Human Engineered Heart Tissue Research Articles

Distinct mechanisms drive divergent phenotypes in hypertrophic and dilated cardiomyopathy associated TPM1 variants.

Hypertrophic and dilated cardiomyopathies (HCM and DCM, respectively) are inherited disorders that may be caused by mutations to the same sarcomeric protein but have completely different clinical phenotypes. The precise mechanisms by which point mutations within the same gene bring about phenotypic diversity remain unclear. Our objective has been to develop a mechanistic explanation of diverging phenotypes in two TPM1 mutations, E62Q (HCM) and E54K (DCM). Drawing on data from the literature and experiments with stem cell-derived cardiomyocytes expressing the TPM1 mutations of interest, we constructed computational simulations that provide plausible explanations of the distinct muscle contractility caused by each variant. In E62Q, increased calcium sensitivity and hypercontractility was explained most accurately by a reduction in effective molecular stiffness of tropomyosin and alterations in its interactions with the actin thin filament that favor the 'closed' regulatory state. By contrast, the E54K mutation appeared to act via long-range allosteric interactions to increase the association rate of the C-terminal troponin I mobile domain to tropomyosin/actin. These mutation-linked molecular events produced diverging alterations in gene expression that can be observed in human engineered heart tissues. Modulators of myosin activity confirmed our proposed mechanisms by rescuing normal contractile behavior in accordance with predictions.

Chronic Activation of Tubulin Tyrosination Improves Heart Function.

Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and associated with left ventricular hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with the findings that microtubule detyrosination (dTyr-MT) is markedly elevated in heart failure. Acute reduction of dTyr-MT by inhibition of the detyrosinase (VASH [vasohibin]/SVBP [small VASH-binding protein] complex) or activation of the tyrosinase (TTL [tubulin tyrosine ligase]) markedly improved contractility and reduced stiffness in human failing cardiomyocytes and thus posed a new perspective for HCM treatment. In this study, we tested the impact of chronic tubulin tyrosination in an HCM mouse model (Mybpc3 knock-in), in human HCM cardiomyocytes, and in SVBP-deficient human engineered heart tissues (EHTs). Adeno-associated virus serotype 9-mediated TTL transfer was applied in neonatal wild-type rodents, in 3-week-old knock-in mice, and in HCM human induced pluripotent stem cell-derived cardiomyocytes. We show (1) TTL for 6 weeks dose dependently reduced dTyr-MT and improved contractility without affecting cytosolic calcium transients in wild-type cardiomyocytes; (2) TTL for 12 weeks reduced the abundance of dTyr-MT in the myocardium, improved diastolic filling, compliance, cardiac output, and stroke volume in knock-in mice; (3) TTL for 10 days normalized cell area in HCM human induced pluripotent stem cell-derived cardiomyocytes; (4) TTL overexpression activated transcription of tubulins and other cytoskeleton components but did not significantly impact the proteome in knock-in mice; (5) SVBP-deficient EHTs exhibited reduced dTyr-MT levels, higher force, and faster relaxation than TTL-deficient and wild-type EHTs. RNA sequencing and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-deficient versus TTL-deficient EHTs. This study provides the first proof of concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the nonsarcomeric cytoskeleton in heart disease.

Engineered model of heart tissue repair for exploring fibrotic processes and therapeutic interventions

Advancements in human-engineered heart tissue have enhanced the understanding of cardiac cellular alteration. Nevertheless, a human model simulating pathological remodeling following myocardial infarction for therapeutic development remains essential. Here we develop an engineered model of myocardial repair that replicates the phased remodeling process, including hypoxic stress, fibrosis, and electrophysiological dysfunction. Transcriptomic analysis identifies nine critical signaling pathways related to cellular fate transitions, leading to the evaluation of seventeen modulators for their therapeutic potential in a mini-repair model. A scoring system quantitatively evaluates the restoration of abnormal electrophysiology, demonstrating that the phased combination of TGFβ inhibitor SB431542, Rho kinase inhibitor Y27632, and WNT activator CHIR99021 yields enhanced functional restoration compared to single factor treatments in both engineered and mouse myocardial infarction model. This engineered heart tissue repair model effectively captures the phased remodeling following myocardial infarction, providing a crucial platform for discovering therapeutic targets for ischemic heart disease.

Abstract We007: High Throughput 3D-Printed Human Engineered Heart Tissues for Cardiac Disease Modeling

While cell models have been used to study cardiac diseases, there is currently no standardization or fully mature in vitro system. Obtaining a mature cardiomyocyte (CM) phenotype is necessary to create a physiologically relevant environment and accurately model and study cardiac diseases. Increased success in maturation of human induced pluripotent stem cell derived CMs (hiPS-CMs) has been achieved in 3D in vitro models in the form of engineered heart tissues (EHTs). Digital light processing (DLP) offers a high throughput and efficient method to fabricate hydrogels that recapitulate properties of native tissues. In this study, we use DLP-printed EHTs to seed and mature differentiated hiPS-CMs and test their potential for modeling pathological cardiac hypertrophy. To further encourage maturity of CMs, we utilized a low glucose maturation medium supplemented with fatty acids. DLP-printed EHTs displayed high reproducibility and increased CM maturity. To determine degree of maturity of our system, we performed RT-qPCR, western blot analysis, and immunofluorescence (IF) microscopy. We found increased expression of fatty-acid transport and beta oxidation genes and improved sarcomere alignment compared to 2D hiPS-CMs. To model pathological cardiac hypertrophy, we used two different approaches: 1. acute adrenergic agonism with isoproterenol and phenylephrine; 2. chronic culturing of EHTs in pathologically stiff conditions. To assess pathology, we conducted RT-qPCR, western blot, ELISA, IF microscopy, and functional analyses. We found that agonist-treated EHTs displayed increased pathology-associated gene expression compared to standard 2D hiPS-CMs. Both agonist-treated and stiff EHTs had increased secreted B-Type Natriuretic Peptide (BNP) and phosphorylation of proteins involved in pathological remodeling, including ERK and P38. Additionally, we observed increased nuclear area, increased calcium handling, and increased contractile force in the treatment groups. In conclusion, we have established a high throughput, reproducible, mature EHT platform that can be used to model pathological cardiac hypertrophy. This platform can be easily adopted by other laboratories to study a wide array of cardiac diseases.

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.

In silico and in vitro models reveal the molecular mechanisms of hypocontractility caused by TPM1 M8R.

Dilated cardiomyopathy (DCM) is an inherited disorder often leading to severe heart failure. Linkage studies in affected families have revealed hundreds of different mutations that can cause DCM, with most occurring in genes associated with the cardiac sarcomere. We have developed an investigational pipeline for discovering mechanistic genotype-phenotype relationships in DCM and here apply it to the DCM-linked tropomyosin mutation TPM1 M8R. Atomistic simulations predict that M8R increases flexibility of the tropomyosin chain and enhances affinity for the blocked or inactive state of tropomyosin on actin. Applying these molecular effects to a Markov model of the cardiac thin filament reproduced the shifts in Ca2+sensitivity, maximum force, and a qualitative drop in cooperativity that were observed in an in vitro system containing TPM1 M8R. The model was then used to simulate the impact of M8R expression on twitch contractions of intact cardiac muscle, predicting that M8R would reduce peak force and duration of contraction in a dose-dependent manner. To evaluate this prediction, TPM1 M8R was expressed via adenovirus in human engineered heart tissues and isometric twitch force was observed. The mutant tissues manifested depressed contractility and twitch duration that agreed in detail with model predictions. Additional exploratory simulations suggest that M8R-mediated alterations in tropomyosin-actin interactions contribute more potently than tropomyosin chain stiffness to cardiac twitch dysfunction, and presumably to the ultimate manifestation of DCM. This study is an example of the growing potential for successful in silico prediction of mutation pathogenicity for inherited cardiac muscle disorders.

Biogenic derived nanoparticles modulate mitochondrial function in cardiomyocytes.

Preservation of mitochondrial functionality is essential for heart hemostasis and cardiovascular diseases treatment. However, the current nanomedicines including liposomes, polymers and inorganic nanomaterials are severely hindered by poor stability, high manufacturing costs and potential biotoxicity. In this research, we present novel polyphenolic nanoparticles (NPs) derived from naturally occurring pomegranate peel (PP, labelled as PPP NPs), which exhibit potent antioxidative and anti-inflammatory properties, serving as a modulator of mitochondrial function. PPP NPs have been identified to improve survival rates in models of mitochondrial depletion through enhancement of cardiomyocyte proliferation and the reduction of DNA damage. Moreover, PPP NPs can effectively inhibit the production of reactive oxygen species and inflammatory mediators in lipopolysaccharide (LPS)-induced mitochondrial damage. Utilizing human engineered heart tissue and mice models, PPP NPs were found to significantly improve contractile function and alleviate inflammation activities after LPS treatment. Mechanically, PPP NPs regulated inflammatory responses via a m6A dependent manner, as determined using RNA-seq and MeRIP-seq analyses. Collectively, these insights underscore the potential of PPP NPs as a novel therapeutic approach for mitochondrial dysfunction.

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