 Tutorial Video
Short introduction of the CardioExcyte 96 (tutorial video, 2 minutes)
2017 – HTS in Cardiac Safety
Presenter: Dr. Sonja Stölzle-Feix, Nanion Technologies GmbH, Source: Satellite symposium during SPS, Berlin, September 22. 2017
Presentation PDF
2020 – Validation of a impedance-based phenotypic screening assay able to detect multiple mechanisms of chronic cardiotoxicity in human stem cell-derived cardiomyocytes
Presenter: Dr. Marc Rogers, CSO at Metrion Biosciences, United Kingdom

Source: The 64th Annual Meeting of the Biophysical Society, San Diego, CA (USA) Feb 15-19. 2020 About: Metrion Biosciences is a UK-based Contract Research Organization (CRO) focussed on delivering a range of high quality ion channel screening and drug discovery services. We provide sophisticated electrophysiology assays to support medicinal chemistry optimisation programmes, CiPA-compliant cardiac safety.

Poster PDF
2019 – High content in vitro cell monitoring effects of adjuvant chemotherapy in breast cancer and cancer treatment-related cardiomyopathy
Poster PDF
2021 – Cell Monitoring using Multi-Frequency Impedance Recordings for Label-free and Time-resolved Cell Response Analysis
Poster PDF
2018 – Assessment of Drug Effects on Cardiomyocyte Function: Comprehensive In Vitro Proarrhythmia Assay (CiPA) Results
Poster PDF
2018 – Investigations into idiosyncratic drug-induced hepatotoxicity and chronic proliferation of cancer cells using a label-free method
Flyer PDF
CardioExcyte 96 Flyer – Pacing
Flyer PDF
CardioExcyte 96 Flyer – SOL
Flyer PDF
CardioExcyte 96 – Application Areas
Flyer PDF
CardioExcyte 96 / FLEXcyte 96 Product Flyer – Plates
Application Note PDF
Hepatocytes – Toxicity analysis in expanded upcyte® liver cells on Nanion’s CardioExcyte 96
CardioExcyte 96 Application Note: Cells were kindly provided by upcyte technologies

Drug induced hepatic toxicity is one of the main reasons for regulatory actions and market withdrawals in the last 50 years [1]. In Europe and the USA, the major cause of acute liver failure is indeed Drug Induced Liver Injury (DILI) [2]. Current existing in vitro models employed to predict DILI mostly focus on hepatocytes. Other cell types of the liver, such as liver sinusoidal endothelial cells (LSECs), are often overlooked. It is thus questionable whether, hepatotoxicity can be sufficiently predicted by analyzing hepatocytes only. LSECs are highly specialized endothelial cells forming the hepatic sinusoidal wall. Besides their high endocytic potential, LSECs have been demonstrated to markedly contribute to liver homeostasis, immunity, and may partially explain unexpected hepatotoxicity of selected drug candidates. Several reports in the literature have highlighted a high sensitivity of LSECs towards hepatotoxic drugs [3]. It has been suggested that LSECs act as an early direct target for acetaminophen (paracetamol) induced toxicity, causing early swelling and loss of uptake activity and fenestrations before effects on hepatocytes are observed [4]. LSECs are further important cellular targets during sinusoidal obstruction syndrome (a distinctive and potentially fatal form of hepatic injury that occurs predominantly, if not only, after drug or toxin exposure). The use of primary LSECs for comprehensive in vitro studies is compromised by poor cell yields, rapid dedifferentiation, contamination with other endothelial cells, and limited proliferation after isolation [5]. In this study upcyte® LSECs have been developed as a complementary tool to predict hepatotoxicity, uptake and drug interactions. In addition to conventional toxicity readouts such as ATP levels, upcyte® LSECs have been successfully tested using an impedance based system.

Application Note PDF
hiPSC-CM – “Conduction velocity with the 2-electrode layout of the CardioExcyte 96”
CardioExcyte 96 Application Note: Cardiosight®-S hiPSC-CMs kindly provided by Nexel

Cardiac conduction is the process by which electrical excitation spreads through the heart, triggering individual myocytes to contract synchronously. Slowed cardiac conduction velocity (CV) is associated with an increased risk of re-entrant excitation, leading to a pre-disposition to life-threatening arrhythmias1. CV is determined by the ion channel properties of cardiac myocytes and by their interconnections. It is strictly dependent on the maximum upstroke velocity of an action potential, which is determined by the sodium current2. In addition, gap junctions play a key role because they ultimately determine how much depolarizing sodium current passes from excited to non-excited regions of the network. Uncoupling of gap junctions causes discontinuities leading to slower CV. Defective intercellular coupling between the cardiomyocytes results in increased subthreshold depolarization, which slowly inactivates the voltage-gated sodium channels, further reducing the sodium current and excitability3.In the case of collagenous scar tissue the uncoupling of myocyte–myocyte connections and subsequent coupling of myocytes with fibroblasts impairs the electrical conduction. In fact, collagen deposition results in electrically isolated fibers of viable myocardium, discontinuing the conduction path and globally reducing the action potential propagation velocity and consequently promoting the onset of re-entrant arrhythmias4. Since CV plays a pivotal role in cardiovascular diseases, it is essential to investigate the effect of a new compound on the cardiac CV.In this study, we have investigated the effect of the sodium channel blocker lidocaine (30 and 100 μM) on CV in hiPSC-CMs (NEXEL Cardiosight®-S) using the CardioExcyte 96.

Application Note PDF
Hepatocytes – “Investigating DILI using MetaHeps cells on Nanion’s CardioExcyte 96”
CardioExcyte 96 Application Note: Cells were kindly provided by Metaheps GmbH.  

Drug Induced Liver Injury (DILI) is the major cause of acute liver failure in the USA and Europe and is one of the main reasons for regulatory actions and market withdrawals. Indeed, hepatic toxicity has accounted for 15 of the 47 drugs withdrawn from the market in the period 1975 - 2007. DILI is classified as intrinsic or dosedependent, acetaminophen (paracetamol) being the most important example of this class, or idiosyncratic which is unpredictable and not directly dependent on dose.  A number of factors contribute to an individual’s susceptibility to develop idiosyncratic DILI including age, sex, alcohol consumption, drug interactions and genetic variability. Although improvements have been made to cellular and animal models to predict intrinsic (dose-dependent) DILI, it is almost impossible to predict idiosyncratic DILI. Withdrawal of compounds at a late stage (or postmarketing) due to idiosyncratic DILI is costly and can lead to incorrect withdrawal of potentially useful compounds. Monocyte-derived hepatocyte-like (MH) cells have been developed as a tool to investigate longterm hepatotoxicity, metabolism and drug interactions. Furthermore, patient-derived MH cells could provide a  tool for diagnosis or exclusion of idiosyncratic DILI and provide the causative agent in polymedicated patients.In this study, MH cells (MetaHeps®) were used on the CardioExcyte 96 and changes in impedance, and therefore confluency, were used as a measure of toxicity. Intrinsic (dose-dependent) effects of paracetamol could be identified consistent with other methods of liver injury detection. Therefore, the CardioExcyte 96 in combination with patient-specific MH cells provides a novel tool for investigating intrinsic and idiosyncratic DILI.

Application Note PDF
Cardiotoxicity – “Assessing cardiotoxic risk of anti-cancer agents on Nanion’s CardioExcyte 96”
CardioExcyte 96 Application Note: Cells were kindly provided by Ncardia and experiments performed by Oliver Reinhardt and Dr. Frauke Alves from the University of Göttingen.

New anticancer agents have led to higher life expectancy for patients surviving cancer. However, treatment related morbidity factors such as cardiac toxicity have become important issues for long-term cancer survivors. Cardiotoxic side effects such as arrhythmia, thromboembolism and myocardial ischemia are common with anti-cancer drugs such as the anthracyclines. This has led to the need for a sub-speciality of medicine, cardio-oncology or oncocardiology, to promote cardiovascular health whilst providing the best therapy to fight cancer. It is important to be able to assess the cardiotoxic risk of new and existing cancer therapies in order to facilitate effective cardiovascular health during chemotherapy. In addition, advances in human stem cell derived cardiomyocytes (hiPSC-CMs) and, indeed, patient-derived hiPSC-CMs offers new possibilities for personalized medicine, being able to assess a patient’s risk of developing cardiovascular complications based on their own cells, thus taking into account their own genetic factors. Using the measurement of electrical impedance coupled with human stem cell-derived cardiomyocytes (hSC-CMs) we could confirm the cardiotoxic effects of paclitaxel, also known as Taxol, a microtuble stabilizing drug approved for the treatment of breast, ovarian and lung cancer. In addition, we investigated different combinations of cylophosphamide (CP), doxorubicin (DOX) and 5-Fluorouracil (5F) and found that any combination which included DOX was highly cardiotoxic.

Application Note PDF
CytoSwitch – “Inducing cell death using a photostatin on the CardioExcyte 96 and SOL”
CardioExcyte 96 and SOL Application Note: Cells were kindly provided by LMU Munich/CytoSwitch.

Microtubules are important for cell support, acting as a kind of internal scaffold providing both shapeand an organized structure. Another important role of microtubules is the formation of the mitotic spindle which is critical in cell division. For this reason,compounds which control microtubule dynamics such as Taxol and Vinca alkaloids are important as anti-cancer therapeutics and biological research tools. Other compounds e.g. combretastin A-4 (CA4), have been shown to be powerful inhibitors of tubule polymerization. Until now, the use of such compounds in both biological research and as anti-cancer agentshas been limited because of their non-specificity; their bioactivity cannot be spatially or temporally directed,e.g. against particular tissues or cells. As an anti-cancer agent, CA4 was taken into Phase III clinical trials but was discontinued due to the cardio- or neurotoxic side effects at high doses as healthy organs were affected. The use of light to control the activity of microtubule inhibitors may offer a solution to combat the specificity problem by controlling the activation of the compound using visible light in a spatial, temporal and reversible fashion. The CytoSwitch startup project at the LMU Munich is developing photostatins as anti-cancer agents with reduced side-effects. Furthermore, CytoSwitch will commercialize photostatins as research tools for biological research.

Application Note PDF
Cardiomyocytes – “Impedance and EFP recordings of Cor.4U cells using Nanion’s CardioExcyte 96”
CardioExcyte 96 Application Note: Cells were kindly provided by Ncardia.  

Cardiomyocytes derived from human inducedpluripotent stem cells (hiPSCs) are gaining interest in cardiac safety screening. Given their relative abundance, availability, ease of use and standardized production, they are likely to provide a viable alternative to acutely isolated cardiomyocytes to assess the pro-arrythmic potentials of drug candidates. Although automated patch clamp can provide excellent information about the effects of compounds on cardiac ion channels and possible effects on the cardiac action potential, other techniques, such as impedance, also provide crucial and complementary information about complex physiological parameters such as beat rate, amplitude and duration. The CardioExcyte 96 is a new hybrid screening tool combining impedance (cell contractility) and extracellular field potential (EFP) recordings. These measurements are non-invasive, label-free and have a temporal resolution of 1 ms. The recordings are made from cells within a network thus providing a physiologically relevant environment for measuring drug-induced changes in beat parameters. This hybrid technology addresses the lack of easy-to-use high-throughput screening for in-vitro assays, and permits the reliable investigation of short- and long-term pharmacological effects. Here we present data recorded on the CardioExcyte 96 using Cor.4U hiPSCs from Axiogenesis. The effects of the compounds blebbistatin, E4031 and nifedipine on the impedance and EFP signals are shown.

Application Note PDF
Cardiomyocytes – “Impedance and EFP recordings of iCell Cardiomyocytes² on the CardioExcyte 96”
CardioExcyte 96 Application Note: Cells were kindly provided by Cellular Dynamics.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) are gaining interest in cardiac safety screening. Given their recapitulation of native behavior, availability, ease of use and standardized production, they are likely to provide a viable alternative to acutely isolated cardiomyocytes to assess the pro-arrhythmic potentials of drug candidates. Although automated patch clamp can provide excellent information about the effects of compounds on cardiac ion channels and possible effects on the cardiac action potential, other outputs such as extracellular field potential (EFP) and impedance, also provide crucial and complementary information about complex physiological parameters such as beat rate, amplitude and duration. The CardioExcyte 96 is a new hybrid screening tool combining impedance (cell contractility) and EFP recordings. These measurements are non-invasive, label free and have a temporal resolution of 1 ms. The recordings are made from cells within a network thus providing a physiologically relevant environment for measuring drug-induced changes in contractileparameters. This hybrid technology is a high-throughput screening tool which permits the reliable investigation of short- and long-term pharmacological effects.Here we present data recorded on the CardioExcyte 96 using iCell® Cardiomyocytes2 from Cellular Dynamics International (CDI). When handled according to the manufacture’s instructions, impedance and EFP signals were stable 4 days afterplating and compound effects could be robustly measured and analyzed.

Publication link
2022 – The potential of remdesivir to affect function, metabolism and proliferation of cardiac and kidney cells in vitro
CardioExcyte 96 Publication in Organ Toxicity and Mechanisms (2022) Authors: Merches K., Breunig L., Fender J., Brand T., Bätz V., Idel S., Kollipara L., Reinders Y., Sickmann A., Mally A., & Lorenz K.

Remdesivir is a prodrug of a nucleoside analog and the first antiviral therapeutic approved for coroNaVirus disease. Recent cardiac safety concerns and reports on remdesivir-related acute kidney injury call for a better characterization of remdesivir toxicity and understanding of the underlying mechanisms. Here, we performed an in vitro toxicity assessment of remdesivir around clinically relevant concentrations (Cmax 9 µM) using H9c2 rat cardiomyoblasts, neonatal mouse cardiomyocytes (NMCM), rat NRK-52E and human RPTEC/TERT1 cells as cell models for the assessment of cardiotoxicity or nephrotoxicity, respectively. Due to the known potential of nucleoside analogs for the induction of mitochondrial toxicity, we assessed mitochondrial function in response to remdesivir treatment, early proteomic changes in NMCM and RPTEC/TERT1 cells and the contractile function of NMCM. Short-term treatments (24 h) of H9c2 and NRK-52E cells with remdesivir adversely affected cell viability by inhibition of proliferation as determined by significantly decreased 3H-thymidine uptake. Mitochondrial toxicity of remdesivir (1.6–3.1 µM) in cardiac cells was evident by a significant decrease in oxygen consumption, a collapse of mitochondrial membrane potential and an increase in lactate secretion after a 24–48-h treatment. This was supported by early proteomic changes of respiratory chain proteins and intermediate filaments that are typically involved in mitochondrial reorganization. Functionally, an impedance-based analysis showed that remdesivir (6.25 µM) affected the beat rate and contractility of NMCM. In conclusion, we identified adverse effects of remdesivir in cardiac and kidney cells at clinically relevant concentrations, suggesting a careful evaluation of therapeutic use in patients at risk for cardiovascular or kidney disease.

Application Note PDF
Cardiomyocytes – “Impedance and EFP recordings of Pluricyte Cardiomyocytes on the CardioExcyte 96”
CardioExcyte 96 Application Note: Cells were kindly provided by Ncardia.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) are gaining interest in cardiac safety screening. Given their recapitulation of native behavior, availability, ease of use and standardized production, they are likely to provide a viable alternative to acutely isolated cardiomyocytes to assess the pro-arrhythmic potentials of drug candidates. Although automated patch clamp can provide excellent information about the effects of compounds on cardiac ion channels and possible effects on the cardiac action potential, other outputs such as extracellular field potential (EFP) and impedance, also provide crucial and complementary information about complex physiological parameters such as beat rate, amplitude and duration. The CardioExcyte 96 is a hybrid screening tool combining impedance (cell contractility) and EFP recordings. These measurements are non-invasive, label-free and have a temporal resolution of 1 ms. The recordings are made from cells within a network thus providing a physiologically relevant environment for measuring drug-induced changes in contractileparameters. This hybrid technology is an easy-to-use screening tool which permits the reliable investigation of short- and long-term pharmacological effects. Here we present data recorded on the CardioExcyte 96 using Pluricyte® Cardiomyocytes from Ncardia. The effects of the CaV blocker, nifedipine, hERG blockers, dofetilide and E4031, and NaV blocker, mexiletine, on EFP and impedance parameters are shown.

Publication link
2022 – NRF-2/HO-1 Pathway-Mediated SHOX2 Activation Is a Key Switch for Heart Rate Acceleration by Yixin-Fumai Granules
CardioExcyte 96 Publication in Oxidative Medicine and Cellular Longevity (2022) Authors: Zhang H., Chen C., Liu Y., Ren L., Qi J., Yang Y., Chen W., Yao Y., Cai X., Liu Z., Hao M., Li L., Deng Z., Sun M., Lu Y., Chen K., Hou P.

Population aging has led to increased sick sinus syndrome (SSS) incidence; however, no effective and safe medical therapy has been reported thus far. Yixin-Fumai granules (YXFMs), a Chinese medicine granule designed for bradyarrhythmia treatment, can effectively increase SSS patients’ heart rate. Senescence-induced sinoatrial node (SAN) degeneration is an important part of SSS pathogenesis, and older people often show high levels of oxidative stress; reactive oxygen species (ROS) accumulation in the SAN causes abnormal SAN pacing or conduction functions. The current study observed the protective effects of YXFMs on senescent SAN and explored the relationship between the NRF-2/HO-1 pathway, SHOX2, and T-type calcium channels. We selected naturally senescent C57BL/6 mice with bradycardia to simulate SSS; electrocardiography, Masson’s trichrome staining, and DHE staining were used to assess SAN function and tissue damage. Immunofluorescence staining and Western blotting were used to assay related proteins. In vitro, we treated human-induced pluripotent stem cell-derived atrial myocytes (hiPSC-AMs) and mouse atrial myocyte-derived cell line HL-1 with D-galactose to simulate senescent SAN-pacemaker cells. CardioExcyte96 was used to evaluate the pulsatile function of the hiPSC-AMs, and the mechanism was verified by DCFH-DA, immunofluorescence staining, RT-qPCR, and Western blotting. The results demonstrated that YXFMs effectively inhibited senescence-induced SAN hypofunction, and this effect possibly originated from sCaVenging of ROS and promotion of NRF-2, SHOX2, and T-type calcium channel expression. In vitro experiment results indicated that ML385, si-SHOX2, LDN193189, and Mibefradil reversed YXFMs’ effects. Moreover, we, for the first time, found that ROS accumulation may hinder SHOX2 expression; YXFMs can activate SHOX2 through the NRF-2/HO-1 pathway-mediated ROS sCaVenging and then regulate CACNA1G through the SHOX2/BMP4/GATA4/NKX2-5 axis, improve T-type calcium channel function, and ameliorate the SAN dysfunction. Finally, through network pharmacology and molecular docking, we screened for the most stable YXFMs compound that docks to NRF-2, laying the foundation for future studies.

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2022 – The G213D variant in NaV1.5 alters sodium current and causes an arrhythmogenic phenotype resulting in a multifocal ectopic Purkinje-related premature contraction phenotype in human-induced pluripotent stem cell-derived cardiomyocytes
CardioExcyte 96 Publication in EP Europace (2022) Authors: Calloe K., Geryk M., Freude K., Treat J.A., Vold V.A., Frederiksen H.R.S., Broendberg A.K., Frederiksen T.C., Jensen H.K., Cordeiro J.M.

Variants in SCN5A encoding NaV1.5 are associated with cardiac arrhythmias. We aimed to determine the mechanism by which c.638G>A in SCNA5 resulting in p.Gly213Asp (G213D) in NaV1.5 altered Na+ channel function and how flecainide corrected the defect in a family with multifocal ectopic Purkinje-related premature contractions (MEPPC)-like syndrome. Methods and results Five patients carrying the G213D variant were treated with flecainide. Gating pore currents were evaluated in Xenopus laevis oocytes. The 638G>A SCN5A variant was introduced to human-induced pluripotent stem cell (hiPSC) by CRISPR–Cas9 gene editing and subsequently differentiated to cardiomyocytes (hiPSC-CM). Action potentials and sodium currents were measured in the absence and presence of flecainide. Ca2+ transients were measured by confocal microscopy. The five patients exhibited premature atrial and ventricular contractions which were suppressed by flecainide treatment. G213D induced gating pore current at potentials negative to −50 mV. Voltage-clamp analysis in hiPSC-CM revealed the activation threshold of INa was shifted in the hyperpolarizing direction resulting in a larger INa window current. The G213D hiPSC-CMs had faster beating rates compared with wild-type and frequently showed Ca2+ waves and alternans. Flecainide applied to G213D hiPSC-CMs decreased window current by shifting the steady-state inactivation curve and slowed the beating rate. Conclusion The G213D variant in NaV1.5 induced gating pore currents and increased window current. The changes in INa resulted in a faster beating rate and Ca2+ transient dysfunction. Flecainide decreased window current and inhibited INa, which is likely responsible for the therapeutic effectiveness of flecainide in MEPPC patients carrying the G213D variant.

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2022 – How Carvedilol activates β2-adrenoceptors
CardioExcyte 96 Publication in Nature Communications (2022) Authors: Benkel T., Zimmermann M., Zeiner J., Bravo S., Merten N., Jun Yu Lim V., Fabienne Matthees E., Drube J., Miess-Tanneberg E., Malan D., Szpakowska M., Monteleone S., Grimes J., Koszegi Z., Lanoiselée Y., O’Brien S., Pavlaki N., Dobberstein N., Inoue A., Nikolaev V., Calebiro D., Chevigné A., Sasse P., Schulz S., Hoffmann C., Kolb P., Waldhoer M., Simon K., Gomeza J., Kostenis E.

Carvedilol is among the most effective β-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of β1-adrenoceptors, arrestin-biased signalling via β2-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol’s cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through β2ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the β-adrenoceptor system.

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2022 – Hypoglycemia-Exacerbated Mitochondrial Connexin 43 Accumulation Aggravates Cardiac Dysfunction in Diabetic Cardiomyopathy
CardioExcyte 96 prePublication in Frontiers in Cardiovascular Medicine (2022) Authors: Wei X., Chang A.C.H., Chang H., Xu S., Xue Y., Zhang Y., Lei M., Chang A.C.Y., Zhang Q.

Diabetic cardiomyopathy (DCM) is a complex multifaceted disease responsible for elevated heart failure (HF) morbidity and mortality in patients with diabetes mellitus (DM). Patients with DCM exhibit subclinical diastolic dysfunction, progression toward systolic impairment, and abnormal electrophysiology. Hypoglycemia events that occur spontaneously or due to excess insulin administration threaten the lives of patients with DM—with the increased risk of sudden death. However, the molecular underpinnings of this fatal disease remain to be elucidated.

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2021 – Towards engineering heart tissues from bioprinted cardiac spheroids
CardioExcyte 96 Publication in IOP Science (2021) Authors: Polonchuk L., Surija L., Lee M.H., Sharma P, Ming C.L.C., Richter F., Ben-Sefer E., Rad M.A., Sarmast H.M.S., Shamery W.A.,Tran H.A., Vettori L., Haeusermann F., Filipe E.C., Rnjak-Kovacina J., Cox T., Tipper J., Kabakova I., and Gentile C.

Current in vivo and in vitro models fail to accurately recapitulate the human heart microenvironment for biomedical applications. This study explores the use of cardiac spheroids (CSs) to biofabricate novel and advanced physiological cardiac models for in vitro testing. CSs were created from human cardiac myocytes, fibroblasts and endothelial cells, mixed within optimal alginate/gelatin (Al/Ge) hydrogels and then bioprinted on a microelectrode plate for drug testing. Bioprinted CSs maintained their structure and viability for at least 30 days after printing. Vascular endothelial growth factor (VEGF) promoted endothelial cell branching from CSs within hydrogels. Alginate/gelatin-based hydrogels enabled spheroids fusion, which was further facilitated by addition of VEGF. Bioprinted CSs contracted spontaneously and under stimulation, allowing to record contractile and electrical signals on the microelectrode plates for industrial applications. Taken together, our findings indicate for the first time that bioprinted CSs within Al/Ge hydrogels can be used to biofabricate durable, viable and functional human heart tissues for long term in vitro testing.

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2021 – Transient Cell Cycle Induction in Cardiomyocytes to Treat Ischemic Heart Failure
CardioExcyte 96 prePublication in Research Square (2021) Authors: Abouleisa R., Ou Q., Tang X-L., Solanki M., Guo Y., Nong Y., Mcnally L., Lorkiewicz P., Kassem K., Ahern B., Choudhary K., Thomas R., Huang Y., Juhardeen H., Siddique A., Ifthikar Z., Salama A-B., Hammad S., Elbaz A., Ivey K., Satin J., Hill B., Srivastava D., Bolli R., Mohamed T.

The regenerative capacity of the heart after myocardial infarction (MI) is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 and Cdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in 15-20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after MI. Here, we aim to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Also, we aim to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure. Temporal bulk and single-cell RNAseq and further biochemical validations of mature hiPS-CMs treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population after 48 h post-infection with 4F, which was associated with sarcomere disassembly and metabolic reprogramming. Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic non-integrating lentivirus (NIL) encoding the 4F; each is driven by a TNNT2 promoter (TNNT2-4F-NIL). TNNT2-4F-NIL or control virus was injected intramyocardially one week after MI in rats or pigs. TNNT2-4F-NIL treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus treated animals four weeks post-injection. In conclusion, the present study provides a mechanistic demonstration of the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors using a novel transient and cardiomyocyte-specific viral construct.

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2021 – Sex-Specific Control of Human Heart Maturation by the Progesterone Receptor
CardioExcyte 96 Publication in Circulation (2021) Authors: Sim C.B., Phipson B., Zienabb N., Rafehi H., Mills R.J., Watt K.I., Abu-Bonsrah K.D., Kalathur R.K.R., Voges H.K., Dinh D.T., ter Huurne M.,Vivien C.J., Kaspi A., Kaipanaickal H., Hidalgo A., Delbridge L.M.D., Robker R.L., Gregorevic P., dos Remedios C.G., Lal S., Piers A.T.,  Konstantinov I.E., Elliott D.A., El-Osta A., Oshlack A., Hudson J.E., Porrello E.R.

Background: Despite in-depth knowledge of the molecular mechanisms controlling embryonic heart development, little is known about the signals governing postnatal maturation of the human heart. Methods: Single nucleus RNA-sequencing (snRNA-seq) of 54,140 nuclei from 9 human donors was used to profile transcriptional changes in diverse cardiac cell types during maturation from fetal stages to adulthood. Bulk RNA-sequencing and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) were used to further validate transcriptional changes and to profile alterations in the chromatin accessibility landscape in purified cardiomyocyte nuclei from 21 human donors. Functional validation studies of sex steroids implicated in cardiac maturation were performed in human pluripotent stem cell-derived cardiac organoids and mice. Results: Our data identify the progesterone receptor as a key mediator of sex-dependent transcriptional programs during cardiomyocyte maturation. Functional validation studies in human cardiac organoids and mice demonstrate the progesterone receptor drives sex-specific metabolic programs and maturation of cardiac contractile properties. Conclusions: These data provide a blueprint for understanding human heart maturation in both sexes and reveal an important role for the progesterone receptor in human heart development.

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2021 – Targeting of CAT and VCAM1 as Novel Therapeutic Targets for DMD Cardiomyopathy
CardioExcyte 96 Publication in Cell & Development Biology (2021) Authors: Li B., Xiong W., Liang W-M., Chiou J-S., Lin Y-J., Chang A.C.Y.

Duchenne muscular dystrophy (DMD) related cardiomyopathy is the leading cause of early mortality in DMD patients. There is an urgent need to gain a better understanding of the disease molecular pathogenesis and develop effective therapies to prevent the onset of heart failure. In the present study, we used DMD human induced pluripotent stem cells (DMD-hiPSCs) derived cardiomyocytes (CMs) as a platform to explore the active compounds in commonly used Chinese herbal medicine (CHM) herbs. Single CHM herb (DaH, ZK, and CQZ) reduced cell beating rate, decreased cellular ROS accumulation, and improved structure of DMD hiPSC-CMs. Cross-comparison of transcriptomic profiling data and active compound library identified nine active chemicals targeting ROS neutralizing Catalase (CAT) and structural protein vascular cell adhesion molecule 1 (VCAM1). Treatment with Quecetin, Kaempferol, and Vitamin C, targeting CAT, conferred ROS protection and improved contraction; treatment with Hesperidin and Allicin, targeting VCAM1, induced structure enhancement via induction of focal adhesion. Lastly, overexpression of CAT or VCAM1 in DMD hiPSC-CMs reconstituted efficacious effects and conferred increase in cardiomyocyte function. Together, our results provide a new insight in treating DMD cardiomyopathy via targeting of CAT and VCAM1, and serves as an example of translating Bed to Bench back to Bed using a muti-omics approach.

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2021 – Long noncoding RNA VENTHEART is required for cardiomyocyte specification and function
CardioExcyte 96 pre-print Publication in bioRxiv (2021) Authors: Dashi A., Tan W.L.W., Anene-Nzelu C.G., Pan B., Ilmari A.M., Tiang Z., Hartman R.J.G., Stenzig J., Wei H., Gao Bin C., Ackers-Johnson M.A., Lim B., Walentinsson A., Iyer V.V., Jonsson M.K.B., Foo R.S.

Long noncoding RNAs (lncRNAs) control cardiac gene expression during heart development and disease. It is accordingly plausible for the same lncRNA to regulate both cardiac development, as well as play a role in adult heart disease progression. lncRNA regulators of early cardiomyocyte (CM) lineage commitment have been identified and characterised, however those controlling later CM specification remain unknown.

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2021 – Overlap Arrhythmia Syndromes Resulting from Multiple Genetic Variations Studied in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
CardioExcyte 96 Publication in International Journal of Molecular Sciences (2021) Authors: Treat J.A., Pfeiffer R., Barajas-Martinez H., Goodrow R.J., Bot C., Haedo R.J., Knox R., Cordeiro J.M.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for genetic models of cardiac diseases. We report an arrhythmia syndrome consisting of Early Repolarization Syndrome (ERS) and Short QT Syndrome (SQTS). The index patient (MMRL1215) developed arrhythmia-mediated syncope after electrocution and was found to carry six mutations. Functional alterations resulting from these mutations were examined in patient-derived hiPSC-CMs. Electrophysiological recordings were made in hiPSC-CMs from MMRL1215 and healthy controls. ECG analysis of the index patient showed slurring of the QRS complex and QTc = 326 ms. Action potential (AP) recordings from MMRL1215 myocytes showed slower spontaneous activity and AP duration was shorter. Field potential recordings from MMRL1215 hiPSC-CMs lack a “pseudo” QRS complex suggesting reduced inward current(s). Voltage clamp analysis of ICa showed no difference in the magnitude of current. Measurements of INa reveal a 60% reduction in INa density in MMRL1215 hiPSC-CMs. Steady inactivation and recovery of INa was unaffected. mRNA analysis revealed ANK2 and SCN5A are significantly reduced in hiPSC-CM derived from MMRL1215, consistent with electrophysiological recordings. The polygenic cause of ERS/SQTS phenotype is likely due to a loss of INa due to a mutation in PKP2 coupled with and a gain of function in IK,ATP due to a mutation in ABCC9.

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2021 – Cardiac Safety of Kinase Inhibitors – Improving Understanding and Prediction of Liabilities in Drug Discovery Using Human Stem Cell-Derived Models
CardioExcyte 96 prePublication in Frontiers in Cardiovascular Medicine (2021) Authors: Ziegler R., Häusermann F., Kirchner S., Polonchuk L

Many small molecule kinase inhibitors (SMKIs) used to fight cancer have been associated with cardiotoxicity in the clinic. Therefore, preventing their failure in clinical development is a priority for preclinical discovery. Our study focused on the integration and concurrent measurement of ATP, apoptosis dynamics and functional cardiac indexes in human stem cell-derived cardiomyocytes (hSC-CMs) to provide further insights into molecular determinants of compromised cardiac function. Ten out of the fourteen tested SMKIs resulted in a biologically relevant decrease in either beating rate or base impedance (cell number index), illustrating cardiotoxicity as one of the major safety liabilities of SMKIs, in particular of those involved in the PI3K–AKT pathway. Pearson's correlation analysis indicated a good correlation between the different read-outs of functional importance. Therefore, measurement of ATP concentrations and apoptosis in vitro could provide important insight into mechanisms of cardiotoxicity. Detailed investigation of the cellular signals facilitated multi-parameter evaluation allowing integrative assessment of cardiomyocyte behavior. The resulting correlation can be used as a tool to highlight changes in cardiac function and potentially to categorize drugs based on their mechanisms of action.

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2021 – Hypoglycemia Induced Mitochondrial Connexin-43 Accumulation Aggravates Diabetic Cardiomyopathy
CardioExcyte 96 pre-Publication in Research Square (2021) Authors: Wei X., Chang A.C.H., Chang H., Xu S., Xue Y., Zhang Y., Lei M., Chang A.C.Y., Zhang Q.

Background: Diabetic cardiomyopathy (DCM) is a complex multifaceted disease responsible for elevated hospitalization and mortality in patients with diabetes mellitus (DM). DCM patients exhibit subclinical diastolic dysfunction, progression towards systolic impairment, and abnormal electrophysiology. Hypoglycemia events that occur spontaneously or due to excess insulin administration threaten the lives of DM patients – with the increased risk of sudden death. However, the molecular underpinnings of hypoglycemia-aggravated DCM remain to be elucidated. Methods and Results: Here we used the established streptozotocin-induced type 1 diabetic cardiomyopathy (T1 DCM) murine model to investigate how hypoglycemia aggravates DCM progression. We showed that chronic hyper- or hypoglycemic challenges dampened cardiac diastolic function in vivo as well as myocardial contractility and calcium handling in isolated cardiomyocytes. Similar contractile defects were recapitulated using neonatal mouse ventricular myocytes (NMVMs) under glucose fluctuation challenges. Using immunoprecipitation mass spectrometry, we identified and validated that hypoglycemia challenge activates the MEK/ERK and PI3K/Akt pathways which results in Cx43 phosphorylation by Src protein in cardiomyocytes. Cx43 dissociation and accumulation at mitochondrial inner membrane was confirmed both in human and murine cardiomyocytes. To determine causality, we overexpressed a mitochondrial targeting Cx43 (mtCx43) using AAV2. At normal blood glucose levels, mtCx43 overexpression recapitulated cardiomyocytes contractile deficiencies, cardiac diastolic dysfunction as well as aberrant electrophysiology both in vitro as well as in vivo. Conclusions: Hypoglycemia challenges results in the accumulation of mtCx43 through the MEK/ERK/Src and PI3K/Akt/Src pathways. We provide evidence that Cx43 mislocalization is present in diabetes mellitus patient hearts, STZ-induced DCM murine model, and glucose fluctuation challenged NMVMs. Mechanistically, we demonstrated that mtCx43 is responsible for inducing aberrant contraction and disrupts electrophysiology in cardiomyocytes and our results support targeting of mtCx43 in treating DCM. Translational perspective: Severe hypoglycemia drives cardiac dysfunction and aggressive ventricular arrhythmias in patients with DCM that leads to sudden cardiac death. Here we demonstrate that Cx43 mislocalization to mitochondria occurs upon hypoglycemic challenge and mtCx43 accumulation is responsible for cardiac diastolic dysfunction, cardiomyocyte contractile dysfunction, and aberrant electrophysiology in vivo. Our findings give support for therapeutic targeting of MEK/ERK/Src and PI3K/Akt/Src pathways to prevent mtCx43-driven DCM.

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2021 – A Novel Mice Model of Catecholaminergic Polymorphic Ventricular Tachycardia Generated by CRISPR/Cas9
CardioExcyte 96 pre-print Publication in bioRxiv (2021) Authors: Hou C., Jiang X., Qiu Q., Zheng J., Lin S., Chen S., Xu M., Zhang Y., Xie L., Xiao T.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) has been considered as one of the most important causes of children’s sudden cardiac death. Mutations in the genes for RyR2 and CASQ2, two mainly subtypes of CPVT, have been identified. However, the mutation in the gene of TECRL was rarely reported, which could be another genetic cause of CPVT. We evaluated myocardial contractility, electrophysiology, calcium handling in Tecrl knockout (Tecrl KO) mice and human induced pluripotent stem cell-derived cardiomyocytes. Immediately after epinephrine plus caffeine injection, Tecrl KO mice showed much more multiple premature ventricular beats and ventricular tachycardia. The Tecrl KO mice demonstrate CPVT phenotypes. Mechanistically, intracellular calcium amplitude was reduced, while time to baseline of 50 was increased in acute isolated cardiomyocytes. RyR2 protein levels decreased significantly upon cycloheximide treatment in TECRL deficiency cardiomyocytes. Overexpression of TECRL and KN93 can partially reverse cardiomyocytes calcium dysfunction, and this is p-CaMKII/CaMKII dependent. Therefore, a new CPVT mouse model was constructed. We propose a previously unrecognized mechanism of TECRL and provide support for the therapeutic targeting of TECRL in treating CPVT.

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2021 – Anthracycline-Induced Cardiotoxicity: Molecular Insights Obtained from Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes (hiPSC-CMs)
CardioExcyte 96 Publication in The AAPS Journal (2021) Authors: Bozza W.P., Takeda K., Alterovitz W-L., Chou C-K., Shen R-F., Zhang B.

Anthracyclines are a class of chemotherapy drugs that are highly effective for the treatment of human cancers, but their clinical use is limited by associated dose-dependent cardiotoxicity. The precise mechanisms by which individual anthracycline induces cardiotoxicity are not fully understood. Human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) are emerging as a physiologically relevant model to assess drugs cardiotoxicity. Here, we describe an assay platform by coupling hiPSC-CMs and impedance measurement, which allows real-time monitoring of cardiomyocyte cellular index, beating amplitude, and beating rate. Using this approach, we have performed comparative studies on a panel of four anthracycline drugs (doxorubicin, epirubicin, idarubicin, and daunorubicin) which share a high degree of structural similarity but are associated with distinct cardiotoxicity profiles and maximum cumulative dose limits. Notably, results from our hiPSC-CMs impedance model (dose-dependent responses and EC50 values) agree well with the recommended clinical dose limits for these drugs. Using time-lapse imaging and RNAseq, we found that the differences in anthracycline cardiotoxicity are closely linked to extent of cardiomyocyte uptake and magnitude of activation/inhibition of several cellular pathways such as death receptor signaling, ROS production, and dysregulation of calcium signaling. The results provide molecular insights into anthracycline cardiac interactions and offer a novel assay system to more robustly assess potential cardiotoxicity during drug development.

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2020 – Molecular Profiling of Human Induced Pluripotent Stem Cell-Derived Cells and their Application for Drug Safety Study
CardioExcyte 96 Review in Current Pharmaceutical Biotechnology (2020) Authors: Matsui T., Miyamoto N., Saito F., Shinozawa T.

Drug-induced toxicity remains one of the leading causes of discontinuation of the drug candidate and post-marketing withdrawal. Thus, early identification of the drug candidates with the potential for toxicity is crucial in the drug development process. With the recent discovery of humanInduced Pluripotent Stem Cells (iPSC) and the establishment of the differentiation protocol of human iPSC into the cell types of interest, the differentiated cells from human iPSC have garnered much attention because of their potential applicability in toxicity evaluation as well as drug screening, disease modeling and cell therapy. In this review, we expanded on current information regarding the feasibility of human iPSC-derived cells for the evaluation of drug-induced toxicity with a focus on human iPSCderived hepatocyte (iPSC-Hep), cardiomyocyte (iPSC-CMs) and neurons (iPSC-Neurons). Further, we CSAHi, Consortium for Safety Assessment using Human iPS Cells, reported our gene expression profiling data with DNA microarray using commercially available human iPSC-derived cells (iPSC-Hep, iPSC-CMs, iPSC-Neurons), their relevant human tissues and primary cultured human cells to discuss the future direction of the three types of human iPSC-derived cells.

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2020 – Single-cell analysis of murine fibroblasts identifies neonatal to adult switching that regulates cardiomyocyte maturation
CardioExcyte 96 Publication in Nature Communications (2020) Authors: Wang Y., Yao F., Wang L., Li Z., Ren Z., Li D., Zhang M., Han L., Wang S., Zhou B., Wang L.

Cardiac maturation lays the foundation for postnatal heart development and disease, yet little is known about the contributions of the microenvironment to cardiomyocyte maturation. By integrating single-cell RNA-sequencing data of mouse hearts at multiple postnatal stages, we construct cellular interactomes and regulatory signaling networks. Here we report switching of fibroblast subtypes from a neonatal to adult state and this drives cardiomyocyte maturation. Molecular and functional maturation of neonatal mouse cardiomyocytes and human embryonic stem cell-derived cardiomyocytes are considerably enhanced upon co-culture with corresponding adult cardiac fibroblasts. Further, single-cell analysis of in vivo and in vitro cardiomyocyte maturation trajectories identify highly conserved signaling pathways, pharmacological targeting of which substantially delays cardiomyocyte maturation in postnatal hearts, and markedly enhances cardiomyocyte proliferation and improves cardiac function in infarcted hearts. Together, we identify cardiac fibroblasts as a key constituent in the microenvironment promoting cardiomyocyte maturation, providing insights into how the manipulation of cardiomyocyte maturity may impact on disease development and regeneration.

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2020 – Maurocalcin and its analogue MCaE12A facilitate Ca2+ mobilization in cardiomyocytes
CardioExcyte 96 Publication in Biochemical Journal (2020) Authors: De Waard S., Montnach J., Cortinovis C., Chkir O., Erfanian M., Hulin P., Gaborit N., Lemarchand P., Mesirca P., Bidaud I., Mangoni M., De Waard M., Ronjat M.

Ryanodine receptors are responsible for the massive release of calcium from the 25 sarcoplasmic reticulum that triggers heart muscle contraction. Maurocalcin (MCa) is a 33 26 amino acid peptide toxin known to target skeletal ryanodine receptor. We investigated the 27 effect of MCa and its analogue MCaE12A on isolated cardiac ryanodine receptor (RyR2), and 28 showed that they increase RyR2 sensitivity to cytoplasmic calcium concentrations 29 promoting channel opening and decreases its sensitivity to inhibiting calcium concentrations. By measuring intracellular Ca2+ 30 transients, calcium sparks and contraction 31 on cardiomyocytes isolated from adult rats or differentiated from human induced 32 pluripotent stem cells, we demonstrated that MCaE12A passively penetrates cardiomyocytes 33 and promotes abnormal opening of RyR2. We also investigated the effect of MCaE12A on 34 pacemaker activity of sinus node cells from different mice lines and showed that, MCaE12A 35 improves pacemaker activity of sinus node cells obtained from mice lacking L-type CaV1.3 36 channel, or following selective pharmacologic inhibition of calcium influx via CaV1.3. Our 37 results identify MCaE12A as a high affinity modulator of RyR2 and make it an important tool for RyR2 structure-to-function studies as well as for manipulating Ca 38 2+ homeostasis and 39 dynamic of cardiac cells.

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2020 – Metformin therapy confers cardioprotection against the remodeling of gap junction in tachycardia-induced atrial fibrillation dog model
CardioExcyte 96 Publication in Frontiers in Life Sciences (2020) Authors: Li J., Li B., Bai F., Ma Y., Liu N., Liu Y., Wang Y., Liu Q.

OBJECTIVE: Metformin, introduced in 1957, is widely used as an anti-diabetic drug and has considerable benefits in cardiovascular disease reportedly, dependent or independent on its glucose-lowering effects. Aim of this study was to investigate the effect of metformin on gap junction and the inducibility of AF. METHODS: Beagle dogs were subjected to acute or chronic pacing at right atrial appendage by a pacemaker to develop an AF model and electrophysiological parameters were measured. In vitro study, a cell fast pacing model was developed by CardioExcyte 96. We performed Western blot, histology immunohistochemical staining and electron microscopy to detect the effect of metformin. RESULTS: In chronic AF model, the inducibility and duration of AF increased obviously after pacing for 6 weeks compared with sham-operated group (Inducibility, 3.33 ± 5.77 vs. 85.33 ± 7.89%, P0.0001; Duration, 0.8 ± 0.84 vs. 11 ± 2.67 ms, P0.0001). Effective refractory periods (ERP) decreased at left and right left atrium and atrial appendages compared with sham-operated group (123.95 ± 6.57 vs. 89.96 ± 7.39 ms P0.0001). Metformin attenuated the pacing-induced increase in EPR (89.96 ± 7.39 vs. 105.83 ± 7.45 ms, P0.05), AF inducibility and AF duration (Inducibility, 85.33 ± 7.89 vs. 64.17 ± 7.36%, Duration, 11 ± 2.67 vs. 8.62 ± 1.15 ms, P0.05). The expression of Cx43 shows a significant downregulation(about 38%, P0.001) after chronic pacing and treating with metformin could alleviate this decrease(P0.01). However, the effect of metformin in acute pacing model is limited. The immunohistochemical staining of cardiac tissue also shown that there is more lateralized Cx43 under pacing condition (87.67 ± 2.52 vs. 60.8 ± 9.13%, P0.005). These pacing-induced lateralize Cx43 could be alleviated by the metformin (48.4 ± 8.62 vs. 60.8 ± 9.13%, P0.05). Additionally, metformin could affect the interactions of ZO-1 with p-Src/Cx43 via decrease the abnormal cAMP level after pacing (84.04 ± 4.58 vs. 69.34 ± 4.5 nmol/L, P0.001). CONCLUSIONS:Metformin could alleviate the vulnerability of AF and attenuate the downregulation of gap junction under pacing condition via AMPK pathway and decreasing the P-Src level.

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2020 – Elevated myocardial SORBS2 and the underlying implications in leftventricular noncompaction cardiomyopathy
CardioExcyte 96 Publication in EBioMedicine (2020) Authors: Li C., Liu F., Liu S., Pan H., Du H., Huang J., Xie Y., Li Y., Zhao R., Wei Y.

Left ventricular noncompaction cardiomyopathy (LVNC) is a hereditary heart disease characterized by an excessive trabecular meshwork of deep intertrabecular recesses within the ventricular myocardium. The guidelines for management of LVNC patients aim to improve quality of life by preventing cardiac heart failure. However, the mechanism underlying LVNC-associated heart failure remains poorly understood.

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2020 – Functional Impact of BeKm-1, a High-Affinity hERG Blocker, on Cardiomyocytes Derived from Human-Induced Pluripotent Stem Cells
CardioExcyte 96 Publication in Molecular Sciences (2020) Authors: De Waard S., Montnach J., Ribeiro B., Nicolas S., Forest V., Charpentier F., Mangoni M.E., Gaborit N., Ronjat M., Loussouarn G., Lemarchand P., De Waard M.

IKr current, a major component of cardiac repolarization, is mediated by human Ether-à-go-go-Related Gene (hERG, KV11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.

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2020 – Advancing physiological maturation in human induced pluripotent stem cell‐derived cardiac muscle by gene editing an inducible adult troponin isoform switch
CardioExcyte 96 Publication in STEM CELLS (2020) Authors: Wheelwright M., Mikkila J., Bedada F.B., Mandegar M.A., Thompson B.R., Metzger J.M.

Advancing maturation of stem cell‐derived cardiac muscle represents a major barrier to progress in cardiac regenerative medicine. Cardiac muscle maturation involves a myriad of gene, protein, and cell‐based transitions, spanning across all aspects of cardiac muscle form and function. We focused here on a key developmentally controlled transition in the cardiac sarcomere, the functional unit of the heart. Using a gene‐editing platform, human induced pluripotent stem cell (hiPSCs) were engineered with a drug‐inducible expression cassette driving the adult cardiac troponin I (cTnI) regulatory isoform, a transition shown to be a rate‐limiting step in advancing sarcomeric maturation of hiPSC cardiac muscle (hiPSC‐CM) toward the adult state. Findings show that induction of the adult cTnI isoform resulted in the physiological acquisition of adult‐like cardiac contractile function in hiPSC‐CMs in vitro. Specifically, cTnI induction accelerated relaxation kinetics at baseline conditions, a result independent of alterations in the kinetics of the intracellular Ca2+ transient. In comparison, isogenic unedited hiPSC‐CMs had no cTnI induction and no change in relaxation function. Temporal control of adult cTnI isoform induction did not alter other developmentally regulated sarcomere transitions, including myosin heavy chain isoform expression, nor did it affect expression of SERCA2a or phospholamban. Taken together, precision genetic targeting of sarcomere maturation via inducible TnI isoform switching enables physiologically relevant adult myocardium‐like contractile adaptations that are essential for beat‐to‐beat modulation of adult human heart performance. These findings have relevance to hiPSC‐CM structure‐function and drug‐discovery studies in vitro, as well as for potential future clinical applications of physiologically optimized hiPSC‐CM in cardiac regeneration/repair.

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2020 – Comprehensive Cardiac Safety Assessment using hiPS-cardiomyocytes
CardioExcyte 96 Publication in the Journal of Current Pharmaceutical Biotechnology (2020) Authors: Takasuna K., Kazusa K., Hayakawa T.

Current cardiac safety assessment platforms (in vitro hERG-centric, APD, and/or in vivo animal QT assays) are not fully predictive of drug-induced Torsades de Pointes (TdP) and do not address other mechanism-based arrhythmia, including ventricular tachycardia or ventricular fibrillation, or cardiac safety liabilities such as contractile and structural cardiotoxicity which are another growing safety concerns. We organized the Consortium for Safety Assessment using Human iPS cells in 2013, based on the Japan Pharmaceutical Manufacturers Association (JPMA), to verify the application of human iPS/ES cell-derived cardiomyocytes for drug safety evaluation. The CSAHi HEART team focused on comprehensive screening strategies to predict a diverse range of cardiotoxicities using recently introduced platforms such as the Multi-Electrode Array (MEA), cellular impedance, Motion Field Imaging (MFI), and optical imaging of Ca transient to identify strengths and weaknesses of each platform. Our study showed that hiPS-CMs used in these platforms could detect pharmacological responses that were more relevant to humans compared to existing hERG, APD, or Langendorff (MAPD/contraction) assays. Further, MEA and other methods such as impedance, MFI, and Ca transient assays provided paradigm changes of platforms for predicting drug-induced QT risk and/or arrhythmia or contractile dysfunctions. In contrast, since discordances such as overestimation(false positive) of arrhythmogenicity, oversight, or opposite conclusions in positive inotropic and negative chronotropic activities to some compounds were also confirmed, possibly due to their functional immaturity of hiPS-CMs, hiPS-CMs should be used in these platforms for cardiac safety assessment based upon their advantages and disadvantages.

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2019 – Transcriptomic profiling reveals p53 as a key regulator of doxorubicin-induced cardiotoxicity
CardioExcyte 96 Publication in Cell Death Discovery (2019) Authors: McSweeney K.M., Bozza W.P., Alterovitz W.-L., Zhang B.

Doxorubicin is an important anticancer drug in the clinic. Unfortunately, it causes cumulative and dose-dependent cardiotoxic side effects. As the population of cancer survivors who have been exposed to treatment continues to grow, there is increased interest in assessing the long-term cardiac effects of doxorubicin and understanding the underlying mechanisms at play. In this study, we investigated doxorubicin-induced transcriptomic changes using RNA-sequencing (RNAseq) and a cellular model comprised of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Analyses of predicted upstream regulators identified the p53 protein as a key regulator of transcriptomic changes induced by doxorubicin. Clustering and pathway analyses showed that increased death receptor (DR) expression and enrichment of the extrinsic apoptotic pathway are significantly associated with doxorubicin-induced cardiotoxicity. Increased expression of p53 and DRs were confirmed via immunoblotting. Our data pinpoints increased DR expression as an early transcriptomic indicator of cardiotoxicity, suggesting that DR expression might function as a predictive biomarker for cardiac damage.

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2020 – A Scalable Approach Reveals Functional Responses of iPSC Cardiomyocyte 3D Spheroids
CardioExcyte 96 Publication in SLAS Discovery (2020) Authors: Burnham M.P., Harvey R., Sargeant R., Fertig N., Haddrick M.

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) provide an in vitro model of the human myocardium. Complex 3D scaffolded culture methods improve the phenotypical maturity of iPSC-CMs, although typically at the expense of throughput. We have developed a novel, scalable approach that enables the use of iPSC-CM 3D spheroid models in a label-free readout system in a standard 96-well plate-based format. Spheroids were accurately positioned onto recording electrodes using a magnetic gold–iron oxide nanoparticle approach. Remarkably, both contractility (impedance) and extracellular field potentials (EFPs) could be detected from the actively beating spheroids over long durations and after automated dosing with pharmacological agents. The effects on these parameters of factors, such as co-culture (including human primary cardiac fibroblasts), extracellular buffer composition, and electrical pacing, were investigated. Beat amplitudes were increased greater than 15-fold by co-culture with fibroblasts. Optimization of extracellular Ca2+ fluxes and electrical pacing promoted the proper physiological response to positive inotropic agonists of increased beat amplitude (force) rather than the increased beat rate often observed in iPSC-CM studies. Mechanistically divergent repolarizations in different spheroid models were indicated by their responses to BaCl2 compared with E-4031. These studies demonstrate a new method that enables the pharmacological responses of 3D iPSC-CM spheroids to be determined in a label-free, standardized, 96-well plate-based system. This approach could have discovery applications across cardiovascular efficacy and safety, where parameters typically sought as readouts of iPSC-CM maturity or physiological relevance have the potential to improve assay predictivity.

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2019 – The N-termini of GRK2 and GRK3 simulate the stimulating effects of RKIP on β-adrenoceptors
CardioExcyte 96 Publication in Biochemical and Biophysical Research Communications (2019) Authors: Maimari T., Krasel C., Bünemann M., Lorenz K.

The Raf kinase inhibitor protein (RKIP) activates β-adrenoceptors (β-AR) and thereby induces a well-tolerated cardiac contractility and prevents heart failure in mice. Different to RKIP-mediated β-AR activation, chronic activation of β-AR by catecholamines was shown to be detrimental for the heart. RKIP is an endogenous inhibitor of G protein coupled receptor kinase 2 (GRK2); it binds GRK2 and thereby inhibits GRK2 mediated β-AR phosphorylation and desensitization. Here, we evaluate RKIP-mediated effects on β-AR to explore new strategies for β-AR modulation. Co-immunoprecipitation assays and pull-down assays revealed subtype specificity of RKIP for the cardiac GRK isoforms GRK2 and GRK3 – not GRK5 – as well as several RKIP binding sites within their N-termini (GRK21−185 and GRK31−185). Overexpression of these N-termini prevented β2-AR phosphorylation and internalization, subsequently increased receptor signaling in HEK293 cells and cardiomyocyte contractility. Co-immunoprecipitation assays of β2-AR with these N-terminal GRK fragments revealed a direct interaction suggesting a steric interference of the fragments with the functional GRK-receptor interaction. Altogether, N-termini of GRK2 and GRK3 efficiently simulate RKIP effects on β-AR signaling in HEK293 cells and in cardiomyocytes by their binding to β2-AR and, thus, provide important insights for the development of new strategies to modulate β2-AR signaling.

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2019 – The patient-independent human iPSC model – a new tool for rapid determination of genetic variant pathogenicity in long QT syndrome
CardioExcyte 96 Publication in Heart Rhythm (2019)Authors: Chavali, N.V., Kryshtal, D.O., Parikh, S.S., Wang, L., Glazer, A.M., Blackwell, D.J., Kroncke, B.M., Shoemaker, M.B., Knollmann, B.C.

Background Commercial genetic testing for Long QT Syndrome (LQTS) has rapidly expanded, but the inability to accurately predict whether a rare variant is pathogenic has limited its clinical benefit. Novel missense variants are routinely reported as “Variant of Unknown Significance (VUS)” and cannot be used to screen family members at-risk for sudden cardiac death. Better approaches to determine pathogenicity of VUS are needed. Objective To rapidly determine the pathogenicity of a CACNA1C variant reported by commercial genetic testing as a VUS using a patient-independent induced pluripotent stem cell (hiPSC) model. Methods Using CRISPR/Cas9 genome editing, CACNA1C-p.N639T was introduced into a previously-established hiPSC from an unrelated healthy volunteer, thereby generating a patient-independent hiPSC model. Three independent heterozygous N639T hiPSC lines were generated and differentiated into cardiomyocytes (CM). Electrophysiological properties of N639T hiPSC-CM were compared to those of isogenic and population control hiPSC-CM by measuring the extracellular field potential (EFP) of 96-well hiPSC-CM monolayers, and by patch-clamp. Results Significant EFP prolongation was observed only in optically-stimulated but not in spontaneously-beating N639T hiPSC-CM. Patch clamp studies revealed that N639T prolonged the ventricular action potential by slowing voltage-dependent inactivation of CaV1.2 currents. Heterologous expression studies confirmed the effect of N639T on CaV1.2 inactivation. Conclusion The patient-independent hiPSC model enabled rapid generation of functional data to support reclassification of a CACNA1C VUS to “likely pathogenic”, thereby establishing a novel LQTS type 8 mutation. Furthermore, our results indicate the importance of controlling beating rates to evaluate functional significance of LQTS VUS in high-throughput hiPSC-CM assays.

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2019 – Particulate matter 2.5 induced arrhythmogenesis mediated by TRPC3 in human induced pluripotent stem cell-derived cardiomyocytes
CardioExcyte 96 Publication in Archives of Toxicology (2019)Authors: Cai C., Huang J., Lin Y., Miao W., Chen P., Chen X., Wang J., Chen M.

Particulate matter (PM) is one of the most important environmental issues worldwide, which is associated with not only pulmonary but also cardiovascular diseases. However, the underlying biological mechanisms of PM related cardiovascular dysfunction remained poorly defined, especially mediated by the pathway of direct impact on vascular and heart. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an ideal platform for studying PM-exposed cellular diseases model in vitro. Here, we investigated the direct effects of particulate matter 2.5 (PM2.5) on hiPSC-CMs and the potential mechanism at non-cytotoxic concentrations. Cell viability, contraction amplitude and spontaneous beating rate of iPSC-CMs after direct exposure to PM2.5 showed that the concentration of lower than 100 µg/ml would not lead to cytotoxic effects. Calcium-mediated optical mapping illustrated that there was a concentration-dependent reduction in quantification of calcium transient amplitude and an increase in the incidence of early after depolarizations due to PM2.5 treatment. Furthermore, there were dramatic dosage-dependent shortening in action potential duration and decrease in L-type calcium peak current density. The Ingenuity Pathway Analysis of our transcriptive study indicated that PM2.5 exposure preferentially influenced the expression of genes involved in calcium signaling. Among them the up-regulation of TRPC3 potentially played an important role in the electrophysiological alteration of PM2.5 on hiPSC-CMs, which could be ameliorated by pretreatment with pyr3, the inhibitor of TRPC3. In conclusion, our results demonstrated that exposure to PM2.5 was capable of increasing propensity to cardiac arrhythmias which could be attenuated with TRPC3 inhibition.

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2019 – Pharmacological enhancement of repolarization reserve in human induced pluripotent stem cells derived cardiomyocytes
CardioExcyte 96 Publication in Biochem. Pharmacol. (2019) Authors: Treat JA, Goodrow RJ, Bot CT, Haedo RJ, Cordeiro JM.

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for many applications including safety pharmacology. However, a deficiency or complete absence of several K+ currents suggests repolarization reserve is low in hiPSC-CMs. We determined whether a dual Ito and IKr activator can improve repolarization reserve in hiPSC-CMs resulting in a more electrophysiologically mature phenotype. METHODS AND RESULTS: Human iPSC were maintained on growth factor and differentiated into the cardiac phenotype by addition of selective Wnt molecules. Current and voltage clamp recordings in single cells were made using patch electrodes. Extracellular field potentials were made using a microelectrode array on hiPSC monolayers. Action potential recordings from hiPSC-CMs following application of an IKr inhibitor resulted in depolarization of the membrane potential and prolongation of the APD. A flattening of the T-wave was noted on the pseudo-ECG. In contrast, application of the IKr and Ito agonist, NS3623, resulted in hyperpolarization of the membrane, slowing of the spontaneous rate and shortening of the APD. Voltage clamp recording showed a significant increase in IKr; no enhancement of Ito in hiPSC-CMs was noted. AP clamp experiments revealed that IKr plays a role in both phase 3 repolarization and phase 4 depolarization. mRNA analysis revealed that KCNH2 is abundantly expressed in hiPSC-CM, consistent with electrophysiological recordings. CONCLUSIONS: Although NS3623 is a dual Ito and IKr activator in ventricular myocytes, application of this compound to hiPSC-CMs enhanced only IKr and no effect on Ito was noted. Our results suggest IKr enhancement can improve repolarization reserve in this cell type. The disconnect between a dramatic increase in Ito in adult myocytes versus the lack of effect in hiPSC-CMs suggest that the translation of pharmacological effects in hiPSC-CM to adult myocytes should be viewed with caution.

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2019 – Downregulation of miR-146a Contributes to Cardiac Dysfunction Induced by the Tyrosine Kinase Inhibitor Sunitinib
CardioExcyte 96 Publication in Frontiers in Pharmacology (2019) Authors: Shen L., Li C., Zhang H., Qiu S., Fu T., Xu Y.

The main adverse effect of tyrosine kinase inhibitors, such as sunitinib, is cardiac contractile dysfunction; however, the molecular mechanisms of this effect remain largely obscure. MicroRNAs (miRNAs) are key regulatory factors in both cardiovascular diseases and the tyrosine kinase pathway. Therefore, we analyzed the differential expression of miRNAs in the myocardium in mice after exposure to sunitinib using miRNA microarray. A significant downregulation of miR-146a was observed in the myocardium of sunitinib-treated mice, along with a 20% decrease in left ventricle ejection fraction (LVEF). The downregulation of miR-146a was further validated by RT-qPCR. Among the potential targets of miR-146a, we focused on Pln and Ank2, which are closely related to cardiac contractile dysfunction. Results of luciferase reporter assay confirmed that miR-146a directly targeted the 3' untranslated region of Pln and Ank2. Significant upregulation of PLN and ANK2 at the mRNA and protein levels was observed in the myocardium of sunitinib-treated mice. Cardiac-specific overexpression of miR-146a prevented the deteriorate effect of SNT on calcium transients, thereby alleviating the decreased contractility of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). SiRNA knockdown of PLN or ANK2 prevented sunitinib-induced suppression of contractility in hiPSC-CMs. Therefore, our in vivo and in vitro results showed that sunitinib downregulated miR-146a, which contributes to cardiac contractile dysfunction by regulating the downstream targets PLN and ANK2, and that upregulation of miR-146a alleviated the inhibitory effect of SNT on cardiac contractility. Thus, miR-146a could be a useful protective agent against sunitinib-induced cardiac dysfunction.

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2019 – Label-Free Monitoring of Cells in vitro
CardioExcyte 96 book chapter in Label-Free Monitoring of Cells in vitro in Bioanalytical Reviews - Springer (2019) Authors: Stolwijk J.A., Wegener J.

This book is dedicated to label-free, non-invasive monitoring of cell-based assays and it comprises the most widely applied techniques. Each approach is described and critically evaluated by an expert in the field such that researchers get an overview on what is possible and where the limitations are. The book provides the theoretical basis for each technique as well as the most successful and exciting applications. Label-free bioanalytical techniques have been known for a long time as valuable tools to monitor adsorption processes at the solid-liquid interface in general – and biomolecular interaction analysis (BIA) in particular. The underlying concepts have been progressively transferred to the analysis of cell-based assays. The strength of these approaches is implicitly given with the name 'label-free': the readout is independent of any label, reagent or additive that contaminates the system under study and potentially affects its properties. Thus, label-free techniques provide an unbiased analytical perspective in the sense that the sample is not manipulated by additives but pure. They are commonly based on physical principles and read changes in integral physical properties of the sample like refractive index, conductivity, capacitance or elastic modulus to mention just a few. Even though it is not implied in the name, label-free approaches usually monitor the cells under study non-invasively meaning that the amplitude of the signal (e.g. electric field strength, mechanical elongation) that is used for the measurement is too low to interfere or affect. In contrast to label-based analytical techniques that are commonly restricted to a single reading at a predefined time point, label-free approaches allow for a continuous observation so that the dynamics of the biological system or reaction become accessible.

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2018 – The opioid oxycodone use‐dependently inhibits the cardiac sodium channel NaV1.5
CardioExcyte 96 Publication in British Journal of Pharmacology (2018) Authors: Meents J.E., Juhasz K., Stölzle-Feix S., Peuckmann-Post V., Rolke R. Lampert A.

BACKGROUND AND PURPOSE: Oxycodone is a potent semi-synthetic opioid that is commonly used for the treatment of severe acute and chronic pain. However, treatment with oxycodone can lead to cardiac electrical changes, such as long-QT syndrome, potentially inducing sudden cardiac arrest. Here, we investigate whether the cardiac side effects of oxycodone can be explained by modulation of the cardiac sodium channel NaV1.5. EXPERIMENTAL APPROACH: Heterologously expressed NaV1.5, NaV1.7 or NaV1.8 were used for whole-cell patch-clamp electrophysiology. A variety of voltage-clamp protocols was used to test the effect of oxycodone on different channel gating modalities. Human stem cell-derived cardiomyocytes were used to measure the effect of oxycodone on cardiomyocyte beating. KEY RESULTS: Oxycodone concentration-dependently and use-dependently inhibits NaV1.5 with an IC50 of 483.2 μM. In addition, oxycodone slows recovery of NaV1.5 from fast inactivation and increases slow inactivation. At high concentrations, these effects lead to a reduced beat rate in cardiomyocytes and to arrhythmia. In contrast, no effects could be observed on NaV1.7 or NaV1.8. CONCLUSION AND IMPLICATIONS: Oxycodone leads to an accumulation of NaV1.5 in inactivated states with a slow time course. While the concentrations needed to elicit cardiac arrhythmia in vitro are comparably high, some patients under long-term treatment with oxycodone as well as drug abusers and addicts might suffer from severe cardiac side effects induced by the slow effects of oxycodone on NaV1.5.

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2019 – An integrated characterization of contractile, electrophysiological, and structural cardiotoxicity of Sophora tonkinensis Gapnep. in human pluripotent stem cell-derived cardiomyocytes
CardioExcyte 96 Publication in Stem Cell Research & Therapy (2019)Authors: Wang R., Wang M., Wang S., Yang K., Zhou P., Xie X., Cheng Q., Ye J., Sun G., Sun X.

Background Cardiotoxicity remains an important concern in drug discovery and clinical medication. Meanwhile, Sophora tonkinensis Gapnep. (S. tonkinensis) held great value in the clinical application of traditional Chinese medicine, but cardiotoxic effects were reported, with matrine, oxymatrine, cytisine, and sophocarpine being the primary toxic components. Methods In this study, impedance and extracellular field potential (EFP) of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were recorded using the cardio non-labeled cell function analysis and culture system (CardioExcyte 96). The effects of matrine, oxymatrine, cytisine, and sophocarpine (2, 10, 50 μM) on cell viability; level of lactate dehydrogenase (LDH), creatine kinase MB isoenzyme (CK-MB), and cardiac troponin I (CTn-I); antioxidant activities; production of reactive oxygen species (ROS) and malondialdehyde (MDA); and disruption of intracellular calcium homeostasis were also added into the integrated assessment. Results The results showed that matrine and sophocarpine dose-dependently affected both impedance and EFP, while oxymatrine and cytisine altered impedance significantly. Our study also indicated that cardiotoxicity of matrine, oxymatrine, cytisine, and sophocarpine was related to the disruption of calcium homeostasis and oxidative stress. Four alkaloids of S. tonkinensis showed significant cardiotoxicity with dose dependence and structural cardiotoxicity synchronized with functional changes of cardiomyocytes. Conclusions This finding may provide guidance for clinical meditation management. Furthermore, this study introduced an efficient and reliable approach, which offers alternative options for evaluating the cardiotoxicity of the listed drugs and novel drug candidates.

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2018 – Safety Pharmacology Methods and Models in an Evolving Regulatory Environment
CardioExcyte 96 CiPA Editorial in Journal of Pharmacological and Toxicological Methods (2018) Authors: Pugsley M.K., Harter M.L., de Korte T., Connaughton C., Authier S., Curtis M.J.

This editorial prefaces the annual themed issue on safety pharmacology (SP) methods published in the Journal of Pharmacological and Toxicological Methods (JPTM). We highlight here the content derived from the recent 2016 Safety Pharmacology Society (SPS), Canadian Society of Pharmacology and Therapeutics (CSPT), and Japanese Safety Pharmacology Society (JSPS) joint meeting held in Vancouver, B.C., Canada. This issue of JPTM continues the tradition of providing a publication summary of articles primarily presented at the joint meeting with direct bearing on the discipline of SP. As the regulatory landscape is expected to evolve with revision announced for the existing guidance document on non-clinical proarrhythmia risk assessment (ICHS7B) there is also imminent inception of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative. Thus, the field of SP is dynamically progressing with characterization and implementation of numerous alternative non-clinical safety models. Novel method development and refinement in all areas of the discipline are reflected in the content.

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2018 – Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells
CardioExcyte 96 Publication in Stem Cells and Development Authors: Kondrashov A., Hoang M.D., Smith J., Bhagwan J., Duncan G., Mosqueira D., Munoz M., Vo N.T.N., Denning C.

Modelling disease with hPSCs is hindered because the impact on cell phenotype from genetic variability between individuals can be greater than from the pathogenic mutation. While ‘footprint-free’ Cas9/CRISPR editing solves this issue, existing approaches are inefficient or lengthy. Here, a simplified PiggyBac strategy shortened hPSC editing by 2 weeks and required one round of clonal expansion and genotyping rather than two, with similar efficiencies to the longer conventional process. Success was shown across 4 cardiac-associated loci (ADRB2, GRK5, RYR2, ACTC1) by genomic cleavage and editing efficiencies of 8-93% and 8-67%, respectively, including mono- and/or bi-allelic events. Pluripotency was retained, as was differentiation into high purity cardiomyocytes (CMs; 88-99%). Using the GRK5 isogenic lines as an exemplar, chronic stimulation with the b-adrenoceptor agonist, isoprenaline, reduced beat rate in hPSC-CMs expressing GRK5-Q41 but not GRK5-L41; this was reversed by the b-blocker, propranolol. This simplified, footprint-free approach will be useful for mechanistic studies.

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2018 – Cross-site comparison of excitation-contraction coupling using impedance and field potential recordings in hiPSC cardiomyocytes
CardioExcyte 96 Publication in Journal of Pharmacological and Toxicological Methods (2018) Authors: Bot C.T., Juhasz K., Haeusermann F., Polonchuk L., Traebert M., Stölzle-Feix S.

Introduction: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines.Methods: Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed.Results: Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPD⁠max was observed, or not a significant change from pre-addition control values.Discussion: With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.

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2018 – Hypertrophic cardiomyopathy-linked mutation in troponin T causes myofibrillar disarray and pro-arrhythmic action potential changes in human iPSC cardiomyocytes
CardioExcyte 96 Publication in Journal of Molecular and Cellular CardiologyAuthors: Wang L., Kim K., Parikh S., Cadar A.G., Bersell K.R., He H., Pinto J.R., Kryshtal D.O., Knollmann B.C.

Background Mutations in cardiac troponin T (TnT) are linked to increased risk of ventricular arrhythmia and sudden death despite causing little to no cardiac hypertrophy. Studies in mice suggest that the hypertrophic cardiomyopathy (HCM)-associated TnT-I79N mutation increases myofilament Ca sensitivity and is arrhythmogenic, but whether findings from mice translate to human cardiomyocyte electrophysiology is not known.ObjectivesTo study the effects of the TnT-I79N mutation in human cardiomyocytes. Methods Using CRISPR/Cas9, the TnT-I79N mutation was introduced into human induced pluripotent stem cells (hiPSCs). We then used the matrigel mattress method to generate single rod-shaped cardiomyocytes (CMs) and studied contractility, Ca handling and electrophysiology. Results Compared to isogenic control hiPSC-CMs, TnT-I79N hiPSC-CMs exhibited sarcomere disorganization, increased systolic function and impaired relaxation. The Ca-dependence of contractility was leftward shifted in mutation containing cardiomyocytes, demonstrating increased myofilament Ca sensitivity. In voltage-clamped hiPSC-CMs, TnT-I79N reduced intracellular Ca transients by enhancing cytosolic Ca buffering. These changes in Ca handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. The myofilament Ca sensitizer EMD57033 produced similar action potential triangulation in control hiPSC-CMs. Conclusions The TnT-I79N hiPSC-CM model not only reproduces key cellular features of TnT-linked HCM such as myofilament disarray, hypercontractility and diastolic dysfunction, but also suggests that this TnT mutation causes pro-arrhythmic changes of the human ventricular action potential.

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2017 – Frequency-Dependent Multi-Well Cardiotoxicity Screening Enabled by Optogenetic Stimulation
CardioExcyte 96 Publication in International Journal of Molecular Sciences Authors: Rehnelt S., Malan D., Juhasz K., Wolters B., Doerr L., Beckler M., Kettenhofen R., Bohlen H., Bruegmann T., Sasse P.

Side effects on cardiac ion channels causing lethal arrhythmias are one major reason for drug withdrawals from the market. Field potential (FP) recording from cardiomyocytes, is a well-suited tool to assess such cardiotoxic effects of drug candidates in preclinical drug development, but it is currently limited to the spontaneous beating of the cardiomyocytes and manual analysis. Herein, we present a novel optogenetic cardiotoxicity screening system suited for the parallel automated frequency-dependent analysis of drug effects on FP recorded from human-induced pluripotent stem cell-derived cardiomyocytes. For the expression of the light-sensitive cation channel Channelrhodopsin-2, we optimised protocols using virus transduction or transient mRNA transfection. Optical stimulation was performed with a new light-emitting diode lid for a 96-well FP recording system. This enabled reliable pacing at physiologically relevant heart rates and robust recording of FP. Thereby we detected rate-dependent effects of drugs on Na+, Ca2+ and K+ channel function indicated by FP prolongation, FP shortening and the slowing of the FP downstroke component, as well as generation of afterdepolarisations. Taken together, we present a scalable approach for preclinical frequency-dependent screening of drug effects on cardiac electrophysiology. Importantly, we show that the recording and analysis can be fully automated and the technology is readily available using commercial products.

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2017 – Structural and electrophysiological dysfunctions due to increased endoplasmic reticulum stress in a long-term pacing model using human induced pluripotent stem cell-derived ventricular cardiomyocytes
CardioExcyte 96 Publication in Stem Cell Research and Therapy (2017)Authors: Cui C., Geng L., Shi J., Zhu Y., Yang G., Wang Z., Wang J., Chen M.

Background: Long-term ventricular pacing has deleterious effects and becomes more significant when cumulative percent ventricular pacing (Cum%VP) exceeds 40% of time. However, cellular disturbances and pathways by which pacing leads to myocardial disorders are not well understood. Attempts to resolve these questions have been hampered by difficulties in obtaining human cardiac tissue and the inability to build a longer-lasting (lasting longer than weeks) pacing model in vitro. Methods: Human induced pluripotent stem cell-derived ventricular cardiomyocytes (VCMs) were cultured in the presence of electrical stimulation for 2 weeks. Quantitative structural and electrophysiological analyses were used to define the functional disturbances of pacing. Results: Compared to controls, paced VCMs exhibited a remarkable reduction in the contractile protein expression, an increased apoptosis ratio and electrophysiological remodelling in a Cum%VP-dependent manner. Investigation of the protein expression levels revealed that long-term pacing universally activated both ER stress and downstream calpain. Moreover, the inhibition of calpain attenuated the adverse effects on the structural remodelling and increased the ICa, L in paced VCMs. Conclusions: The results demonstrated that pacing VCMs for 2 weeks in vitro led to a series of structural and electrophysiological dysfunctions. The increased ER stress and downstream calpain could be a central mechanism underlying the disease pathogenesis. This finding could represent a new therapeutic target in the management of long-term pacing patients.

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2017 – An impedance-based approach using human iPSC-derived cardiomyocytes significantly improves in vitro prediction of in vivo cardiotox liabilities
CardioExcyte 96 Publication in Toxicology and Applied Pharmacology (2017)Authors: Authors: 

Current in vitro approaches to cardiac safety testing typically focus on mechanistic ion channel testing to predict in vivo proarrhythmic potential. Outside of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, structural and functional cardiotoxicity related to chronic dosing effects are of great concern as these effects can impact compound attrition. Development and implementation of an in vitro cardiotoxicity screening platform that effectively identifies these liabilities early in the discovery process should reduce costly attrition and decrease preclinical development time. Impedence platforms have the potential to accurately identify structural and functional cardiotoxicity and have sufficient throughput to be included in a multi-parametric optimization approach. Human induced pluripotent stem cell cardiomyocytes (hIPSC-CMs) have demonstrated utility in cardiac safety and toxicity screening. The work described here leverages these advantages to assess the predictive value of data generated by two impedance platforms. The response of hIPSC-CMs to compounds with known or predicted cardiac functional or structural toxicity was determined. The compounds elicited cardiac activities and/or effects on “macro” impedance often associated with overt structural or cellular toxicity, detachment, or hypertrophy. These assays correctly predicted in vivo cardiotox findings for 81% of the compounds tested and did not identify false positives. In addition, internal or literature Cmax values from in vivo studies correlated within 4 fold of the in vitro observations. The work presented here demonstrates the predictive power of impedance platforms with hIPSC-CMs and provides a means toward accelerating lead candidate selection by assessing preclinical cardiac safety earlier in the drug discovery process.

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2017 – Combined Impedance and Extracellular Field Potential Recordings from Human Stem Cell-Derived Cardiomyocytes 
CardioExcyte 96 book chapter in Stem Cell-Derived Models in Toxicology (2017) Authors: Obergrussberger A., Thomas U., Stölzle-Feix S., Becker N., Juhasz K, Doerr L., Beckler M., George M., Fertig N.

Measurement of contractility using impedance is a novel method for gaining information about a drug candidate’s potential to disturb cardiac cell contraction. The impedance signal is recorded from a monolayer of cardiac cells, most commonly derived from human-induced pluripotent stem cells (hiPSCs), which are becoming an attractive model for safety testing, especially in the light of the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative introduced in 2013. The goal of this initiative is, in part, to standardize assays, targets, and cell types but also to evaluate the potential of new technologies, in this context, such as impedance. The CardioExcyte 96 is a hybrid system that combines the impedance readout (a measure of cell contractility) with extracellular field potential (EFP) recordings. This chapter focuses on cell handling of hiPSC cardiomyocytes (CMs) and the short- and long-term investigation into pharmacological effects of a wide range of pharmacological agents, including flecainide, nifedipine, isoproterenol, and E4031 using the CardioExcyte 96.

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2016 – Identification of Drug-Drug Interactions In Vitro: A Case Study Evaluating the Effects of Sofosbuvir and Amiodarone on hiPSC-Derived Cardiomyocytes
CardioExcyte 96 Publication in Toxicological Sciences (2016)Authors: Millard DC, Strock CJ, Carlson CB, Aoyama N, Juhasz K, Goetze TA, Stoelzle-Feix S, Becker N, Fertig N, January CT, Anson BD, Ross JD.

Drug–drug interactions pose a difficult drug safety problem, given the increasing number of individuals taking multiple medications and the relative complexity of assessing the potential for interactions. For example, sofosbuvir-based drug treatments have significantly advanced care for hepatitis C virus-infected patients, yet recent reports suggest interactions with amiodarone may cause severe symptomatic bradycardia and thus limit an otherwise extremely effective treatment. Here, we evaluated the ability of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to recapitulate the interaction between sofosbuvir and amiodarone in vitro, and more generally assessed the feasibility of hiPSC-CMs as a model system for drug–drug interactions. Sofosbuvir alone had negligible effects on cardiomyocyte electrophysiology, whereas the sofosbuvir-amiodarone combination produced dose-dependent effects beyond that of amiodarone alone. By comparison, GS-331007, the primary circulating metabolite of sofosbuvir, had no effect alone or in combination with amiodarone. Further mechanistic studies revealed that the sofosbuvir-amiodarone combination disrupted intracellular calcium (Ca2+) handling and cellular electrophysiology at pharmacologically relevant concentrations, and mechanical activity at supra-pharmacological (30x Cmax) concentrations. These effects were independent of the common mechanisms of direct ion channel block and P-glycoprotein activity. These results support hiPSC-CMs as a comprehensive, yet scalable model system for the identification and evaluation of cardioactive pharmacodynamic drug–drug interactions. 

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2016 – Safety pharmacology studies using EFP and impedance
CardioExcyte 96 Publication in Journal of Pharmacological and Toxicological Methods (2016) Authors: Obergrussberger A., Juhasz K., Thomas U., Stölzle-Feix S., Becker N., Dörr L., Beckler M., Bot C., George M., Fertig N.

Introduction While extracellular field potential (EFP) recordings using multi-electrode arrays (MEAs) are a well-established technique for monitoring changes in cardiac and neuronal function, impedance is a relatively unexploited technology. The combination of EFP, impedance and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has important implications for safety pharmacology as functional information about contraction and field potentials can be gleaned from human cardiomyocytes in a beating monolayer. The main objectives of this study were to demonstrate, using a range of different compounds, that drug effects on contraction and electrophysiology can be detected using a beating monolayer of hiPSC-CMs on the CardioExcyte 96. Methods hiPSC-CMs were grown as a monolayer on NSP-96 plates for the CardioExcyte 96 (Nanion Technologies) and recordings were made in combined EFP and impedance mode at physiological temperature. The effect of the hERG blockers, E4031 and dofetilide, hERG trafficking inhibitor, pentamidine, β-adrenergic receptor agonist, isoproterenol, and calcium channel blocker, nifedipine, was tested on the EFP and impedance signals. Results Combined impedance and EFP measurements were made from hiPSC-CMs using the CardioExcyte 96 (Nanion Technologies). E4031 and dofetilide, known to cause arrhythmia and Torsades de Pointes (TdP) in humans, decreased beat rate in impedance and EFP modes. Early afterdepolarization (EAD)-like events, an in vitro marker of TdP, could also be detected using this system. Isoproterenol and nifedipine caused an increase in beat rate. A long-term study (over 30 h) of pentamidine, a hERG trafficking inhibitor, showed a concentration and time-dependent effect of pentamidine. Discussion In the light of the new Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative to improve guidelines and standardize assays and protocols, the use of EFP and impedance measurements from hiPSCs may become critical in determining the proarrhythmic risk of potential drug candidates. The combination of EFP offering information about cardiac electrophysiology, and impedance, providing information about contractility from the same area of a synchronously beating monolayer of human cardiomyocytes in a 96-well plate format has important implications for future cardiac safety testing.

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2023 – Establishment and validation of a torsade de pointes prediction model based on human iPSC‑derived cardiomyocytes
CardioExcyte 96 Publication in Exp. & Ther. Med. (2023) Authors: Pan D., Li B., Wang S.

Drug‑induced cardiotoxicity is one of the main causes of drug failure, which leads to subsequent withdrawal from pharmaceutical development. Therefore, identifying the potential toxic candidate in the early stages of drug development is important. Human induced pluripotent stem cell‑derived cardiomyocytes (hiPSC‑CMs) are a useful tool for assessing candidate compounds for arrhythmias. However, a suitable model using hiPSC‑CMs to predict the risk of torsade de pointes (TdP) has not been fully established. The present study aimed to establish a predictive TdP model based on hiPSC‑CMs. In the current study, 28 compounds recommended by the Comprehensive in vitro Proarrhythmia Assay (CiPA) were used as training set and models were established in different risk groups, high‑ and intermediate‑risk versus low‑risk groups. Subsequently, six endpoints of electrophysiological responses were used as potential model predictors. Accuracy, sensitivity and area under the curve (AUC) were used as evaluation indices of the models and seven compounds with known TdP risk were used to verify model differentiation and calibration. The results showed that among the seven models, the AUC of logistic regression and AdaBoost model was higher and had little difference in both training and test sets, which indicated that the discriminative ability and model stability was good and excellent, respectively. Therefore, these two models were taken as submodels, similar weight was configured and a new TdP risk prediction model was constructed using a soft voting strategy. The classification accuracy, sensitivity and AUC of the new model were 0.93, 0.95 and 0.92 on the training set, respectively and all 1.00 on the test set, which indicated good discrimination ability on both training and test sets. The risk threshold was defined as 0.50 and the consistency between the predicted and observed results were 92.8 and 100% on the training and test sets, respectively. Overall, the present study established a risk prediction model for TdP based on hiPSC‑CMs which could be an effective predictive tool for compound‑induced arrhythmias.

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