Human-induced Pluripotent Stem Cell-derived Cardiomyocytes Research Articles

Association of genetically predicted human metabolomic gene expression with incident heart failure

Abstract Background Heart failure (HF) represents a significant challenge both for patients and healthcare systems worldwide. Despite ongoing efforts, therapeutic options are limited. The underlying pathophysiology involves intricate changes in cardiac metabolism that strongly influence the clinical phenotype, thus potentially offering novel options for therapeutic targeting. Systematic prioritisation of metabolic genes regarding their phenotypic impact on HF is outstanding. Purpose We investigate the association of genetically predicted metabolic gene expression with the onset of HF within UK Biobank (UKB). We followed up on identified candidate genes using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) models of cardiomyocyte hypertrophy. Methods Utilising genomic data of 484,372 UKB participants and left ventricle-specific expression quantitative trait loci (eQTL) information from Genotype-Tissue Expression (GTEx) project, we predicted genetically regulated gene expression using established methods (PrediXcan) for all metabolic genes. Subsequently, we employed per-gene Cox proportional hazards (COX-PH) models to assess the association of genetically predicted metabolic gene expression with incident HF. We adjusted models for clinical risk using established risk models (Pooled Cohort Equations to Prevent HF (PCP-HF)). To validate our findings in vitro, we subjected hiPSC-CMs to a sympathomimetic stimulus via treatment with endothelin-1 (ET-1; 10 nM). We evaluate alterations in expression via real-time quantitative polymerase chain reaction (RT-qPCR) and establish methods for assessing hypertrophic remodelling. Results COX-PH models revealed the predicted expression of 104/1515 metabolic genes to be independently associated with the onset of HF. Subsequent refinement with a meta-analysis of commonly dysregulated genes in end-stage HF narrowed our findings to 46 candidate genes, of which several were known for their role in HF pathophysiology. We further traced nine genes for validation in hiPSC-CM models and showed significant dysregulation of several genes following the treatment with ET-1. Flow cytometry measured hiPSC-CM hypertrophy following sympathomimetic stimulation (p=0.036). Extracellular flux analysis revealed canonical changes following treatment with ET-1. Conclusions The study revealed several metabolic genes associated with incident HF, thus confirming previous findings and highlighting novel, putative therapeutic target genes. We confirmed transcriptional changes in an in vitro model. Ongoing work includes further validation via silencing candidate genes in hiPSC-CMs and subjection to the established phenotypic in vitro models.

The cardiac sympathetic co-transmitter neuropeptide-Y is pro-arrhythmic in human cardiomyocytes by lengthening activation-recovery interval

Abstract Background Myocardial infarction (MI) is associated with sympathetic overactivity, during which the co-release of neuropeptide-Y (NPY) is prominent. NPY is associated with arrhythmia risk and mortality following ST-elevation MI, although the exact mechanisms for this in human cardiomyocytes remains unclear. Purpose We therefore tested the hypothesis that NPY signalling would alter the electrophysiology of human induced-pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in a way that would promote arrhythmia, and that in patients being treated for MI, high NPY concentrations previously associated with ventricular arrhythmia (>27.3 pg.mL-1) would be associated with comparable ECG changes. Methods Immunohistochemistry and western blot were performed on hiPSC-CMs and ventricular biopsies from human patients undergoing valve surgery. The FRET-based sensor Epac-SH187 was expressed in hiPSC-CMs to monitor the cyclic adenosine 3’,5’-monophosphate (cAMP) response to NPY. A micro-electrode array was used to investigate the effects of NPY on a confluent layer of iPSC-CMs, measuring changes in activation, recovery, conduction velocity and spontaneous automaticity. Additionally, patients presenting with ST-elevation MI had peripheral venous [NPY] measured and compared to 12-lead ECGs recorded following stenting. Results hiPSC-CMs expressed Y1 and Y5 receptor mRNA and protein, which are also expressed in ventricular biopsies from human patients. NPY (1-100 nM) significantly reduced intracellular cAMP levels (n=65), most effectively prevented by Y5 receptor inhibition (1µM CGP71683A, n=40). In a confluent layer of hiPSC-CM, NPY (100 nM) slowed late-repolarisation represented by T-wave peak-end duration (p=0.04), which could be prevented by Y1-receptor inhibition (1 μM BIBO 3304, n=6), and caused prolongation of rate corrected activation-recovery interval (ARIc) (p=0.009, n=6) which could be abolished by Y5-receptor inhibition (n=7). NPY significantly increased automaticity to more than one site of initiation in 4/6 (66.7%) compared to 2/14 (14.3%) control plates (p=0.037), despite no change in conduction velocity. Moreover, in 65 patients being treated for ST elevation MI, those with high peripheral venous NPY concentrations were well matched in terms of cardiovascular risk factors, medications on admission, and pain to balloon times, but had significantly longer corrected QT interval (QTc) on the 12 lead ECG (p=0.04) despite no differences in PR interval, heart rate or electrolyte concentrations. Conclusions NPY slowed late repolarisation and caused ARIc prolongation, which was associated with increased automaticity in human iPSC-CMs. NPY also correlated with QTc-prolongation in patients being treated for MI and may contribute to the increased incidence of ventricular arrhythmia observed in these patients. Antagonists of Y1 and Y5 receptors may therefore offer an adjunct to β-blockade therapy to reduce the risk of ventricular arrhythmia.

Modified mRNA-based gene editing reveals sarcomere-based regulation of gene expression in human induced-pluripotent stem cell-derived cardiomyocytes

Mutations in genes coding sarcomere components are the major causes of human inherited cardiomyopathy. Genome editing is widely applied to genetic modification of human pluripotent stem cells (hPSCs) before hPSCs were differentiated into cardiomyocytes to model cardiomyopathy. Whether genetic mutations influence the early hPSC differentiation process or solely the terminally differentiated cardiomyocytes during cardiac pathogenesis remains challenging to distinguish. To solve this problem, here we harnessed chemically modified mRNA (modRNA) and synthetic single-guide RNA to develop an efficient genome editing approach in hPSC-derived cardiomyocytes (hPSC-CMs). We showed that modRNA-based CRISPR/Cas9 mutagenesis of TNNT2, the coding gene for cardiac troponin T, results in sarcomere disassembly and contractile dysfunction in hPSC-CMs. These structural and functional phenotypes were associated with profound downregulation of oxidative phosphorylation genes and upregulation of cardiac stress markers NPPA and NPPB. These data confirmed that sarcomeres regulate gene expression in hPSC-CMs and highlighted the RNA technology as a powerful tool to achieve stage-specific genome editing during hPSC differentiation.

SARS-CoV-2 ORF 3a-mediated currents are inhibited by antiarrhythmic drugs.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to cardiovascular complications, notably cardiac arrhythmias. The open reading frame (ORF) 3a of the coronavirus genome encodes for a transmembrane protein that can function as an ion channel. The aim of this study was to investigate the role of the SARS-CoV-2 ORF 3a protein in COVID-19-associated arrhythmias and its potential as a pharmacological target. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and cultured human fibroblasts were infected with SARS-CoV-2. Subsequent immunoblotting assays revealed the expression of ORF 3a protein in hiPSC-CM but not in fibroblasts. After intracytoplasmic injection of RNA encoding ORF 3a proteins into Xenopus laevis oocytes, macroscopic outward currents could be measured. While class I, II, and IV antiarrhythmic drugs showed minor effects on ORF 3a-mediated currents, a robust inhibition was detected after application of class III antiarrhythmics. The strongest effects were observed with dofetilide and amiodarone. Finally, molecular docking simulations and mutagenesis studies identified key amino acid residues involved in drug binding. Class III antiarrhythmic drugs are potential inhibitors of ORF 3a-mediated currents, offering new options for the treatment of COVID-19-related cardiac complications.

Recording and Interpretation of Active Calcium Transients in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Calcium plays a pivotal role in the excitation-contraction coupling process in cardiomyocytes, a critical multi-parametric event leading to rhythmic contraction. Over the past few decades, calcium signaling in cardiomyocytes has been extensively studied in cardiovascular sciences. However, a standard methodology is needed not only to trace the calcium within cells but also to remove signal processing biases and to accurately interpret the features of calcium transient signals in relation to cardiomyocyte electrophysiology. This article outlines the use of genetically encoded calcium indicator (GCaMP) human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to record calcium transients. These cells express a green fluorescent signal when calcium binds to intracellular calmodulin, a key regulator of calcium signaling. The extraction and processing of calcium transient waveforms are performed using ImageJ and MATLAB software. Key features of these waveforms are then identified and categorized based on their physiological relevance to cardiomyocyte function. Additionally, this work includes a Support Protocol for the successful replating of cardiomyocytes onto non-traditional culture platforms, such as metallic sensors and polymer-based substrates, to facilitate data multiplexing. The three Basic Protocols outlined here provide a comprehensive approach for maintaining, expanding, and differentiating the GCaMP hiPSCs, video recording of calcium transients, and the subsequent signal extraction, preprocessing, analysis, and data visualization. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance, expansion, and differentiation of genetically encoded calcium indicator hiPSCs Support Protocol: Replating GCaMP hiPSC-CMs for stimulation and multielectrode array studies Basic Protocol 2: Video recording from calcium transients of GCaMP hiPSC-CMs Basic Protocol 3: Signal extraction, preprocessing, analysis, and data visualization.

Highly conductive, stretchable, and biocompatible graphene oxide biocomposite hydrogel for advanced tissue engineering

The importance of hydrogels in tissue engineering cannot be overemphasized due to their resemblance to the native extracellular matrix. However, natural hydrogels with satisfactory biocompatibility exhibit poor mechanical behavior, which hampers their application in stress-bearing soft tissue engineering. Here, we describe the fabrication of a double methacrylated gelatin bioink covalently linked to graphene oxide (GO) via a zero-length crosslinker, digitally light-processed (DLP) printable into 3D complex structures with high fidelity. The resultant natural hydrogel (GelGOMA) exhibits a conductivity of 15.0 S m-1as a result of the delocalization of theπ-orbital from the covalently linked GO. Furthermore, the hydrogel shows a compressive strength of 1.6 MPa, and a 2.0 mm thick GelGOMA can withstand a 1.0 kg ms-1momentum. The printability and mechanical strengths of GelGOMAs were demonstrated by printing a fish heart with a functional fluid pumping mechanism and tricuspid valves. Its biocompatibility, electroconductivity, and physiological relevance enhanced the proliferation and differentiation of myoblasts and neuroblasts and the contraction of human-induced pluripotent stem cell-derived cardiomyocytes. GelGOMA demonstrates the potential for the tissue engineering of functional hearts and wearable electronic devices.

Abstract Mo053: Acacetin's Antiarrhythmic Potential in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Harboring Hypertrophic and Dilated Cardiomyopathy-Related Mutations

Background: Hypertrophic cardiomyopathy (HCM) is often caused by mutations such as p.R943X in the Myosin Binding Protein C3 (MYBPC3), leading to early and delayed afterdepolarizations, myofibrillar disarray, and calcium dysfunction, which may induce lethal arrhythmias. Dilated cardiomyopathy (DCM) can also result from mutations, such as the p.R14del mutation in the phospholamban (PLN) protein, which is crucial for calcium signaling regulation, leading to arrhythmogenesis when provoked. This study explores the arrhythmogenic potential in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the HCM MYBPC3 p.R943X mutation and the DCM PLN p.R14del mutation. Methods: We utilized a 384-well optical physiological system to record action potentials (APs) in hiPSC-CMs carrying the MYBPC3 p.R943X mutation and the DCM PLN p.R14del mutation, including their isogenic controls. Action potential metrics were measured under spontaneous and electrically paced conditions. The effects of Acacetin, NS-5806, nifedipine, and GS-967 on the recorded AP types were analyzed to understand the underlying channelopathies and arrhythmogenic potential. Special attention was given to the variability between the HCM MYBPC3 p.R943X and DCM PLN p.R14del cell lines' susceptibility to arrhythmogenesis and their isogenic controls. Results: The HCM MYBPC3 p.R943X hiPSC-CMs exhibited a significant increase in early afterdepolarizations, non-sustained and sustained ventricular tachycardia, and AP notches compared to their isogenic control. AP notches and arrhythmogenic events were notably provoked by NS-5806 in both the HCM MYBPC3 p.R943X and DCM PLN p.R14del hiPSC-CMs. However, these were significantly ameliorated (p<0.05) by Acacetin administration (5 µM), highlighting its potential as an effective antiarrhythmic in managing both HCM- and DCM-induced arrhythmias. GS-967 also showed efficacy in reducing early afterdepolarizations (EADs) in the HCM model. Conclusions: This study provides novel insights into the arrhythmogenic potential of hiPSC-CMs carrying MYBPC3 p.R943X and PLN p.R14del mutations, underscoring the significant role of Acacetin in attenuating these effects. It paves the way for developing targeted therapies that address the complex molecular mechanisms underlying arrhythmogenesis in cardiomyopathies. The findings suggest that Acacetin could be a promising candidate for the pharmacologic treatment of cardiomyopathies characterized by arrhythmogenic risks.

Exosomal Preconditioning of Human iPSC-Derived Cardiomyocytes Beneficially Alters Cardiac Electrophysiology and Micro RNA Expression.

Experimental evidence, both in vitro and in vivo, has indicated cardioprotective effects of extracellular vesicles (EVs) derived from various cell types, including induced pluripotent stem cell-derived cardiomyocytes. The biological effects of EV secretion, particularly in the context of ischemia and cardiac electrophysiology, remain to be fully explored. Therefore, the goal of this study was to unveil the effects of exosome (EXO)-mediated cell-cell signaling during hypoxia by employing a simulated preconditioning approach on human-induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs). Electrophysiological activity of hIPSC-CMs was measured using a multielectrode array (MEA) system. A total of 16 h of hypoxic stress drastically increased the beat period. Moreover, hIPSC-CMs preconditioned with EXOs displayed significantly longer beat periods compared with non-treated cells after 16 h of hypoxia (+15.7%, p < 0.05). Furthermore, preconditioning with hypoxic EXOs resulted in faster excitation-contraction (EC) coupling compared with non-treated hIPSC-CMs after 16 h of hypoxia (-25.3%, p < 0.05). Additionally, microRNA (miR) sequencing and gene target prediction analysis of the non-treated and pre-conditioned hIPSC-CMs identified 10 differentially regulated miRs and 44 gene targets. These results shed light on the intricate involvement of miRs, emphasizing gene targets associated with cell survival, contraction, apoptosis, reactive oxygen species (ROS) regulation, and ion channel modulation. Overall, this study demonstrates that EXOs secreted by hIPSC-CM during hypoxia beneficially alter electrophysiological properties in recipient cells exposed to hypoxic stress, which could play a crucial role in the development of targeted interventions to improve outcomes in ischemic heart conditions.

Abstract Mo080: Clinically Variable Penetrant MYH7 G256E Mutation Shows Gene-Dose-Dependent HCM Disease Phenotype on a Transcriptomic, Proteomic, and Functional Level

Introduction: Hypertrophic cardiomyopathy (HCM) is a monogenic disorder caused by heterozygous (HET) mutations in sarcomere genes such as MYH7 . Recently, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying HET HCM mutations have shed new insights into the molecular mechanisms of HCM. However, the cause of phenotype variability and the role of allelic imbalance in phenotype penetrance of HET missense mutations such as MYH7 G256E remain unclear. Hypothesis: We hypothesize that hiPSC CMs carrying clinically variable penetrant HCM missense mutations such as MYH7 G256E show a gene dose-dependent disease phenotype indicating their susceptibility for allelic imbalance as a cause for variable disease phenotype penetrance. Methods: We generated HET and homozygous (HOM) hiPSC-lines for variable penetrant HCM mutation MYH7 G256E. We performed multiplexed single-cell RNA sequencing and quantitative proteomics analyses on wildtype (WT), HET, and HOM iPSC-CMs at day 30 of cardiac differentiation. Additional Digital Droplet PCR studies were performed to investigate the MYH7 mutant allele expression. The functional contractile phenotype was assessed with Sony SI8000 Cell motion imaging system. Results: We detected a large clone-to-clone and batch-to-batch variability in HET iPSC-CMs on a transcriptomic and proteomic level that obscured the identification of a consistent mutant phenotype in HET MYH7 G256E vs WT CMs. These phenotypes correlated with a 1:1 bulk ratio of mutant vs WT mRNA and protein expression, while overall MYH7 gene and protein expression remained comparable. Transcriptomic and proteomic analyses of WT, HET, and HOM CMs show a gene dose-dependent regulation of known hypertrophy response markers such as NPPB (mRNA: HET vs WT: +1.76x p .adj=7.0*10 -7 , HOM vs WT: +5.42x p.adj=1.5*10 -48 ; protein: HET vs WT: +1.15x, 95% CI [0.86, 1.58]; HOM vs WT: +1.51x, 95% CI [1.12, 2.01]) which functionally correlated with enhanced contractility (contraction velocity in μm/s: WT: 5.84 +/-2.24, HET: 6.04 +/-2.89, HOM: 7.77 +/-3.352, p=0.3*10 -2 ANOVA) and the more frequent occurrence of arrhythmias (WT: 1/46, HET: 0/55, HOM: 5/41). Conclusions: Hypertrophic disease phenotype in MYH7 G256E mutation iPSC-CMs shows a gene dose-dependent penetrance on a transcriptomic, proteomic, and functional level. This indicates susceptibility to allelic imbalance as a potential cause for variable disease penetrance.

Computational approaches identify a transcriptomic fingerprint of drug-induced structural cardiotoxicity

Structural cardiotoxicity (SCT) presents a high-impact risk that is poorly tolerated in drug discovery unless significant benefit is anticipated. Therefore, we aimed to improve the mechanistic understanding of SCT. First, we combined machine learning methods with a modified calcium transient assay in human-induced pluripotent stem cell-derived cardiomyocytes to identify nine parameters that could predict SCT. Next, we applied transcriptomic profiling to human cardiac microtissues exposed to structural and non-structural cardiotoxins. Fifty-two genes expressed across the three main cell types in the heart (cardiomyocytes, endothelial cells, and fibroblasts) were prioritised in differential expression and network clustering analyses and could be linked to known mechanisms of SCT. This transcriptomic fingerprint may prove useful for generating strategies to mitigate SCT risk in early drug discovery.Graphical

Innovative approaches to cardiovascular safety pharmacology assessment

This editorial prefaces the annual themed issue on safety pharmacology (SP) methods which has been published since 2004 in the Journal of Pharmacological and Toxicological Methods (JPTM). Here we highlight content derived from the 2023 Safety Pharmacology Society (SPS) meeting held in Brussels, Belgium. The meeting generated 138 abstracts, reproduced in the current volume of JPTM. As in prior years, the manuscripts reflect various areas of innovation in SP including in silico modeling of stroke volume, cardiac output and systemic vascular resistance, computational approaches that compare drug-induced proarrhythmic sensitivity of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), an evaluation of the utility of the corrected J-Tpeak and Tpeak-to-Tend parameters from the ECG as potential proarrhythmia biomarkers, and the applicability of nonclinical concentration-QTc (C-QTc) modeling of data derived from the conduct of the in vivo QTc study as a component of the core battery of safety pharmacology studies.

A Possible Role of Tetrodotoxin-Sensitive Na+ Channels for Oxidation-Induced Late Na+ Currents in Cardiomyocytes.

An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na+ currents by removing the inactivation of voltage-gated Na+ channels including the tetrodotoxin (TTX)-resistant cardiac α-subunit Nav1.5 as well as TTX-sensitive α-subunits like Nav1.2 and Nav1.3. Here, we explored oxidant-induced late Na+ currents in mouse cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in HEK 293 cells expressing Nav1.2, Nav1.3, or Nav1.5. Na+ currents in mouse cardiomyocytes and hiPSC-CMs treated with the oxidant chloramine T (ChT) developed a moderate reduction in peak current amplitudes accompanied by large late Na+ currents. While ChT induced a strong reduction in peak current amplitudes but only small persistent currents on Nav1.5, both Nav1.2 and Nav1.3 produced increased peak current amplitudes and large persistent currents following oxidation. TTX (300 nM) blocked ChT-induced late Na+ currents significantly stronger as compared to peak Na+ currents in both mouse cardiomyocytes and hiPSC-CMs. Similar differences between Nav1.2, Nav1.3, and Nav1.5 regarding ROS sensitivity were also evident when oxidation was induced with UVA-light (380 nm) or the cysteine-selective oxidant nitroxyl (HNO). To conclude, our data on TTX-sensitive Na+ channels expressed in cardiomyocytes may be relevant for the generation of late Na+ currents following oxidative stress.

Human cardiomyocyte derived extracellular vesicles regulate cardiac fibroblast activation through miR-24

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondazione Leonardo Introduction Cardiovascular diseases are associated with increase in interstitial fibrosis due to cardiac fibroblast (CF) activation into myofibroblast leading to the secretion and deposition of extracellular matrix (ECM). The intricate functional activation of these processes is not well understood, especially concerning the contribution of the microenvironment. One of the most important mechanisms of paracrine communication are due to Extracellular vesicles (EVs). EVs are cell-released nanoparticles carrying proteins, lipids, and nucleic acids, particularly miRs. To note, previous transcriptomic analysis of healthy human heart demonstrated that miR24-3p is one of the most abundant miRs [1], suggesting an important role associated with its expression. Aim of the work Here we aim to investigate the role of miR24-3p as regulator of cardiac fibroblast activation, focusing on healthy and pathological conditions. Materials and methods Bioinformatic analysis of open-data, single-cell RNA sequencing from heart tissue was conducted to assess the expression of miR24-3p and its specific targets in both healthy and infarcted myocardium. To explore the functional role of miR24-3p, loss- and gain-of-function experiments were performed by transfecting cardiac fibroblasts (CF) with specific antimiR or mimic oligonucleotides, respectively. In investigating the influence of cardiomyocytes (CM) on CF regulation, extracellular vesicles (EVs) from human-induced pluripotent stem cell-derived CM were isolated and utilized in in vitro experiments. The expression levels of miRNA24-3p were also evaluated in the plasma of healthy individuals and patients who had experienced myocardial infarction. Finally, cardiac slices from human tissue were employed as ex-vivo microtissues to functionally validate the in vitro findings. Results RealTime analysis of CF, CM and cardiac endothelial cells (EC) revealed a CM specific expression of miR24-3p, not only at cellular level but also in EVs. Upregulation of miR24-3p in CF was able to significantly reduce protein expression of FURIN, CYCLD1 and SMAD4, resulting in decrease of CF activation and ECM secretion. Interestingly, treatment of CF with CM-EVs recapitulate miR24-3p transfection, while EVs derived from hypoxia cultured CM (MI-like) does not. RNAScope and ddPCR analysis on human cardiac slice cultured under hypoxia conditions confirmed decrease in miR24-3p expression upon induction of stress. Ultimately, a significant decrease in miR24-3p in circulating serum EVs of MI patients’ was observed. Conclusions These data demonstrate the role of miR24-3p in CF regulation through the paracrine activity mediated by CM-EVs. Further analysis, including the use of a 3D human heart microtissue, will confirm the mechanism of cross-talk between CM and CF.

Reduced Graphene-Oxide-Doped Elastic Biodegradable Polyurethane Fibers for Cardiomyocyte Maturation.

Conductive biomaterials offer promising solutions to enhance the maturity of cultured cardiomyocytes. While the conventional culture of cardiomyocytes on nonconductive materials leads to more immature characteristics, conductive microenvironments have the potential to support sarcomere development, gap junction formation, and beating of cardiomyocytes in vitro. In this study, we systematically investigated the behaviors of cardiomyocytes on aligned electrospun fibrous membranes composed of elastic and biodegradable polyurethane (PU) doped with varying concentrations of reduced graphene oxide (rGO). Compared to PU and PU-4%rGO membranes, the PU-10%rGO membrane exhibited the highest conductivity, approaching levels close to those of native heart tissue. The PU-rGO membranes retained anisotropic viscoelastic behavior similar to that of the porcine left ventricle and a superior tensile strength. Neonatal rat cardiomyocytes (NRCMs) and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on the PU-rGO membranes displayed enhanced maturation with cell alignment and enhanced sarcomere structure and gap junction formation with PU-10%rGO having the most improved sarcomere structure and CX-43 presence. hiPSC-CMs on the PU-rGO membranes exhibited a uniform and synchronous beating pattern compared with that on PU membranes. Overall, PU-10%rGO exhibited the best performance for cardiomyocyte maturation. The conductive PU-rGO membranes provide a promising matrix for in vitro cardiomyocyte culture with promoted cell maturation/functionality and the potential for cardiac disease treatment.

HiPSC-CM electrophysiology: impact of temporal changes and study parameters on experimental reproducibility.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are frequently used for preclinical cardiotoxicity testing and remain an important tool for confirming model-based predictions of drug effects in accordance with the comprehensive in vitro proarrhythmia assay (CiPA). Despite the considerable benefits hiPSC-CMs provide, concerns surrounding experimental reproducibility have emerged. We investigated the effects of temporal changes and experimental parameters on hiPSC-CM electrophysiology. iCell cardiomyocytes2 were cultured and biosignals were acquired using a microelectrode array (MEA) system (2-14 days). Continuous recordings revealed a 22.6% increase in the beating rate and 7.7% decrease in the field potential duration (FPD) during a 20-min equilibration period. Location-specific differences across a multiwell plate were also observed, with iCell cardiomyocytes2 in the outer rows beating 8.8 beats/min faster than the inner rows. Cardiac endpoints were also impacted by cell culture duration; from 2 to 14 days, the beating rate decreased (-12.7 beats/min), FPD lengthened (+257 ms), and spike amplitude increased (+3.3 mV). Cell culture duration (4-10 days) also impacted cardiomyocyte drug responsiveness (E-4031, nifedipine, isoproterenol). qRT-PCR results suggest that daily variations in cardiac metrics may be linked to the continued maturation of hiPSC-CMs in culture (2-30 days). Daily experiments were also repeated using a second cell line (Cor.4U). Collectively, our study highlights multiple sources of variability to consider and address when performing hiPSC-CM MEA studies. To improve reproducibility and data interpretation, MEA-based studies should establish a standardized protocol and report key experimental conditions (e.g., cell line, culture time, equilibration time, electrical stimulation settings, and raw data values).NEW & NOTEWORTHY We demonstrate that iCell cardiomyocytes2 electrophysiology measurements are impacted by deviations in experimental techniques including electrical stimulation protocols, equilibration time, well-to-well variability, and length of hiPSC-CM culture. Furthermore, our results indicate that hiPSC-CM drug responsiveness changes within the first 2 wk following defrost.

Inhibition of TBL1 cleavage alleviates doxorubicin-induced cardiomyocytes death by regulating the Wnt/β-catenin signal pathway.

Doxorubicin (DOX) is a widely used anthracycline anticancer agent; however, its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately, the pathophysiology of DICT has not yet been fully elucidated, and there are no effective strategies for its prevention or treatment. In this investigation, the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored. We observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites-D125, D136, and D215. Interestingly, overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis, effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with β-catenin. As a result, Wnt reporter activity and Wnt target gene expression collectively indicate a decrease in Wnt/β-catenin signalling, leading to DICT progression. Furthermore, the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However, these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally, in a DICT mouse model, TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation, ultimately protecting cardiomyocytes from cell death. Our findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function, even in the context of DOX administration. Consequently, this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT.

CD8+ T cell-mediated rejection of allogenic human-induced pluripotent stem cell-derived cardiomyocyte sheets in human PBMC-transferred NOG MHC double knockout mice

BackgroundTransplantation of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has emerged as a promising therapy to treat end-stage heart failure. However, immunogenicity of hiPS-CMs in transplanted patients has not been fully elucidated. Thus, in vivo models are required to estimate immune responses against hiPS-CMs in transplant recipients. MethodsWe transferred human peripheral blood mononuclear cells (hPBMCs) into NOD/Shi-scid IL2rgnull (NOG) MHC class I/II double knockout (NOG-ΔMHC) mice, which were reported to accept hPBMCs without xenogeneic-graft-versus-host disease (xeno-GVHD). Then, hiPS-CM sheets generated from the hiPS cell line 201B7 harboring a luciferase transgene were transplanted into the subcutaneous space of NOG-ΔMHC mice. Graft survival was monitored by bioluminescent images using a Xenogen In Vivo Imaging System. ResultsThe human immune cells were engrafted for more than three months in NOG-ΔMHC mice without lethal xeno-GVHD. The hiPS-CMs expressed a moderate level of human leukocyte antigen (HLA)-class I, but not HLA-class II, molecules even after interferon-gamma (IFN-γ) stimulation. Consistently, the allogenic IFN-γ-treated hiPS-CMs induced weak CD8+ but not CD4+, T cell responses in vitro. hiPS-CM sheets disappeared approximately 17–24 days after transplantation in hPBMC-transferred NOG-ΔMHC mice, and CD8+ T cell depletion significantly prolonged graft survival, similar to what was observed following tacrolimus treatment. ConclusionhiPS-CMs are less immunogenic in vitro but induce sufficient CD8+ T cell-mediated immune responses for graft rejection in vivo.

Evaluation of Contractile Function Using Human iPS Cell-derived Cardiomyocytes

Cardiotoxicity induced by anti-cancer drugs is a significant concern for patients undergoing cancer treatment. Some anti-cancer drugs can damage cardiac muscle cells directly or indirectly, potentially leading to severe heart failure. Various risk factors, including the type and dosage of chemotherapy agents as well as patient background, contribute to the development of cardiotoxicity. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which enable patient-specific toxicity prediction, hold great promise in this regard. However, the practical implementation of hiPSC-CMs-based prediction of anti-cancer drug-induced cardiotoxicity still faces hurdles. One major challenge involves establishing and optimizing experimental systems for evaluating contractile dysfunction, the ultimate output of heart failure, using hiPSC-CMs. Such efforts are currently underway globally, focusing on tailoring functional evaluation systems to the characteristics of hiPSC-CMs. In this paper, we provide an overview of the contraction mechanisms of cardiac cells and introduce a method of measuring contraction that we have developed, and discuss the current status of contractile function evaluation methods using hiPSC-CMs.

3D Aligned Nanofiber Scaffold Fabrication with Trench-Guided Electrospinning for Cardiac Tissue Engineering.

Constructing three-dimensional (3D) aligned nanofiber scaffolds is significant for the development of cardiac tissue engineering, which is promising in the field of drug discovery and disease mechanism study. However, the current nanofiber scaffold preparation strategy, which mainly includes manual assembly and hybrid 3D printing, faces the challenge of integrated fabrication of morphology-controllable nanofibers due to its cross-scale structural feature. In this research, a trench-guided electrospinning (ES) strategy was proposed to directly fabricate 3D aligned nanofiber scaffolds with alternative ES and a direct ink writing (DIW) process. The electric field effect of DIW poly(dimethylsiloxane) (PDMS) side walls on guiding whipping ES nanofibers was investigated to construct trench design rules. It was found that the width/height ratio of trenches greatly affected the nanofiber alignment, and the trench width/height ratio of 1.5 provided the nanofiber alignment degree over 60%. As a proof of principle, 3D nanofiber scaffolds with controllable porosity (60-80%) and alignment (30-60%) were fabricated. The effect of the scaffolds was verified by culturing human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which resulted in the uniform 3D distribution of aligned hiPSC-CMs with ∼1000 μm thickness. Therefore, this printing strategy shows great potential for the efficient engineered tissue construction.

Generation of a human iPSC-derived cardiomyocyte/fibroblast engineered heart tissue model

Animal models have proven integral to broadening our understanding of complex cardiac diseases but have been hampered by significant species-dependent differences in cellular physiology. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown great promise in the modelling of cardiac diseases despite limitations in functional and structural maturity. 3D stem cell-derived cardiac models represent a step towards mimicking the intricate microenvironment present in the heart as an in vitro model. Incorporation of non-myocyte cell types, such as cardiac fibroblasts, into engineered heart tissue models (EHTs) can help better recapitulate the cell-to-cell and cell-to-matrix interactions present in the human myocardium. Integration of human-induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CFs) and hiPSC-CM into EHT models enables the generation of a genetically homogeneous modelling system capable of exploring the abstruse structural and electrophysiological interplay present in cardiac pathophysiology. Furthermore, the construction of more physiologically relevant 3D cardiac models offers great potential in the replacement of animals in heart disease research. Here we describe efficient and reproducible protocols for the differentiation of hiPSC-CMs and hiPSC-CFs and their subsequent assimilation into EHTs. The resultant EHT consists of longitudinally arranged iPSC-CMs, incorporated alongside hiPSC-CFs. EHTs with both hiPSC-CMs and hiPSC-CFs exhibit slower beating frequencies and enhanced contractile force compared to those composed of hiPSC-CMs alone. The modified protocol may help better characterise the interplay between different cell types in the myocardium and their contribution to structural remodelling and cardiac fibrosis.