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Abstract Activation of cardiac fibroblasts and differentiation to myofibroblasts underlies development of pathological cardiac fibrosis, leading to arrhythmias and heart failure. Myofibroblasts are characterised by increased α-smooth muscle actin (α-SMA) fibre expression, secretion of collagens and changes in proliferation. Transforming growth factor-beta (TGF-β) and increased mechanical stress can initiate myofibroblast activation. Reversibility of the myofibroblast phenotype has been observed in murine cells but has not been explored in human cardiac fibroblasts. In this study, chronically activated adult primary human ventricular cardiac fibroblasts and human induced pluripotent stem cell derived cFbs (hiPSC-cFbs) were used to investigate the potential for reversal of the myofibroblast phenotype using either subculture on soft substrates or TGF-β receptor inhibition. Culture on softer plates (25 or 2 kPa Young’s modulus) did not alter proliferation or reduce expression of α-SMA and collagen 1. Similarly, culture of myofibroblasts in the presence of TGF-β inhibitor did not reverse myofibroblasts back to a quiescent phenotype. Chronically activated hiPSC-cFbs also showed attenuated response to TGF-β receptor inhibition and inability to reverse to quiescent fibroblast phenotype. Our data demonstrate substantial loss of TGF-β signalling plasticity as well as a loss of feedback from the surrounding mechanical environment in chronically activated human myofibroblasts.

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.

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

Summary: Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/−) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/− hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.

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.

Abstract Polymerase chain reaction (PCR) has proven to be the gold-standard for SARS-CoV-2 detection in clinical settings. The most common approaches rely on nasopharyngeal specimens obtained from swabs, followed by RNA extraction, reverse transcription and quantitative PCR. Although swab-based PCR is sensitive, swabbing is invasive and unpleasant to administer, reducing patient compliance for regular testing and resulting in an increased risk of improper sampling. To overcome these obstacles, we developed a non-invasive one-step RT-qPCR assay performed directly on saliva specimens. The University of Nottingham Asymptomatic Testing Service protocol simplifies sample collection and bypasses the need for RNA extraction, or additives, thus helping to encourage more regular testing and reducing processing time and costs. We have evaluated the assay against the performance criteria specified by the UK regulatory bodies and attained accreditation (BS EN ISO/IEC 17,025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service. We observed a sensitivity of 1 viral copy per microlitre of saliva, and demonstrated a concordance of > 99.4% between our results and those of other accredited testing facilities. We concluded that saliva is a stable medium that allows for a highly precise, repeatable, and robust testing method.

Abstract The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+‐load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow‐up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end‐stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non‐failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca2+‐binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+‐scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.

During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (β2AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions. To overcome these limitations, we used CRISPR/Cas9 gene editing technology in human pluripotent stem cells (hPSCs) to create a unique suite of four isogenic homozygous variants at amino acid positions 16(G/R) and 27(G/Q), which reside in the N terminus of the β2AR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level β2AR signaling dominated over β1AR . Examining changes in beat rates and responses to isoprenaline, Gi coupling, cyclic AMP (cAMP) production, downregulation, and desensitization indicated that responses were often heightened for the GE variant, implying differential dominance of both polymorphic location and amino acid substitution. This finding was corroborated, since GE showed hypersensitivity to doxorubicin-induced cardiotoxicity relative to GQ and RQ variants. Thus, understanding the effect of β2AR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact.

Summary: Force measurements in ex vivo and engineered heart tissues are well established. Analysis of calcium transients (CaT) is complementary to force, and the combined analysis is meaningful to the study of cardiomyocyte biology and disease. This article describes a model of human induced pluripotent stem cell cardiomyocyte-derived engineered heart tissues (hiPSC-CM EHTs) transduced with the calcium sensor GCaMP6f followed by sequential analysis of force and CaT. Average peak analysis demonstrated the temporal sequence of the CaT preceding the contraction twitch. The pharmacological relevance of the test system was demonstrated with inotropic indicator compounds. Force-frequency relationship was analyzed in the presence of ivabradine (300 nM), which reduced spontaneous frequency and unmasked a positive correlation of force and CaT at physiological human heart beating frequency with stimulation frequency between 0.75 and 2.5 Hz (force +96%; CaT +102%). This work demonstrates the usefulness of combined force/CaT analysis and demonstrates a positive force-frequency relationship in hiPSC-CM EHTs. : In this article, Hansen and colleagues establish a system using human induced pluripotent stem cell-derived cardiomyocytes to sequentially analyze force and calcium transients (CaTs) in an engineered heart tissue model by using genetically encoded calcium indicator (GCaMP6f) and demonstrate a positive force- and CaT-frequency relationship at physiological human heart beat frequencies. Keywords: positive force-frequency relationship, positive calcium transient-frequency relationship, omecamtiv mecarbil, engineered heart tissues, human induced pluripotent stem cell-derived cardiomyocytes

Patients with heart failure often develop cardiac arrhythmias. The mechanisms and interrelations linking heart failure and arrhythmias are not fully understood. Historically, research into arrhythmias has been performed on affected individuals or in vivo (animal) models. The latter however is constrained by interspecies variation, demands to reduce animal experiments and cost. Recent developments in in vitro induced pluripotent stem cell technology and in silico modelling have expanded the number of models available for the evaluation of heart failure and arrhythmia. An agnostic approach, combining the modalities discussed here, has the potential to improve our understanding for appraising the pathology and interactions between heart failure and arrhythmia and can provide robust and validated outcomes in a variety of research settings. This review discusses the state of the art models, methodologies and techniques used in the evaluation of heart failure and arrhythmia and will highlight the benefits of using them in combination. Special consideration is paid to assessing the pivotal role calcium handling has in the development of heart failure and arrhythmia.

During the COVID-19 pandemic, countries with robust population-based asymptomatic testing were generally successful in controlling virus spread, hence reducing hospitalizations and deaths. This effectiveness inspired widespread asymptomatic surveillance for COVID-19/SARS-CoV-2 globally. Polarized vaccination programs, coupled with the relatively short-lived immunity vaccines provide, mean that reciprocal cross-border exchanges of each new variant are likely, as evidenced by Delta and Gamma, and asymptomatic testing will be required for the foreseeable future. Reliance on nasopharyngeal swabs contributes to “testing fatigue” arising due to difficulties in standardizing administration, unpleasantness, and inappropriateness of use in younger people or individuals with special needs. There has also been erosion in confidence of testing due to variable and/or poor accuracy of lateral flow devices to detect COVID-19. Here, we question why saliva-based PCR assays are not being used more widely, given that standardization is easy and this non-invasive test is suitable for everyone, providing high sensitivity and accuracy. We reflect on our experience with the University of Nottingham COVID-19 Asymptomatic Testing, where (as of October 2021) 96,317 samples have been processed by RT-qPCR from 23,740 repeat saliva donors, yielding 465 positive cases. We challenge myths that saliva is difficult to process, concluding that it is an undervalued resource for both asymptomatic and symptomatic detection of SARS-CoV-2 genomes to an accuracy of >99% and a sensitivity of 1–10 viral copies/μl. In July 2021, our data enabled Nottingham to become the first UK University to gain accreditation and the first UK institute to gain this accolade for saliva.

Cardiomyocytes obtained from human induced pluripotent stem cells are increasingly used for drug testing, but they are not always predictive of the heart contractile responses. Here the authors develop a method to measure cytosolic calcium, action potentials and contraction simultaneously, to achieve higher sensitivity for drug screenings.

Abstract Cardiac fibroblasts are the primary cell type responsible for deposition of extracellular matrix in the heart, providing support to the contracting myocardium and contributing to a myriad of physiological signaling processes. Despite the importance of fibrosis in processes of wound healing, excessive fibroblast proliferation and activation can lead to pathological remodeling, driving heart failure and the onset of arrhythmias. Our understanding of the mechanisms driving the cardiac fibroblast activation and proliferation is expanding, and evidence for their direct and indirect effects on cardiac myocyte function is accumulating. In this review, we focus on the importance of the fibroblast‐to‐myofibroblast transition and the cross talk of cardiac fibroblasts with cardiac myocytes. We also consider the current use of models used to explore these questions.

Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal ‘testing fatigue’ and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed.

Summary: Hypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs. Aberrant phenotypes were most common in hiPSC-CMs produced from the heterozygote father. Unexpectedly, pathological phenotypes were less evident in E99K-expressing hiPSC-CMs from the two sons. Mechanistic insight from Ca2+ handling expression studies prompted pharmacological rescue experiments, wherein dual dantroline/ranolazine treatment was most effective. Our data are consistent with E99K mutant protein being a central cause of HCM but the three-way interaction between the primary genetic lesion, background (epi)genetics, and donor patient age may influence the pathogenic phenotype. This illustrates the value of isogenic hiPSC-CMs in genotype-phenotype correlations. : In this article Smith, Denning and colleagues show that the E99K-ACTC1 cardiac actin mutation is a central cause of HCM, but the three-way interaction between the primary genetic lesion, background genetics, and donor patient age may influence the pathogenic phenotype. Pharmacological rescue experiments demonstrated dual dantroline/ranolazine to be an effective treatment. Keywords: arrhythmia, contractile function, hypertrophy, cardiomyopathy

Abstract Background Stem cells are increasingly seen as a solution for many health challenges for an ageing population. However, their potential benefits in the clinic are currently curtailed by technical challenges such as high cell dose requirements and point of care delivery, which pose sourcing and logistics challenges. Cell manufacturing solutions are currently in development to address the supply issue, and ancillary technologies such as nanoparticle-based labelling are being developed to improve stem cell delivery and enable post-treatment follow-up. Methods The application of magnetic particle (MP) labelling to potentially scalable cell manufacturing processes was investigated in a range of therapeutically relevant cells, including mesenchymal stromal cells (MSC), cardiomyocytes (CMC) and neural progenitor cells (ReN). The efficiency and the biological effect of particle labelling were analysed using fluorescent imaging and cellular assays. Results Flow cytometry and fluorescent microscopy confirmed efficient labelling of monolayer cultures. Viability was shown to be retained post labelling for all three cell types. MSC and CMC demonstrated higher tolerance to MP doses up to 100× the standard concentration. This approach was also successful for MP labelling of suspension cultures, demonstrating efficient MP uptake within 3 h, while cell viability was unaffected by this suspension labelling process. Furthermore, a procedure to enable the storing of MP-labelled cell populations to facilitate cold chain transport to the site of clinical use was investigated. When MP-labelled cells were stored in hypothermic conditions using HypoThermosol solution for 24 h, cell viability and differentiation potential were retained post storage for ReN, MSC and beating CMC. Conclusions Our results show that a generic MP labelling strategy was successfully developed for a range of clinically relevant cell populations, in both monolayer and suspension cultures. MP-labelled cell populations were able to undergo transient low-temperature storage whilst maintaining functional capacity in vitro. These results suggest that this MP labelling approach can be integrated into cell manufacturing and cold chain transport processes required for future cell therapy approaches.

Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operational in this system; however, it is still unknown if TDG/BER-dependent demethylation is used during other types of cellular differentiation. Here we analyze dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells toward hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment, and drop in differentiating cells concurrently with the onset of expression of α fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the beginning of their expression. Our data indicate that transient 5caC accumulation is a common feature of 2 different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.

The congenital long QT syndrome (LQTS) is a cardiac electrophysiological disorder that can cause sudden cardiac death. LQT1 is a subtype of LQTS caused by mutations in KCNQ1, affecting the slow delayed-rectifier potassium current (IKs), which is essential for cardiac repolarization. Paradoxically, gain-of-function mutations in KCNQ1 have been reported to cause borderline QT prolongation, atrial fibrillation (AF), sinus bradycardia, and sudden death, however, the mechanisms are not well understood. The goal of the study is to investigate the ionic, cellular and tissue mechanisms underlying the complex phenotype of a gain-of-function mutation in KCNQ1, c.686G > A (p.G229D) using computer modeling and simulations informed by in vitro measurements. Previous studies have shown this mutation to cause AF and borderline QT prolongation. We report a clinical description of a family that carry this mutation and that a member of the family died suddenly during sleep at 21 years old. Using patch-clamp experiments, we confirm that KCNQ1-G229D causes a significant gain in channel function. We introduce the effect of the mutation in populations of atrial, ventricular and sinus node (SN) cell models to investigate mechanisms underlying phenotypic variability. In a population of human atrial and ventricular cell models and tissue, the presence of KCNQ1-G229D predominantly shortens atrial action potential duration (APD). However, in a subset of models, KCNQ1-G229D can act to prolong ventricular APD by up to 7% (19 ms) and underlie depolarization abnormalities, which could promote QT prolongation and conduction delays. Interestingly, APD prolongations were predominantly seen at slow pacing cycle lengths (CL > 1,000 ms), which suggests a greater arrhythmic risk during bradycardia, and is consistent with the observed sudden death during sleep. In a population of human SN cell models, the KCNQ1-G229D mutation results in slow/abnormal sinus rhythm, and we identify that a stronger L-type calcium current enables the SN to be more robust to the mutation. In conclusion, our computational modeling experiments provide novel mechanistic explanations for the observed borderline QT prolongation, and predict that KCNQ1-G229D could underlie SN dysfunction and conduction delays. The mechanisms revealed in the study can potentially inform management and treatment of KCNQ1 gain-of-function mutation carriers.

Twenty years since their first derivation, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have shown promise in disease modelling research, while their potential for cardiac repair is being investigated. However, low transfection efficiency is a barrier to wider realisation of the potential this model system has to offer. We endeavoured to produce a protocol for improved transfection of hPSC-CMs using the ViafectTM reagent by Promega. Through optimisation of four essential parameters: (i) serum supplementation, (ii) time between replating and transfection, (iii) reagent to DNA ratio and (iv) cell density, we were able to successfully transfect hPSC-CMs to ~95% efficiencies. Transfected hPSC-CMs retained high purity and structural integrity despite a mild reduction in viability, and preserved compatibility with phenotyping assays of hypertrophy. This protocol greatly adds value to the field by overcoming limited transfection efficiencies of hPSC-CMs in a simple and quick approach that ensures sustained expression of transfected genes for at least 14 days, opening new opportunities in mechanistic discovery for cardiac-related diseases.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), and is characterized by progressive weakness in skeletal and cardiac muscles. Currently, dilated cardiomyopathy due to cardiac muscle loss is one of the major causes of lethality in late-stage DMD patients. To study the molecular mechanisms underlying dilated cardiomyopathy in DMD heart, we generated cardiomyocytes (CMs) from DMD and healthy control induced pluripotent stem cells (iPSCs). DMD iPSC-derived CMs (iPSC-CMs) displayed dystrophin deficiency, as well as the elevated levels of resting Ca2+, mitochondrial damage and cell apoptosis. Additionally, we found an activated mitochondria-mediated signaling network underlying the enhanced apoptosis in DMD iPSC-CMs. Furthermore, when we treated DMD iPSC-CMs with the membrane sealant Poloxamer 188, it significantly decreased the resting cytosolic Ca2+ level, repressed caspase-3 (CASP3) activation and consequently suppressed apoptosis in DMD iPSC-CMs. Taken together, using DMD patient-derived iPSC-CMs, we established an in vitro model that manifests the major phenotypes of dilated cardiomyopathy in DMD patients, and uncovered a potential new disease mechanism. Our model could be used for the mechanistic study of human muscular dystrophy, as well as future preclinical testing of novel therapeutic compounds for dilated cardiomyopathy in DMD patients.

Cardiomyocytes from human embryonic stem cells (hESC-CMs) and induced pluripotent stem cells (hiPSC-CMs) represent new models for drug discovery. Although hypertrophy is a high-priority target, we found that hiPSC-CMs were systematically unresponsive to hypertrophic signals such as the α-adrenoceptor (αAR) agonist phenylephrine (PE) compared to hESC-CMs. We investigated signaling at multiple levels to understand the underlying mechanism of this differential responsiveness. The expression of the normal α1AR gene, ADRA1A, was reversibly silenced during differentiation, accompanied by ADRA1B upregulation in either cell type. ADRA1B signaling was intact in hESC-CMs, but not in hiPSC-CMs. We observed an increased tonic activity of inhibitory kinase pathways in hiPSC-CMs, and inhibition of antihypertrophic kinases revealed hypertrophic increases. There is tonic suppression of cell growth in hiPSC-CMs, but not hESC-CMs, limiting their use in investigation of hypertrophic signaling. These data raise questions regarding the hiPSC-CM as a valid model for certain aspects of cardiac disease.

Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular disease characterised by multifarious hallmarks, a heterogeneous set of clinical manifestations, and several molecular mechanisms. Various disease models have been developed to study this condition, but they often show contradictory results, due to technical constraints and/or model limitations. Therefore, new tools are needed to better investigate pathological features in an unbiased and technically refined approach, towards improving understanding of disease progression. Herein, we describe three simple protocols to phenotype cellular models of HCM in vitro, in a high-throughput manner where technical artefacts are minimized. These are aimed at investigating: (1) Hypertrophy, by measuring cell volume by flow cytometry; (2) HCM molecular features, through the analysis of a hypertrophic marker, multinucleation, and sarcomeric disarray by high-content imaging; and (3) mitochondrial respiration and content via the Seahorse™ platform. Collectively, these protocols comprise straightforward tools to evaluate molecular and functional parameters of HCM phenotypes in cardiomyocytes in vitro. These facilitate greater understanding of HCM and high-throughput drug screening approaches and are accessible to all researchers of cardiac disease modelling. Whilst HCM is used as an exemplar, the approaches described are applicable to other cellular models where the investigation of identical biological changes is paramount.

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.

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.

Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca 2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.