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Abstract Background Abnormalities in T cell activation play an important role in the pathogenesis of myocarditis, and persistent T cell responses can lead to autoimmunity and chronic cardiac inflammation, as well as even dilated cardiomyopathy. Although previous work has examined the role of T cells in myocarditis in animal models, the specific mechanism for human cardiomyocytes has not been investigated. Methods In this study, we constructed the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and established the T cell-mediated cardiac injury model by co-culturing with activated CD4 + T or CD8 + T cells that were isolated from peripheral mononuclear blood to elucidate the pathogenesis of myocardial cell injury caused by inflammation. Results By combination of quantitative proteomics with tissue and cell immunofluorescence examination, we established a proteome profile of inflammatory myocardia from hiPSC-CMs with obvious cardiomyocyte injury and increased levels of lactate dehydrogenase content, creatine kinase isoenzyme MB and cardiac troponin. A series of molecular dysfunctions of hiPSC-CMs was observed and indicated that CD4 + cells could produce direct cardiomyocyte injury by activating the NOD-like receptor signals pathway. Conclusions The data presented in our study established a proteome map of inflammatory myocardial based on hiPSC-CMs injury model. These results can provide guidance in the discovery of improved clinical treatments for myocarditis.

Background: Abnormalities in T cell activation play an important role in the pathogenesis of myocarditis, and persistent T cell responses can lead to autoimmunity and chronic cardiac inflammation, as well as even dilated cardiomyopathy. Although previous work has examined the role of T cells in myocarditis in animal models, the specific mechanism for human cardiomyocytes has not been investigated. Methods: In this study, we constructed the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and established the T cell-mediated cardiac injury model by co-culturing with activated CD4 + T or CD8 + T cells that were isolated from peripheral mononuclear blood to elucidate the pathogenesis of myocardial cell injury caused by inflammation. Results: By combination of quantitative proteomics with tissue and cell immunofluorescence examination, we established a proteome profile of inflammatory myocardia from hiPSC-CMs with obvious cardiomyocyte injury and increased levels of lactate dehydrogenase content, creatine kinase isoenzyme MB and cardiac troponin. A series of molecular dysfunctions of hiPSC-CMs was observed and indicated that CD4 + cells could produce direct cardiomyocyte injury by activating the NOD-like receptor signals pathway. Conclusions: The data presented in our study established a proteome map of inflammatory myocardial based on hiPSC-CMs injury model. These results can provide guidance in the discovery of improved clinical treatments for myocarditis. [ABSTRACT FROM AUTHOR]

Abstract Background Duchenne muscular dystrophy (DMD) is an X‐linked disorder characterized by progressive muscle weakness due to the absence of functional dystrophin. DMD patients also develop dilated cardiomyopathy (DCM). We have previously shown that DMD (mdx) mice and a canine DMD model (GRMD) exhibit abnormal intracellular calcium (Ca2+) cycling related to early‐stage pathological remodelling of the ryanodine receptor intracellular calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) contributing to age‐dependent DCM. Methods Here, we used hiPSC‐CMs from DMD patients selected by Speckle‐tracking echocardiography and canine DMD cardiac biopsies to assess key early‐stage Duchenne DCM features. Results Dystrophin deficiency was associated with RyR2 remodelling and SR Ca2+ leak (RyR2 Po of 0.03 ± 0.01 for HC vs. 0.16 ± 0.01 for DMD, P

Abstract Background The ongoing coronavirus disease 2019 (COVID-19) pandemic has had an enormous impact on our societies. Moreover, the disease’s extensive and sustained symptoms are now becoming a nonnegligible medical challenge. In this respect, data indicate that heart failure is one of the most common readmission diagnoses among COVID-19 patients. Methods In this study, we used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes to develop an in vitro model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and studied the dynamic changes occurring in cardiomyocytes after SARS-CoV-2 infection. Results To this end, we have created an effective time series SARS-CoV-2 infection model exhibiting different functional patterns of up- and downregulated proteins, and demonstrating that SARS-CoV-2 mainly affects (i) the lipid and the energy metabolism of hiPSC-derived cardiomyocytes during the early infection stage, and (ii) the DNA repair ability of cardiomyocytes during the late infection stage. By analyzing the proteome changes occurring at different infection timepoints, we were able to observe that the simulated disease (COVID-19) course developed rapidly, and that each of the studied timepoints was characterized by a distinct protein expression pattern. Conclusions Our findings highlight the importance of early detection and personalized treatment based on the disease stage. Finally, by combing the proteomics data with virus-host interaction network analysis, we were able to identify several potential drug targets for the disease.

Abstract Background Polymorphic ventricular tachycardia (PMVT) is a rare genetic disease associated with structurally normal hearts which in 8% of cases can lead to sudden cardiac death, typically exercise-induced. We previously showed a link between the RyR2-H29D mutation and a clinical phenotype of short-coupled PMVT at rest using patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs). In the present study, we evaluated the effects of clinical and experimental anti-arrhythmic drugs on the intracellular Ca2+ handling, contractile and molecular properties in PMVT hiPSC-CMs in order to model a personalized medicine approach in vitro. Methods Previously, a blood sample from a patient carrying the RyR2-H29D mutation was collected and reprogrammed into several clones of RyR2-H29D hiPSCs, and in addition we generated an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. Here, we tested 4 drugs with anti-arrhythmic properties: propranolol, verapamil, flecainide, and the Rycal S107. We performed fluorescence confocal microscopy, video-image-based analyses and biochemical analyses to investigate the impact of these drugs on the functional and molecular features of the PMVT RyR2-H29D hiPSC-CMs. Results The voltage-dependent Ca2+ channel inhibitor verapamil did not prevent the aberrant release of sarcoplasmic reticulum (SR) Ca2+ in the RyR2-H29D hiPSC-CMs, whereas it was prevented by S107, flecainide or propranolol. Cardiac tissue comprised of RyR2-H29D hiPSC-CMs exhibited aberrant contractile properties that were largely prevented by S107, flecainide and propranolol. These 3 drugs also recovered synchronous contraction in RyR2-H29D cardiac tissue, while verapamil did not. At the biochemical level, S107 was the only drug able to restore calstabin2 binding to RyR2 as observed in the isogenic control. Conclusions By testing 4 drugs on patient-specific PMVT hiPSC-CMs, we concluded that S107 and flecainide are the most potent molecules in terms of preventing the abnormal SR Ca2+ release and contractile properties in RyR2-H29D hiPSC-CMs, whereas the effect of propranolol is partial, and verapamil appears ineffective. In contrast with the 3 other drugs, S107 was able to prevent a major post-translational modification of RyR2-H29D mutant channels, the loss of calstabin2 binding to RyR2. Using patient-specific hiPSC and CRISPR/Cas9 technologies, we showed that S107 is the most efficient in vitro candidate for treating the short-coupled PMVT at rest.

In vitro reconstruction of highly mature engineered heart tissues (EHTs) is attempted for the selection of cardiotoxic drugs suitable for individual patients before administration. Mechanical contractile force generated in the EHTs is known to be a critical indicator for evaluating the EHT response. However, measuring contractile force requires anchoring the EHT in a tailored force‐sensing cell culture chamber, causing technical difficulties in the stable evaluation of contractile force in long‐term culture. This paper proposes a hydrogel‐sheathed human induced pluripotent stem cell (hiPSC)‐derived heart microtissue (H3M) that can provide an anchor‐free contractile force measurement platform in commonly used multi‐well plates. The contractile force associated with tissue formation and drug response is calculated by motion tracking and finite element analysis on the bending angle of the hydrogel sheath. From the experiment of the drug response, H3M is an excellent drug screening platform with high sensitivity and early testing capability compared to conventionally anchored EHT. This unique platform would be useful and versatile for regenerative therapy and drug discovery research in EHT. [ABSTRACT FROM AUTHOR]

Background: The ongoing coronavirus disease 2019 (COVID-19) pandemic has had an enormous impact on our societies. Moreover, the disease's extensive and sustained symptoms are now becoming a nonnegligible medical challenge. In this respect, data indicate that heart failure is one of the most common readmission diagnoses among COVID-19 patients. Methods: In this study, we used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes to develop an in vitro model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and studied the dynamic changes occurring in cardiomyocytes after SARS-CoV-2 infection. Results: To this end, we have created an effective time series SARS-CoV-2 infection model exhibiting different functional patterns of up- and downregulated proteins, and demonstrating that SARS-CoV-2 mainly affects (i) the lipid and the energy metabolism of hiPSC-derived cardiomyocytes during the early infection stage, and (ii) the DNA repair ability of cardiomyocytes during the late infection stage. By analyzing the proteome changes occurring at different infection timepoints, we were able to observe that the simulated disease (COVID-19) course developed rapidly, and that each of the studied timepoints was characterized by a distinct protein expression pattern. Conclusions: Our findings highlight the importance of early detection and personalized treatment based on the disease stage. Finally, by combing the proteomics data with virus-host interaction network analysis, we were able to identify several potential drug targets for the disease. [ABSTRACT FROM AUTHOR]

Background: Polymorphic ventricular tachycardia (PMVT) is a rare genetic disease associated with structurally normal hearts which in 8% of cases can lead to sudden cardiac death, typically exercise-induced. We previously showed a link between the RyR2-H29D mutation and a clinical phenotype of short-coupled PMVT at rest using patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs). In the present study, we evaluated the effects of clinical and experimental anti-arrhythmic drugs on the intracellular Ca2+ handling, contractile and molecular properties in PMVT hiPSC-CMs in order to model a personalized medicine approach in vitro. Methods: Previously, a blood sample from a patient carrying the RyR2-H29D mutation was collected and reprogrammed into several clones of RyR2-H29D hiPSCs, and in addition we generated an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. Here, we tested 4 drugs with anti-arrhythmic properties: propranolol, verapamil, flecainide, and the Rycal S107. We performed fluorescence confocal microscopy, video-image-based analyses and biochemical analyses to investigate the impact of these drugs on the functional and molecular features of the PMVT RyR2-H29D hiPSC-CMs. Results: The voltage-dependent Ca2+ channel inhibitor verapamil did not prevent the aberrant release of sarcoplasmic reticulum (SR) Ca2+ in the RyR2-H29D hiPSC-CMs, whereas it was prevented by S107, flecainide or propranolol. Cardiac tissue comprised of RyR2-H29D hiPSC-CMs exhibited aberrant contractile properties that were largely prevented by S107, flecainide and propranolol. These 3 drugs also recovered synchronous contraction in RyR2-H29D cardiac tissue, while verapamil did not. At the biochemical level, S107 was the only drug able to restore calstabin2 binding to RyR2 as observed in the isogenic control. Conclusions: By testing 4 drugs on patient-specific PMVT hiPSC-CMs, we concluded that S107 and flecainide are the most potent molecules in terms of preventing the abnormal SR Ca2+ release and contractile properties in RyR2-H29D hiPSC-CMs, whereas the effect of propranolol is partial, and verapamil appears ineffective. In contrast with the 3 other drugs, S107 was able to prevent a major post-translational modification of RyR2-H29D mutant channels, the loss of calstabin2 binding to RyR2. Using patient-specific hiPSC and CRISPR/Cas9 technologies, we showed that S107 is the most efficient in vitro candidate for treating the short-coupled PMVT at rest. [ABSTRACT FROM AUTHOR]

Heart regeneration after myocardial infarction (MI) using human stem cell-derived cardiomyocytes (CMs) is rapidly accelerating with large animal and human clinical trials. However, vascularization methods to support the engraftment, survival, and development of implanted CMs in the ischemic environment of the infarcted heart remain a key and timely challenge. To this end, we developed a dual remuscularization-revascularization therapy that is evaluated in a rat model of ischemia-reperfusion MI. This study details the differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for engineering cardiac tissue containing patterned engineered vessels 400 μm in diameter. Vascularized engineered human myocardial tissues (vEHMs) are cultured in static conditions or perfused in vitro prior to implantation and evaluated after two weeks. Immunohistochemical staining indicates improved engraftment of hiPSC-CMs in in vitro-perfused vEHMs with greater expression of SMA+ vessels and evidence of inosculation. Three-dimensional vascular reconstructions reveal less tortuous and larger intra-implant vessels, as well as an improved branching hierarchy in in vitro-perfused vEHMs relative to non-perfused controls. Exploratory RNA sequencing of explanted vEHMs supports the hypothesis that co-revascularization impacts hiPSC-CM development in vivo. Our approach provides a strong foundation to enhance vEHM integration, develop hierarchical vascular perfusion, and maximize hiPSC-CM engraftment for future regenerative therapy. [ABSTRACT FROM AUTHOR]

Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

Electrospun nanofiber constructs represent a promising alternative for mimicking the natural extracellular matrix in vitro and have significant potential for cardiac patch applications. While the effect of fiber orientation on the morphological structure of cardiomyocytes has been investigated, fibers only provide contact guidance without accounting for substrate stiffness due to their deposition on rigid substrates (e.g., glass or polystyrene). This paper introduces an in situ fabrication method for suspended and well aligned nanofibrous scaffolds via roller electrospinning, providing an anisotropic microenvironment with reduced stiffness for cardiac tissue engineering. A fiber surface modification strategy, utilizing oxygen plasma treatment combined with sodium dodecyl sulfate solution, was proposed to maintain the hydrophilicity of polycaprolactone (PCL) fibers, promoting cellular adhesion. Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), cultured on aligned fibers, exhibited an elongated morphology with extension along the fiber axis. In comparison to Petri dishes and suspended random fiber scaffolds, hiPSC-CMs on suspended aligned fiber scaffolds demonstrated enhanced sarcomere organization, spontaneous synchronous contraction, and gene expression indicative of maturation. This work demonstrates the suspended and aligned nano-fibrous scaffold provides a more realistic biomimetic environment for hiPSC-CMs, which promoted further research on the inducing effect of fiber scaffolds on hiPSC-CMs microstructure and gene-level expression. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells (hPSCs), derived from individuals or genetically modified with disease-related mutations and variants, have revolutionised studies of human disease. Researchers are beginning to exploit the extraordinary potential of stem cell technology to screen for newdrugs to treat intractable diseases, ideally without sideeffects. However, a major problem is that the differentiated cell types on which these models are based are immature; they resemble fetal and not adult cells. Here, we discuss the nature and hurdles of hPSC maturation, using cardiomyocytes as an example. We review methods used to induce cardiomyocyte maturation in culture and consider remaining challenges for their integration into research on human disease and drug development pipelines. [ABSTRACT FROM AUTHOR]

BackgroundSarcoplasmic reticulum Ca2+ leak and post-translational modifications under stress have been implicated in catecholaminergic polymorphic ventricular tachycardia (CPVT), a highly lethal inherited arrhythmogenic disorder. Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling.ObjectiveThe aims were to obtain functional hiPSC-derived cardiomyocytes from a CPVT patient harboring a novel ryanodine receptor (RyR2) mutation and model the syndrome, drug responses and investigate the molecular mechanisms associated to the CPVT syndrome.MethodsPatient-specific cardiomyocytes were generated from a young athletic female diagnosed with CPVT. The contractile, intracellular Ca2+ handling and electrophysiological properties as well as the RyR2 macromolecular remodeling were studied.ResultsExercise stress electrocardiography revealed polymorphic ventricular tachycardia when treated with metoprolol and marked improvement with flecainide alone. We found abnormal stress-induced contractile and electrophysiological properties associated with sarcoplasmic reticulum Ca2+ leak in CPVT hiPSC-derived cardiomyocytes. We found inadequate response to metoprolol and a potent response of flecainide. Stabilizing RyR2 with a Rycal compound prevents those abnormalities specifically in CPVT hiPSC-derived cardiomyocytes. The RyR2-D3638A mutation is located in the conformational change inducing-central core domain and leads to RyR2 macromolecular remodeling including depletion of PP2A and Calstabin2.ConclusionWe identified a novel RyR2-D3638A mutation causing 3D conformational defects and aberrant biophysical properties associated to RyR2 macromolecular complex post-translational remodeling. The molecular remodeling is for the first time revealed using patient-specific hiPSC-derived cardiomyocytes which may explain the CPVT proband's resistance. Our study promotes hiPSC-derived cardiomyocytes as a suitable model for disease modeling, testing new therapeutic compounds, personalized medicine and deciphering underlying molecular mechanisms.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) represent an excellent in vitro model in cardiovascular research. Changes in their action potential (AP) dynamics convey information that is essential for disease modeling, drug screening and toxicity evaluation. High-throughput optical AP recordings utilizing intramolecular Förster resonance energy transfer (FRET) of the voltage-sensitive fluorescent protein (VSFP) have emerged as a substitute or complement to the resource-intensive patch clamp technique. Here, we functionally validated our recently generated voltage indicator hiPSC lines stably expressing CAG-promoter-driven VSFP in the AAVS1 safe harbor locus. By combining subtype-specific cardiomyocyte differentiation protocols, we established optical AP recordings in ventricular, atrial, and nodal CMs in 2D monolayers using fluorescence microscopy. Moreover, we achieved high-throughput optical AP measurements in single hiPSC-derived CMs in a 3D context. Overall, this system greatly expands the spectrum of possibilities for high-throughput, non-invasive and long-term AP analyses in cardiovascular research and drug discovery.

Abstract Background Cardiotoxicity, characterized by severe cardiac dysfunction, is a major problem in patients treated with different classes of anticancer drugs. Development of predictable human-based models and assays for drug screening are crucial for preventing potential drug-induced adverse effects. Current animal in vivo models and cell lines are not always adequate to represent human biology. Alternatively, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show great potential for disease modelling and drug-induced toxicity screenings. Fully automated high-throughput screening of drug toxicity on hiPSC-CMs by fluorescence image analysis is, however, very challenging, due to clustered cell growth patterns and strong intracellular and intercellular variation in the expression of fluorescent markers. Results In this paper, we report on the development of a fully automated image analysis system for quantification of cardiotoxic phenotypes from hiPSC-CMs that are treated with various concentrations of anticancer drugs doxorubicin or crizotinib. This high-throughput system relies on single-cell segmentation by nuclear signal extraction, fuzzy C-mean clustering of cardiac α-actinin signal, and finally nuclear signal propagation. When compared to manual segmentation, it generates precision and recall scores of 0.81 and 0.93, respectively. Conclusions Our results show that our fully automated image analysis system can reliably segment cardiomyocytes even with heterogeneous α-actinin signals.

Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) are increasingly used to study genetic diseases on a human background. However, the lack of a fully mature adult cardiomyocyte phenotype of hiPSC-CM may be limiting the scope of these studies. Muscular dystrophies and concomitant cardiomyopathies result from mutations in genes encoding proteins of the dystrophin-associated protein complex (DAPC), which is a multi-protein membrane-spanning complex. We examined the expression of DAPC components in hiPSC-CM, which underwent maturation in 2D and 3D culture protocols. The results were compared with human adult cardiac tissue and isolated cardiomyocytes. We found that similarly to adult cardiomyocytes, hiPSC-CM express dystrophin, in line with previous studies on Duchenne’s disease. β-dystroglycan was also expressed, but, contrary to findings in adult cardiomyocytes, none of the sarcoglycans nor α-dystroglycan were, despite the presence of their mRNA. In conclusion, despite the robust expression of dystrophin, the absence of several other DAPC protein components cautions for reliance on commonly used protocols for hiPSC-CM maturation for functional assessment of the complete DAPC.

Introduction: Although atrial fibrillation is the most prevalent disorder of electrical conduction, the mechanisms behind atrial arrhythmias remain elusive. To address this challenge, we developed a robust in vitro model of 3D atrial microtissue from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and evaluated chamber-specific chemical responses experimentally and computationally. Methods: We differentiated atrial and ventricular cardiomyocytes (aCMs/vCMs) from GCaMP6f-expressing hiPSCs and assessed spontaneous AP activity using fluorescence imaging. Self-assembling 3D microtissues were formed with lactate purified CMs and 5% human cardiac fibroblasts and electrically stimulated for one week before high resolution action potential (AP) optical mapping. AP responses to the atrial-specific potassium repolarizing current IKur-blocker 4-Aminopyridine (4-AP) and funny current If-blocker Ivabradine were characterized within their therapeutic window. Finally, we expanded upon a published hiPSC-CM computational model by incorporating the atrial-specific IKur current, modifying ion channel conductances to match the AP waveforms of our microtissues, and employing the updated model to reinforce our experimental findings. Results: High purity CMs (> 75% cTnT+) demonstrated subtype specification by MLC2v expression. Spontaneous beating rates significantly decreased following 3D microtissue formation, with atrial microtissues characterized by their faster spontaneous beating rate, slower AP rise time, and shorter AP duration (APD) compared to ventricular microtissues. We measured atrial-specific responses, including dose-dependent APD prolongation with 4-AP treatment and dose-dependent reduction in spontaneous activity post-Ivabradine treatment. Conclusion: The presented in vitro platform for screening atrial-specific responses is both robust and sensitive, with high throughput, enabling studies focused at elucidating the mechanisms underlying atrial arrhythmias. [ABSTRACT FROM AUTHOR]

The formulation of in silico biophysical models generally requires optimization strategies for reproducing experimentally observed phenomena. In electrophysiological modeling, robust nonlinear regressive methods are often crucial for guaranteeing high fidelity models. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), though nascent, have proven to be useful in cardiac safety pharmacology, regenerative medicine, and in the implementation of patient-specific test benches for investigating inherited cardiac disorders. This study demonstrates the potency of heuristic techniques at formulating biophysical models, with emphasis on a hiPSC-CM model using a novel genetic algorithm (GA) recipe we proposed. The proposed GA protocol was used to develop a hiPSC-CM biophysical computer model by fitting mathematical formulations to experimental data for five ionic currents recorded in hiPSC-CMs. The maximum conductances of the remaining ionic channels were scaled based on recommendations from literature to accurately reproduce the experimentally observed hiPSC-CM action potential (AP) metrics. Near-optimal parameter fitting was achieved for the GA-fitted ionic currents. The resulting model recapitulated experimental AP parameters such as AP durations (APD50, APD75, and APD90), maximum diastolic potential, and frequency of automaticity. The outcome of this work has implications for validating the biophysics of hiPSC-CMs in their use as viable substitutes for human cardiomyocytes, particularly in cardiac safety pharmacology and in the study of inherited cardiac disorders. This study presents a novel GA protocol useful for formulating robust numerical biophysical models. The proposed protocol is used to develop a hiPSC-CM model with implications for cardiac safety pharmacology.

The formulation of in silico biophysical models generally requires optimization strategies for reproducing experimentally observed phenomena. In electrophysiological modeling, robust nonlinear regressive methods are often crucial for guaranteeing high fidelity models. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), though nascent, have proven to be useful in cardiac safety pharmacology, regenerative medicine, and in the implementation of patient-specific test benches for investigating inherited cardiac disorders. This study demonstrates the potency of heuristic techniques at formulating biophysical models, with emphasis on a hiPSC-CM model using a novel genetic algorithm (GA) recipe we proposed. The proposed GA protocol was used to develop a hiPSC-CM biophysical computer model by fitting mathematical formulations to experimental data for five ionic currents recorded in hiPSC-CMs. The maximum conductances of the remaining ionic channels were scaled based on recommendations from literature to accurately reproduce the experimentally observed hiPSC-CM action potential (AP) metrics. Near-optimal parameter fitting was achieved for the GA-fitted ionic currents. The resulting model recapitulated experimental AP parameters such as AP durations (APD50, APD75, and APD90), maximum diastolic potential, and frequency of automaticity. The outcome of this work has implications for validating the biophysics of hiPSC-CMs in their use as viable substitutes for human cardiomyocytes, particularly in cardiac safety pharmacology and in the study of inherited cardiac disorders. This study presents a novel GA protocol useful for formulating robust numerical biophysical models. The proposed protocol is used to develop a hiPSC-CM model with implications for cardiac safety pharmacology. [ABSTRACT FROM AUTHOR]

Background: While mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to exercise-induced or catecholaminergic polymorphic ventricular tachycardia (CPVT), its association with polymorphic ventricular tachycardia (PMVT) occurring at rest is unclear. We aimed at constructing a patient-specific human-induced pluripotent stem cell (hiPSC) model of PMVT occurring at rest linked to a single point mutation in RyR2. Methods: Blood samples were obtained from a patient with PMVT at rest due to a heterozygous RyR2-H29D mutation. Patient-specific hiPSCs were generated from the blood samples, and the hiPSC-derived cardiomyocytes (CMs) were generated via directed differentiation. Using CRIPSR/Cas9 technology, isogenic controls were generated by correcting the RyR2-H29D mutation. Using patch-clamp, fluorescent confocal microscopy and video-image-based analysis, the molecular and functional properties of RyR2-H29D hiPSCCMs and control hiPSCCMs were compared. Findings: RyR2-H29D hiPSCCMs exhibit intracellular sarcoplasmic reticulum (SR) Ca2+ leak through RyR2 under physiological pacing. RyR2-H29D enhances the contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling (ECC) that exacerbates abnormal Ca2+ release in RyR2-H29D hiPSCCMs. RyR2-H29D hiPSCCMs exhibit shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. The RyR2-H29D mutation causes post-translational remodeling that is fully reversed with isogenic controls. Interpretation: To conclude, in a model based on a RyR2 point mutation that is associated with short-coupled PMVT at rest, RyR2-H29D hiPSCCMs exhibited aberrant intracellular Ca2+ homeostasis, shortened action potentials, arrhythmias and abnormal contractile properties. Funding: French Muscular Dystrophy Association (AFM; project 16,073, MNM2 2012 and 20,225), “Fondation de la Recherche Médicale” (FRM; SPF20130526710), “Institut National pour la Santé et la Recherche Médicale” (INSERM), National Institutes of Health (ARM; R01 HL145473) and New York State Department of Health (NYSTEM C029156).

Abstract Empagliflozin, a sodium-glucose co-transporter (SGLT) inhibitor, reduces heart failure and sudden cardiac death but the underlying mechanisms remain elusive. In cardiomyocytes, SGLT1 and SGLT2 expression is upregulated in diabetes mellitus, heart failure, and myocardial infarction. We hypothesise that empagliflozin exerts direct effects on cardiomyocytes that attenuate diabetic cardiomyopathy. To test this hypothesis, cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) were used to test the potential effects of empagliflozin on neutralization of cardiac dysfunction induced by diabetic-like cultures. Our results indicated that insulin-free high glucose culture significantly increased the size of and NPPB, SGLT1 and SGLT2 expression of hiPSC-derived cardiomyocytes. In addition, high glucose-treated hiPSC-derived cardiomyocytes exhibited reduced contractility regardless of the increased calcium transient capacity. Interestingly, application of empagliflozin before or after high glucose treatment effectively reduced the high glucose-induced cardiac abnormalities. Since application of empagliflozin did not significantly alter viability or glycolytic capacity of the hiPSC-derived cardiomyocytes, it is plausible that empagliflozin exerts its effects via the down-regulation of SGLT1, SGLT2 and GLUT1 expression. These observations provide supportive evidence that may help explain its unexpected benefit observed in the EMPA-REG trial.

Aims  NOS1AP single-nucleotide polymorphisms (SNPs) correlate with QT prolongation and cardiac sudden death in patients affected by long QT syndrome type 1 (LQT1). NOS1AP targets NOS1 to intracellular effectors. We hypothesize that NOS1AP SNPs cause NOS1 dysfunction and this may converge with prolonged action-potential duration (APD) to facilitate arrhythmias. Here we test (i) the effects of NOS1 inhibition and their interaction with prolonged APD in a guinea pig cardiomyocyte (GP-CMs) LQT1 model; (ii) whether pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from LQT1 patients differing for NOS1AP variants and mutation penetrance display a phenotype compatible with NOS1 deficiency. Methods and results  In GP-CMs, NOS1 was inhibited by S-Methyl-L-thiocitrulline acetate (SMTC) or Vinyl-L-NIO hydrochloride (L-VNIO); LQT1 was mimicked by I Ks blockade (JNJ303) and β-adrenergic stimulation (isoproterenol). hiPSC-CMs were obtained from symptomatic (S) and asymptomatic (AS) KCNQ1 -A341V carriers, harbouring the minor and major alleles of NOS1AP SNPs (rs16847548 and rs4657139), respectively. In GP-CMs, NOS1 inhibition prolonged APD, enhanced I CaL and I NaL, slowed Ca2+ decay, and induced delayed afterdepolarizations. Under action-potential clamp, switching to shorter APD suppressed 'transient inward current' events induced by NOS1 inhibition and reduced cytosolic Ca2+. In S (vs. AS) hiPSC-CMs, APD was longer and I CaL larger; NOS1AP and NOS1 expression and co-localization were decreased. Conclusion  The minor NOS1AP alleles are associated with NOS1 loss of function. The latter likely contributes to APD prolongation in LQT1 and converges with it to perturb Ca2+ handling. This establishes a mechanistic link between NOS1AP SNPs and aggravation of the arrhythmia phenotype in prolonged repolarization syndromes. [ABSTRACT FROM AUTHOR]

Background: Cardiotoxicity, characterized by severe cardiac dysfunction, is a major problem in patients treated with different classes of anticancer drugs. Development of predictable human-based models and assays for drug screening are crucial for preventing potential drug-induced adverse effects. Current animal in vivo models and cell lines are not always adequate to represent human biology. Alternatively, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show great potential for disease modelling and drug-induced toxicity screenings. Fully automated high-throughput screening of drug toxicity on hiPSC-CMs by fluorescence image analysis is, however, very challenging, due to clustered cell growth patterns and strong intracellular and intercellular variation in the expression of fluorescent markers. Results: In this paper, we report on the development of a fully automated image analysis system for quantification of cardiotoxic phenotypes from hiPSC-CMs that are treated with various concentrations of anticancer drugs doxorubicin or crizotinib. This high-throughput system relies on single-cell segmentation by nuclear signal extraction, fuzzy C-mean clustering of cardiac α-actinin signal, and finally nuclear signal propagation. When compared to manual segmentation, it generates precision and recall scores of 0.81 and 0.93, respectively. Conclusions: Our results show that our fully automated image analysis system can reliably segment cardiomyocytes even with heterogeneous α-actinin signals. [ABSTRACT FROM AUTHOR]

The fight-or-flight response (FFR), a physiological acute stress reaction, involves positive chronotropic and inotropic effects on heart muscle cells mediated through β-adrenoceptor activation. Increased systolic calcium is required to enable stronger heart contractions whereas elevated potassium currents are to limit the duration of the action potentials and prevent arrhythmia. The latter effect is accomplished by an increased functional activity of the Kv7.1 channel encoded by KCNQ1. Current knowledge, however, does not sufficiently explain the full extent of rapid Kv7.1 activation and may hence be incomplete. Using inducible genetic KCNQ1 complementation in KCNQ1-deficient human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we here reinvestigate the functional role of Kv7.1 in adapting human CMs to adrenergic stress. Under baseline conditions, Kv7.1 was barely detectable at the plasma membrane of hiPSC-CMs, yet it fully protected these from adrenergic stress-induced beat-to-beat variability of repolarization and torsade des pointes-like arrhythmia. Furthermore, isoprenaline treatment increased field potential durations specifically in KCNQ1-deficient CMs to cause these adverse macroscopic effects. Mechanistically, we find that the protective action by Kv7.1 resides in a rapid translocation of channel proteins from intracellular stores to the plasma membrane, induced by adrenergic signaling. Gene silencing experiments targeting RAB GTPases, mediators of intracellular vesicle trafficking, showed that fast Kv7.1 recycling under acute stress conditions is RAB4A-dependent.Our data reveal a key mechanism underlying the rapid adaptation of human cardiomyocytes to adrenergic stress. These findings moreover aid to the understanding of disease pathology in long QT syndrome and bear important implications for safety pharmacological screening.

The fight-or-flight response (FFR), a physiological acute stress reaction, involves positive chronotropic and inotropic effects on heart muscle cells mediated through b-adrenoceptor activation. Increased systolic calcium is required to enable stronger heart contractions whereas elevated potassium currents are to limit the duration of the action potentials and prevent arrhythmia. The latter effect is accomplished by an increased functional activity of the Kv7.1 channel encoded by KCNQ1. Current knowledge, however, does not sufficiently explain the full extent of rapid Kv7.1 activation and may hence be incomplete. Using inducible genetic KCNQ1 complementation in KCNQ1-deficient human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we here reinvestigate the functional role of Kv7.1 in adapting human CMs to adrenergic stress. Under baseline conditions, Kv7.1 was barely detectable at the plasma membrane of hiPSC-CMs, yet it fully protected these from adrenergic stress-induced beat-to-beat variability of repolarization and torsade des pointes-like arrhythmia. Furthermore, isoprenaline treatment increased field potential durations specifically in KCNQ1-deficient CMs to cause these adverse macroscopic effects. Mechanistically, we find that the protective action by Kv7.1 resides in a rapid translocation of channel proteins from intracellular stores to the plasma membrane, induced by adrenergic signaling. Gene silencing experiments targeting RAB GTPases, mediators of intracellular vesicle trafficking, showed that fast Kv7.1 recycling under acute stress conditions is RAB4A-dependent. Our data reveal a key mechanism underlying the rapid adaptation of human cardiomyocytes to adrenergic stress. These findings moreover aid to the understanding of disease pathology in long QT syndrome and bear important implications for safety pharmacological screening. [ABSTRACT FROM AUTHOR]

Functional vasculature is essential for delivering nutrients, oxygen, and cells to the heart and removing waste products. Here, we developed an in vitro vascularized human cardiac microtissue (MT) model based on human induced pluripotent stem cells (hiPSCs) in a microfluidic organ-on-chip by coculturing hiPSC-derived, pre-vascularized, cardiac MTs with vascular cells within a fibrin hydrogel. We showed that vascular networks spontaneously formed in and around these MTs and were lumenized and interconnected through anastomosis. Anastomosis was fluid flow dependent: continuous perfusion increased vessel density and thus enhanced the formation of the hybrid vessels. Vascularization further improved endothelial cell (EC)-cardiomyocyte communication via EC-derived paracrine factors, such as nitric oxide, and resulted in an enhanced inflammatory response. The platform sets the stage for studies on how organ-specific EC barriers respond to drugs or inflammatory stimuli., Competing Interests: Conflict of interests C.L.M. is co-founder of Ncardia bv., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polymorphic ventricular tachycardia (PMVT) can occur in patients with structurally normal hearts and in 8% of cases can lead to sudden cardiac death, typically exercise-induced. The role of the cardiac type 2 ryanodine receptor (RyR2) in pathogenesis of PMVT presenting at rest is unclear. We aimed here at modelling PMVT observed in a patient harboring the RyR2-H29D mutation by comparing the molecular and functional properties of RyR2-H29D hiPSC-derived cardiomyocytes (hiPSC-CMs) with their isogenic control counterparts with a particular focus on the RyR2 properties. We collected blood samples from the patient and generated several clones of RyR2-H29D hiPSC, in addition to generating an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. We used fluorescent confocal microscopy, patch-clamp and video-image-based analysis to investigate the molecular and functional consequences of the RyR2-H29D mutation. We first hypothesized that PMVT hiPSC-CMs expressing the RyR2-H29D mutation would exhibit abnormal Ca2+ homeostasis. Thus, we measured and analyzed the intracellular Ca2+ variation. We found that the RyR2-H29D hiPSC-CMs exhibit clone-independent aberrant properties including intracellular sarcoplasmic reticulum (SR) Ca2+ leak through RyR2 under physiological pacing. The contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling (ECC) exacerbate the abnormal intracellular Ca2+ release in the RyR2-H29D hiPSC-CMs. Moreover, the RyR2-H29D hiPSC-CMs exhibit RyR2 post-translational remodeling, shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. These abnormalities are fully reversed with isogenic control. Our results suggest that RyR2-mediated Ca2+ leak induces an impairment of Ca2+ homeostasis and provide support to decipher the molecular mechanisms of short-coupled PMVT at rest. [ABSTRACT FROM AUTHOR]

Heart regeneration after myocardial infarction requires new cardiomyocytes and a supportive vascular network. Here, we evaluate the efficacy of localized delivery of angiogenic factors from biomaterials within the implanted muscle tissue to guide growth of a more dense, organized, and perfused vascular supply into implanted engineered human cardiac tissue on an ischemia/reperfusion injured rat heart. We use large, aligned 3-dimensional engineered tissue with cardiomyocytes derived from human induced pluripotent stem cells in a collagen matrix that contains dispersed alginate microspheres as local protein depots. Release of angiogenic growth factors VEGF and bFGF in combination with morphogen sonic hedgehog from the microspheres into the local microenvironment occurs from the epicardial implant site. Analysis of the 3D vascular network in the engineered tissue via Microfil® perfusion and microCT imaging at 30 days shows increased volumetric network density with a wider distribution of vessel diameters, proportionally increased branching and length, and reduced tortuosity. Global heart function is increased in the angiogenic factor-loaded cardiac implants versus sham. These findings demonstrate for the first time the efficacy of a combined remuscularization and revascularization therapy for heart regeneration after myocardial infarction. Image 1 • Heart regeneration requires new muscle, which we deliver with engineered tissue. • We created a dual therapy to deliver hiPSC-cardiomyocytes and vasculogenic proteins. • Alginate microspheres embedded in the engineered tissue are local protein depots. • New vessels perfuse the human cardiac implant at 1 month, shown by 3D analysis. • This new dual therapy improves heart function. [ABSTRACT FROM AUTHOR]

Relevant, predictive normal, or disease model systems are of vital importance for drug development. The difference between nonhuman models and humans could contribute to clinical trial failures despite ideal nonhuman results. As a potential substitute for animal models, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) provide a powerful tool for drug toxicity screening, modeling cardiovascular diseases, and drug discovery. Here, we review recent hiPSC-CM disease models and discuss the features of hiPSC-CMs, including subtype and maturation and the tissue engineering technologies for drug assessment. Updates from the international multisite collaborators/administrations for development of novel drug discovery paradigms are also summarized.