Human Engineered Heart Tissue Research Articles

Fabrication of chitin-fibrin hydrogels to construct the 3D artificial extracellular matrix scaffold for vascular regeneration and cardiac tissue engineering.

As the cornerstone of tissue engineering and regeneration medicine research, developing a cost-effective and bionic extracellular matrix (ECM) that can precisely modulate cellular behavior and form functional tissue remains challenging. An artificial ECM combining polysaccharides and fibrillar proteins to mimic the structure and composition of natural ECM provides a promising solution for cardiac tissue regeneration. In this study, we developed a bionic hydrogel scaffold by combining a quaternized β-chitin derivative (QC) and fibrin-matrigel (FM) in different ratios to mimic a natural ECM. We evaluated the stiffness of those composite hydrogels with different mixing ratios and their effects on the growth of human umbilical vein endothelial cells (HUVECs). The optimal hydrogels, QCFM1 hydrogels were further applied to load HUVECs into nude mice for in vivo angiogenesis. Besides, we encapsulated human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) into QCFM hydrogels and employed 3D bioprinting to achieve batch fabrication of human-engineered heart tissue (hEHT). Finally, the myocardial structure and electrophysiological function of hEHT were evaluated by immunofluorescence and optical mapping. Designed artificial ECM has a tunable modulus (220-1380 Pa), which determines the different cellular behavior of HUVECs when encapsulated in these. QCFM1 composite hydrogels with optimal stiffness (800 Pa) and porous architecture were finally identified, which could adapt for in vitro cell spreading and in vivo angiogenesis of HUVECs. Moreover, QCFM1 hydrogels were applied in 3D bioprinting successfully to achieve batch fabrication of both ring-shaped and patch-shaped hEHT. These QCFM1 hydrogels-based hEHTs possess organized sarcomeres and advanced function characteristics comparable to reported hEHTs. The chitin-derived hydrogels are first used for cardiac tissue engineering and achieve the batch fabrication of functionalized artificial myocardium. Specifically, these novel QCFM1 hydrogels provided a reliable and economical choice serving as ideal ECM for application in tissue engineering and regeneration medicine.

CDK4/6 inhibitor ribociclib induces cardiotoxicity in dynamic engineered heart tissues, mitigated by E2F1 overexpression

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council CDK4/6 inhibitor Ribociclib induces cardiotoxicity in dynamic engineered heart tissues, mitigated by E2F1 overexpression Background Ribociclib, a novel chemotherapeutic agent for metastatic breast cancer, functions as a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor1. Its mechanism of action involves the CDK4/6-Rb-E2F1 pathway. Ribociclib use has very recently been associated with development of heart failure (HF)2, yet the causality of its cardiac effects remains to be explored. Aims This study aims to investigate ribociclib's cardiotoxic effects in dynamic human engineered heart tissues (dyn-EHTs), focusing on the CDK4/6-Rb-E2F1 axis. Methods and Results Dyn-EHTs were generated using human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) and treated with repeated doses of ribociclib (7 µM) to mimic clinical drug administration. Post-treatment, dyn-EHTs exhibited a 17.0% (p<0.001) increase in tissue dilatation, an 8.9% (p<0.001) decrease in systolic force generation, and a 22.1% (p<0.001) increase in systolic stress, indicating significant cardiac dysfunction. Additionally, an increase in phosphorylation levels of Rb protein (Ser249 and Thr252) was observed (FC=0.445, p=0.009). Overexpression of E2F1 in iPSCs resulted in a threefold increase in E2F1 protein expression and successfully mitigated the ribociclib-induced tissue dilatation (p=0.007), decreased systolic force generation (p=0.005), and increase in systolic stress (p=0.004) making their performance comparable to healthy controls, as shown by linear regression analysis. Conclusions Our study demonstrates that ribociclib induces significant cardiotoxic effects in dyn-EHTs and iPSC-CMs through the CDK4/6-Rb-E2F1 pathway. The prevention of these effects by E2F1 overexpression highlights the pathway's involvement in ribociclib's cardiotoxic profile. These findings emphasize the need for careful cardiac monitoring in patients treated with CDK4/6 inhibitors and suggest further research into specific mechanisms of cardiotoxicity in cancer therapeutics.Graphical Abstract

A novel bioreactor to apply dynamic load to neonatal lamb living myocardial slices and human engineered heart tissue for the study of regenerative properties of the heart

A novel bioreactor to apply dynamic load to neonatal lamb living myocardial slices and human engineered heart tissue for the study of regenerative properties of the heart

Hypertrophic cardiomyopathy dysfunction mimicked in human engineered heart tissue and improved by sodium-glucose cotransporter 2 inhibitors.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy, often caused by pathogenic sarcomere mutations. Early characteristics of HCM are diastolic dysfunction and hypercontractility. Treatment to prevent mutation-induced cardiac dysfunction is lacking. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are a group of antidiabetic drugs that recently showed beneficial cardiovascular outcomes in patients with acquired forms of heart failure. We here studied if SGLT2i represent a potential therapy to correct cardiomyocyte dysfunction induced by an HCM sarcomere mutation. Contractility was measured of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) harbouring an HCM mutation cultured in 2D and in 3D engineered heart tissue (EHT). Mutations in the gene encoding β-myosin heavy chain (MYH7-R403Q) or cardiac troponin T (TNNT2-R92Q) were investigated. In 2D, intracellular [Ca2+], action potential and ion currents were determined. HCM mutations in hiPSC-CMs impaired relaxation or increased force, mimicking early features observed in human HCM. SGLT2i enhance the relaxation of hiPSC-CMs, to a larger extent in HCM compared to control hiPSC-CMs. Moreover, SGLT2i-effects on relaxation in R403Q EHT increased with culture duration, i.e. hiPSC-CMs maturation. Canagliflozin's effects on relaxation were more pronounced than empagliflozin and dapagliflozin. SGLT2i acutely altered Ca2+ handling in HCM hiPSC-CMs. Analyses of SGLT2i-mediated mechanisms that may underlie enhanced relaxation in mutant hiPSC-CMs excluded SGLT2, Na+/H+ exchanger, peak and late Nav1.5 currents, and L-type Ca2+ current, but indicate an important role for the Na+/Ca2+ exchanger. Indeed, electrophysiological measurements in mutant hiPSC-CM indicate that SGLT2i altered Na+/Ca2+ exchange current. SGLT2i (canagliflozin > dapagliflozin > empagliflozin) acutely enhance relaxation in human EHT, especially in HCM and upon prolonged culture. SGLT2i may represent a potential therapy to correct early cardiac dysfunction in HCM.

Abstract 18445: Rigorous Mechanics in Engineered Heart Tissues

Introduction: Heart failure can develop or be exacerbated by mechanical stressors, including increased diastolic strain (preload) and increased systolic impedance (afterload). Recent advances in human induced pluripotent stem cell (iPSC) technology and tissue engineering have enabled long term culture and maturation of engineered heart tissues (EHTs). Most current platforms do not allow for mechanical assessment under a range of loading conditions. Here, we develop a technique to engineer cardiac tissues that allow for non-destructive rigorous phenotyping of tissue mechanics. Methods: Custom tissue anchors were 3D printed using Boston Micro Fabrication HTL Resin and placed within polydimethylsiloxane tissue casting molds. iPS cardiomyocytes and ventricular fibroblasts were mixed 5:1 in 2 mg/mL collagen and cast in rectangular molds. Tissues were compacted around custom tissue anchors over 14 days. Tissues were dismounted from the molds and fixed for immunostaining or hooked onto a force transducer for length-tension, force-frequency, and work-loop analysis. Results: 3D printed tissue anchors accommodated EHT molding and compaction. EHTs spontaneously contracted after 3 days in culture, and compacted to 15.9±0.8% their initial tissue area by 14 days of culture. Engineered tissues were cell dense and mature, with bands of aligned sarcomeres. During isometric testing, tissues display a physiologic length-tension relationship, with linear increases in force with stretch between L 0 (slack length) and L max (length at maximum active force generation, 4.3±0.7 mN/mm 2 ; r 2 =0.96). These are coupled with increases in contraction and relaxation velocities. EHTs were capable of mimicking physiologic work loops. Conclusions: In summary, we have shown the ability to grow and mature three-dimensional human engineered heart tissues capable of rigorous mechanical assessment, enabling future studies on load-dependent cardiac remodeling.

Abstract P1086: Induced Pluripotent Stem Cell-based Cardiac Tissue Modeling Of Mitogenic Cardiomyopathy In Alström Syndrome

Introduction: Cardiomyopathies are a prominent cause of heart failure and affect millions worldwide. Mutation in the Alström syndrome 1 (ALMS1) gene causing Alström syndrome (ALMS) , reported an extremely rare type of dilated cardiomyopathy - called mitogenic cardiomyopathy - leading to neonatal heart failure and death in early infancy due to delayed postnatal cardiomyocyte (CM) cell cycle arrest. Methods: Induced pluripotent stem cells (iPSCs) from ALMS patients with or without mitogenic cardiomyopathy were used to generate ALMS patient-specific cardiomyocytes (iPSC-CMs) . The iPSC-CMs were divided into two main groups based on the presence or absence of the mitogenic phenotype. After transcriptome sequencing, differentially expressed genes (FDR < 0.05, logFC > 0.5 | < -0.5) were found by testing (QL F-test) a robust fitted linear model (edgeR). Harnessing recent advances in stem cell differentiation and tissue engineering, we aimed at creating a human engineered heart tissue model as an unprecedented in vitro disease system to study mechanisms responsible for persistent CM proliferation. Results: ALMS1-deficient mitogenic iPSC-CMs exhibited an impaired ability to undergo cell cycle arrest, evidenced by a higher cell percentage in the G2/M phase (19.3% in mitogenic vs . 7.5% in non-mitogenic iPSC-CMs). Furthermore, increased extracellular matrix levels of the fibroblast-derived protein periostin (POSTN) were observed in co-cultures of ALMS1-deficient iPSC-CMs and ALMS1 fibroblasts. Finally, we found preliminary evidence of dysregulation of the Hippo signaling pathway, observing altered cellular localization of its key downstream effector Yes-associated protein (YAP) and dysregulated ratio between phosphor and total YAP protein levels that in combination with transcript alteration of PPIC, SH3BP4, NTN4, LRIG3 suggest perturbations in YAP signaling in ALMS patients. Conclusion: ALMS1-deficient mitogenic iPSC-CMs recapitulate postnatal proliferation, observed in ALMS patients with mitogenic cardiomyopathy. Preliminary molecular analyses in these in vitro (multicellular) models point to an important role for altered YAP signaling and provide novel cues to enhance CM proliferation, a much-sought objective in cardiac repair.

Immature human engineered heart tissues engraft in a guinea pig chronic injury model.

Engineered heart tissue (EHT) transplantation represents an innovative, regenerative approach for heart failure patients. Late preclinical trials are underway, and a first clinical trial started recently. Preceding studies revealed functional recovery after implantation of in vitro-matured EHT in the subacute stage, whereas transplantation in a chronic injury setting was less efficient. When transplanting matured EHTs, we noticed that cardiomyocytes undergo a dedifferentiation step before eventually forming structured grafts. Therefore, we wanted to evaluate whether immature EHT (EHTIm) patches can be used for transplantation. Chronic myocardial injury was induced in a guinea pig model. EHTIm (15×106 cells) were transplanted within hours after casting. Cryo-injury led to large transmural scars amounting to 26% of the left ventricle. Grafts remuscularized 9% of the scar area on average. Echocardiographic analysis showed some evidence of improvement of left-ventricular function after EHTIm transplantation. In a small translational proof-of-concept study, human scale EHTIm patches (4.5×108 cells) were epicardially implanted on healthy pig hearts (n=2). In summary, we provide evidence that transplantation of EHTIm patches, i.e. without precultivation, is feasible, with similar engraftment results to those obtained using matured EHT.

Automated assessment of human engineered heart tissues using deep learning and template matching for segmentation and tracking.

The high rate of drug withdrawal from the market due to cardiovascular toxicity or lack of efficacy, the economic burden, and extremely long time before a compound reaches the market, have increased the relevance of human in vitro models like human (patient-derived) pluripotent stem cell (hPSC)-derived engineered heart tissues (EHTs) for the evaluation of the efficacy and toxicity of compounds at the early phase in the drug development pipeline. Consequently, the EHT contractile properties are highly relevant parameters for the analysis of cardiotoxicity, disease phenotype, and longitudinal measurements of cardiac function over time. In this study, we developed and validated the software HAARTA (Highly Accurate, Automatic and Robust Tracking Algorithm), which automatically analyzes contractile properties of EHTs by segmenting and tracking brightfield videos, using deep learning and template matching with sub-pixel precision. We demonstrate the robustness, accuracy, and computational efficiency of the software by comparing it to the state-of-the-art method (MUSCLEMOTION), and by testing it with a data set of EHTs from three different hPSC lines. HAARTA will facilitate standardized analysis of contractile properties of EHTs, which will be beneficial for in vitro drug screening and longitudinal measurements of cardiac function.

Overexpression of KCNJ2 enhances maturation of human-induced pluripotent stem cell-derived cardiomyocytes

BackgroundAlthough human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel Kir2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking.MethodsiPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs).ResultsWe showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of Kir2.1 and current density of Kir2.1-encoded IK1. The larger IK1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by IK1 blocker BaCl2. KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca2+ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction.ConclusionsOverexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca2+ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine.

Establishment of an in vivo human engineered heart tissue platform to model iron overload cardiomyopathy.

Iron homeostasis is very tightly regulated in the body and its imbalance has adverse effects. Iron overload affects multiple organ systems, including the heart where it can cause cardiomyopathy, heart failure, and arrhythmias. Iron overload can be caused by repeated blood transfusions or genetic mutations in iron-handling genes such as ferroportin. Establishing a model system to study the cardiac effects of iron overload in humans has been challenging since animal models have failed to recapitulate key aspects of the human disease. To overcome this challenge, we have leveraged stem cell technologies to generate human engineered heart tissues (EHTs) comprised of human cardiac cells, including cardiomyocytes and cardiac fibroblasts. We also developed assays to probe key biophysical and physiological responses in the tissues, including tissue mechanics, contractility, calcium handling, and gene expression. We applied this system to model iron overload. We found that iron overload increases the production of reactive oxygen species and reduced cell viability in both human cardiomyocytes and cardiac fibroblasts. EHTs treated with iron were able to recapitulate key aspects of the human disease, including reduced systolic function, with reduced contractile force and slower kinetics. Finally, we provide a roadmap for how this system can be applied to study genetic forms of iron overload and to test novel therapeutic strategies.

Predictive genotype-phenotype correlation of four tropomyosin-1 variants of unknown significance.

Cardiomyopathies affect nearly 20% of humanity worldwide and are often caused by mutations in heart muscle genes that encode cardiac thin filament (TF) proteins. The on-off switching of cardiac muscle contraction is controlled by the TF proteins troponin and tropomyosin responding to alterations in sarcoplasmic calcium levels. In the absence of calcium, troponin-tropomyosin inhibits actomyosin interactions and induces muscle relaxation, whereas the addition of calcium relieves this constraint leading to contraction. Many mutations in TF proteins that are linked to cardiomyopathy appear to disrupt this regulatory switching. Here, we applied an interdisciplinary approach to correlate atomic-level alterations in tropomyosin structure to myofilament and tissue-level function to better understand mechanisms of disease. Twenty variants of unknown significance (VUS) in TPM1 from ClinVar were screened computationally using molecular dynamics to predict effects on tropomyosin structure and dynamics. Four candidate variants (A102D, D258E, K233N, and A239T) were identified and selected for further functional studies using in vitro motility assays and human engineered heart tissue (EHT) mechanics. The variants associated with hypertrophic cardiomyopathy, D258E and A102D, increased calcium sensitivity, while K233N, a VUS associated with dilated cardiomyopathy, lead to a decrease in calcium sensitivity. EHTs expressing A102D displayed a nearly 50% increase in peak isometric twitch force and delayed relaxation and D258E-EHTs also slowed relaxation. These phenotypes are consistent with the increased calcium sensitivity observed in the in vitro motility assay and with clinical manifestations of hypertrophic cardiomyopathy. Meanwhile, DCM-associated variants K233N and A239T caused dramatic and significant reductions in isometric twitch force when expressed in EHTs. Our approach reveals an important step toward predictive genotype-phenotype correlation and has the potential to benefit patients by accelerating the characterization and classification of tropomyosin variants.

Cardiac splicing as a diagnostic and therapeutic target.

Despite advances in therapeutics for heart failure and arrhythmias, a substantial proportion of patients with cardiomyopathy do not respond to interventions, indicating a need to identify novel modifiable myocardial pathobiology. Human genetic variation associated with severe forms of cardiomyopathy and arrhythmias has highlighted the crucial role of alternative splicing in myocardial health and disease, given that it determines which mature RNA transcripts drive the mechanical, structural, signalling and metabolic properties of the heart. In this Review, we discuss how the analysis of cardiac isoform expression has been facilitated by technical advances in multiomics and long-read and single-cell sequencing technologies. The resulting insights into the regulation of alternative splicing - including theidentification of cardiac splice regulators as therapeutic targets and the development of a translational pipeline to evaluate splice modulators in human engineered heart tissue, animal models and clinical trials - provide a basis for improved diagnosis and therapy. Finally, we consider how the medical and scientific communities can benefit from facilitated acquisition and interpretation of splicing data towards improved clinical decision-making and patient care.

Increased length-dependent activation of human engineered heart tissue after chronic α1A-adrenergic agonist treatment: testing a novel heart failure therapy.

Chronic stimulation of cardiac α1A-adrenergic receptors (α1A-ARs) improves symptoms in multiple preclinical models of heart failure. However, the translational significance remains unclear. Human engineered heart tissues (EHTs) provide a means of quantifying the effects of chronic α1A-AR stimulation on human cardiomyocyte physiology. EHTs were created from thin slices of decellularized pig myocardium seeded with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and fibroblasts. With a paired experimental design, EHTs were cultured for 3 wk, mechanically tested, cultured again for 2 wk with α1A-AR agonist A61603 (10 nM) or vehicle control, and retested after drug washout for 24 h. Separate control experiments determined the effects of EHT age (3-5 wk) or repeat mechanical testing. We found that chronic A61603 treatment caused a 25% increase of length-dependent activation (LDA) of contraction compared with vehicle treatment (n = 7/group, P = 0.035). EHT force was not increased after chronic A61603 treatment. However, after vehicle treatment, EHT force was increased by 35% relative to baseline testing (n = 7/group, P = 0.022), suggesting EHT maturation. Control experiments suggested that increased EHT force resulted from repeat mechanical testing, not from EHT aging. RNA-seq analysis confirmed that the α1A-AR is expressed in human EHTs and found chronic A61603 treatment affected gene expression in biological pathways known to be activated by α1A-ARs, including the MAP kinase signaling pathway. In conclusion, increased LDA in human EHT after chronic A61603 treatment raises the possibility that chronic stimulation of the α1A-AR might be beneficial for increasing LDA in human myocardium and might be beneficial for treating human heart failure by restoring LDA.NEW & NOTEWORTHY Chronic stimulation of α1A-adrenergic receptors (α1A-ARs) is known to mediate therapeutic effects in animal heart failure models. To investigate the effects of chronic α1A-AR stimulation in human cardiomyocytes, we tested engineered heart tissue (EHT) created with iPSC-derived cardiomyocytes. RNA-seq analysis confirmed human EHT expressed α1A-ARs. Chronic (2 wk) α1A-AR stimulation with A61603 (10 nM) increased length-dependent activation (LDA) of contraction. Chronic α1A-AR stimulation might be beneficial for treating human heart failure by restoring LDA.

Functional characterisation of a patient-derived laminopathy model in human engineered heart tissues recapitulates fibrosis and mechanical decoupling defect

Functional characterisation of a patient-derived laminopathy model in human engineered heart tissues recapitulates fibrosis and mechanical decoupling defect

Generation of human engineered heart tissue with a chemo- and optogenetic Off-On switch

Generation of human engineered heart tissue with a chemo- and optogenetic Off-On switch

Abstract 14720: Marked Increase in Mitochondria Degradation in Engineered Heart Tissue During Reperfusion

Introduction: Engineered heart tissue (EHT) consisting of beating cardiomyocytes differentiated from human inducible pluripotent stem cells (hiPSCs) has the potential to recapitulate molecular and functional features of the human heart. Cardiac homeostasis is dependent on lysosomal removal of damaged mitochondria through selective autophagy (mitophagy). The level of mitophagy and cardioprotective impact during ischemia and reperfusion remains elusive and demands further investigation. Objective: To assess mitophagy during ischemia-reperfusion simulation in human EHTs. Methods and results: A hiPSC line was genetically modified using CRISPR/Cas9 knock-in to insert a fluorescent tag (mCherry or EGFP) on Translocase of the Outer Mitochondrial Membrane 20 ( TOMM20) gene resulting in hiPSCs with fluorescent mitochondria. The hiPSCs were differentiated to cardiomyocytes that were embedded in a fibrin hydrogel between two flexible silicone posts to generate EHTs. The EHTs (21 days or older) were subjected to 90 min of ischemia simulation followed by 120 min of simulated reperfusion with and without the lysosomal inhibitor Bafilomycin A1 (BafA1) to measure flux. Time-matched control EHTs remained in normal conditions (with and without BafA1). The EHTs were fixed and sections were immunostained with the lysosomal marker LAMP1A and analyzed by confocal microscopy. Co-localization of fluorescent mitochondria and stained lysosomes was observed after 120 min of reperfusion. To further assess this co-localization we used proximity ligation assay (PLA), an antibody-based assay allowing verification of close proximity (within 40 nm) of two proteins resulting in a single fluorescent signal (PLA dot). We performed the assay using anti-TOMM20 and anti-LAMP1A antibodies to localize mitochondrial content within lysosomes. Notably, there was a significant increase in the number of PLA dots in response to 120 min of reperfusion in the presence of BafA1 (3.41±0.56 fold vs. control, p<0.01; n=4), indicative of induced mitophagy flux. Conclusion: We observed a marked increase in lysosomal degradation of mitochondria in EHTs during reperfusion. The functional impact on EHTs during ischemia and reperfusion needs further research.

Investigating and Resolving Cardiotoxicity Induced by COVID‐19 Treatments using Human Pluripotent Stem Cell‐Derived Cardiomyocytes and Engineered Heart Tissues

Coronavirus disease 2019 continues to spread worldwide. Given the urgent need for effective treatments, many clinical trials are ongoing through repurposing approved drugs. However, clinical data regarding the cardiotoxicity of these drugs are limited. Human pluripotent stem cell‐derived cardiomyocytes (hCMs) represent a powerful tool for assessing drug‐induced cardiotoxicity. Here, by using hCMs, it is demonstrated that four antiviral drugs, namely, apilimod, remdesivir, ritonavir, and lopinavir, exhibit cardiotoxicity in terms of inducing cell death, sarcomere disarray, and dysregulation of calcium handling and contraction, at clinically relevant concentrations. Human engineered heart tissue (hEHT) model is used to further evaluate the cardiotoxic effects of these drugs and it is found that they weaken hEHT contractile function. RNA‐seq analysis reveals that the expression of genes that regulate cardiomyocyte function, such as sarcomere organization (TNNT2, MYH6) and ion homeostasis (ATP2A2, HCN4), is significantly altered after drug treatments. Using high‐throughput screening of approved drugs, it is found that ceftiofur hydrochloride, astaxanthin, and quetiapine fumarate can ameliorate the cardiotoxicity of remdesivir, with astaxanthin being the most prominent one. These results warrant caution and careful monitoring when prescribing these therapies in patients and provide drug candidates to limit remdesivir‐induced cardiotoxicity.

Physiological calcium combined with electrical pacing accelerates maturation of human engineered heart tissue.

SummaryHuman-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have wide potential application in basic research, drug discovery, and regenerative medicine, but functional maturation remains challenging. Here, we present a method whereby maturation of hiPSC-CMs can be accelerated by simultaneous application of physiological Ca2+ and frequency-ramped electrical pacing in culture. This combination produces positive force-frequency behavior, physiological twitch kinetics, robust β-adrenergic response, improved Ca2+ handling, and cardiac troponin I expression within 25 days. This study provides insights into the role of Ca2+ in hiPSC-CM maturation and offers a scalable platform for translational and clinical research.

Human Engineered Heart Tissue Models for Disease Modeling and Drug Discovery.

The emergence of human induced pluripotent stem cells (hiPSCs) and efficient differentiation of hiPSC-derived cardiomyocytes (hiPSC-CMs) induced from diseased donors have the potential to recapitulate the molecular and functional features of the human heart. Although the immaturity of hiPSC-CMs, including the structure, gene expression, conduct, ion channel density, and Ca2+ kinetics, is a major challenge, various attempts to promote maturation have been effective. Three-dimensional cardiac models using hiPSC-CMs have achieved these functional and morphological maturations, and disease models using patient-specific hiPSC-CMs have furthered our understanding of the underlying mechanisms and effective therapies for diseases. Aside from the mechanisms of diseases and drug responses, hiPSC-CMs also have the potential to evaluate the safety and efficacy of drugs in a human context before a candidate drug enters the market and many phases of clinical trials. In fact, novel drug testing paradigms have suggested that these cells can be used to better predict the proarrhythmic risk of candidate drugs. In this review, we overview the current strategies of human engineered heart tissue models with a focus on major cardiac diseases and discuss perspectives and future directions for the real application of hiPSC-CMs and human engineered heart tissue for disease modeling, drug development, clinical trials, and cardiotoxicity tests.

GSK-3β Localizes to the Cardiac Z-Disc to Maintain Length Dependent Activation.

Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. Inducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3β. Agreeing with the localization of its targets, GSK-3β that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. We identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.