Tissue-engineered Heart Research Articles

The Role of Human-Induced Pluripotent Stem Cells in Studying Cardiac Channelopathies

Cardiac channelopathies are inherited diseases that increase the risk of sudden cardiac death. While different genes have been associated with inherited channelopathies, there are still subtypes, e.g., catecholaminergic polymorphic ventricular tachycardia and Brugada syndrome, where the genetic cause remains unknown. Various models, including animal models, heterologous expression systems, and the human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSCs-CMs) model, have been used to study the pathophysiological mechanisms of channelopathies. Recently, researchers have focused on using hiPSCs-CMs to understand the genotype–phenotype correlation and screen drugs. By combining innovative techniques such as Clustered Regularly Interspaced Short Palindromic Repeats/Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9)-mediated genome editing, and three-dimensional (3D) engineered heart tissues, we can gain new insights into the pathophysiological mechanisms of channelopathies. This approach holds promise for improving personalized drug treatment. This review highlights the role of hiPSCs-CMs in understanding the pathomechanism of Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia and how these models can be utilized for drug screening.

CRISPRi/a screens in human iPSC-cardiomyocytes identify glycolytic activation as a druggable target for doxorubicin-induced cardiotoxicity.

Doxorubicin is limited in its therapeutic utility due to its life-threatening cardiovascular side effects. Here, we present an integrated drug discovery pipeline combining human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs), CRISPR interference and activation (CRISPRi/a) bidirectional pooled screens, and a small-molecule screening to identify therapeutic targets mitigating doxorubicin-induced cardiotoxicity (DIC) without compromising its oncological effects. The screens revealed several previously unreported candidate genes contributing to DIC, including carbonic anhydrase 12 (CA12). Genetic inhibition of CA12 protected iCMs against DIC by improving cell survival, sarcomere structural integrity, contractile function, and calcium handling. Indisulam, a CA12 antagonist, can effectively attenuate DIC in iCMs, engineered heart tissue, and animal models. Mechanistically, doxorubicin-induced CA12 potentiated a glycolytic activation in cardiomyocytes, contributing to DIC by interfering with cellular metabolism and functions. Collectively, our study provides a roadmap for future drug discovery efforts, potentially leading to more targeted therapies with minimal off-target toxicity.

In Vitro Vascularization Improves In Vivo Functionality of Human Engineered Cardiac Tissues

Engineered human cardiac tissues hold great promise for disease modeling, drug development, and regenerative therapy. For regenerative applications, successful engineered tissue engraftment in vivo requires rapid vascularization and blood perfusion post-implantation. In the present study, we engineered highly functional, vascularized cardiac tissues (“cardiopatches”) by co-culturing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and endothelial cells (hiPSC-ECs) in optimized serum-free media. The vascularized cardiopatches displayed stable capillary networks over 4 weeks of culture, the longest reported in the field, while maintaining high contractile stress (>15 mN/mm2) and fast conduction velocity (>20 cm/s). Robustness of the method was confirmed using two distinct hiPSC-EC sources. Upon implantation into dorsal-skinfold chambers in immunocompromised mice, in vitro vascularized cardiopatches exhibited improved angiogenesis compared to avascular implants. Significant lumenization of the engineered human vasculature and anastomosis with host mouse vessels yielded the formation of hybrid human-mouse capillaries and robust cardiopatch perfusion by blood. Moreover, compared to avascular tissues, the implanted vascularized cardiopatches exhibited significantly higher conduction velocity and Ca2+ transient amplitude, longitudinally monitored in live mice for the first time. Overall, we demonstrate successful 4-week vascularization of engineered human cardiac tissues without loss of function in vitro, which promotes tissue functionality upon implantation in vivo. Statement of SignificanceComplex interactions between cardiac muscle fibers and surrounding capillaries are critical for everyday function of the heart. Tissue engineering is a powerful method to recreate functional cardiac muscle and its vascular network, which are both lost during a heart attack. Our study demonstrates in vitro engineering of dense capillary networks within highly functional engineered heart tissues that successfully maintain the structure, electrical, and mechanical function long-term. In mice, human capillaries from these engineered tissues integrate with host mouse capillaries to allow blood perfusion and support improved implant function. In the future, the developed vascularized engineered heart tissues will be used for in vitro studies of cardiac development and disease and as a potential regenerative therapy for heart attack.

Characterisation of the novel SCN5A-P1891A variant, implicated in left ventricular noncompaction cardiomyopathy in a Finnish family, using human induced pluripotent stem cell-derived cardiomyocytes

Abstract Introduction Left ventricular noncompaction (LVNC) is a heterogenous cardiomyopathy characterised by an extensively trabeculated left ventricle (LV). Several genetic variants that perturb trabeculae compaction, and thus LV maturation, have been reported [1]. However, the precise mechanisms underpinning this have proven contentious with, for example, differentially aberrant cardiomyocyte proliferation implicated in the development of LVNC [2,3]. Purpose In this study, we investigate a novel SCN5A variant, P1891A, which is associated with LVNC in a Finnish family. We ascertain the effects of this variant through the characterisation of variant-carrying human induced pluripotent stem cell-derived cardiomyocytes (P1891A-hiPSC-CMs). Methods We isolated dermal fibroblasts from a symptomatic member of a Finnish family carrying the SCN5A-P1891A variant; derived P1891A-hiPSCs via nucleofection with reprogramming plasmids [4]; and differentiated these into hiPSC-CMs alongside healthy control-hiPSCs. We determined the action potential (AP) and sodium- (INa) and calcium-current (ICa) densities of hiPSC-CMs via patch-clamp. We investigated gene expression in hiPSC-CMs through qPCR and employed Multiple Approaches Combined (MCA) proteomics [5] in SCN5A-transfected HEK293-like cells to elucidate protein-protein interactions (PPI). We subjected hiPSC-CMs to mechanical stretch [6] to examine their stress response and assessed their proliferation capacity via high-content analysis for the percentage of BrdU positive hiPSC-CMs following mitogenic stimulation (5 µM CHIR99021 and 1 µM SB203580). Finally, we ascertained contractile kinetics and apparent contractile force using fibrin hydrogel-based engineered heart tissues (EHTs) [7]. Results The P1891A-hiPSC-CMs demonstrated elevated INa density alongside enhanced arrhythmogenicity relative to healthy control hiPSC-CMs although, AP kinetics and ICa density were unperturbed (Fig. 1). Under baseline conditions, P1891A-hiPSC-CMs exhibited higher SCN5A gene expression and proteomics revealed a reduction in PPI. Baseline proliferation was unchanged; however, aged P1891A-hiPSC-CMs had enhanced proliferative capacity following mitogenic stimulation. Further, the P1891A-hiPSC-CMs displayed impaired tolerance to mechanical stretch as evidenced by upregulated NPPB and ACTA1 expression. In the context of the EHT, P1891A-hiPSC-CMs yielded disparate phenotypes. Whilst a subset compacted fully and yielded weaker contractile parameters relative to healthy control EHTs, the majority of P1891A-hiPSC-CM EHTs failed to fully compact thereby recapitulating the LVNC phenotype (Fig. 2). Conclusions This study presents a unique aetiology of LVNC and links, for the first time, SCN5A mutations with enhanced human cardiomyocyte proliferation. The ability to recapture the noncompaction phenotype in EHTs presents a powerful model for future mechanistic research and drug development.

Mature cardiac tissues modeling Duchenne muscular dystrophy related cardiomyopathy

Abstract Background The major cause of mortality in patients with Duchenne muscular dystrophy (DMD) is cardiomyopathy. This disease is caused by dystrophin gene mutations that result in the loss of dystrophin protein. Gene replacement therapies have been approved for skeletal muscle symptoms of DMD. However, these therapeutic modalities are not indicated for cardiomyopathy due to their low efficiency of delivery to the heart. Despite the significant unmet needs, a limitation in this area of research is the divergence of cardiomyopathy symptoms in model animals from those in patients. Alternatively, cardiomyocytes differentiated from patient-derived iPS cells have recently been analyzed. However, existing results are based on a single time point analysis and do not model disease progression or non-cardiomyocyte involvement. Purpose Our research aims to establish an in vitro model that can reproduce the pathological progression of DMD cardiomyopathy. Methods We have generated cardiac organoids and engineered heart tissues (EHTs) from patient-derived iPS cells, consisting of cardiomyocytes and epicardial-derived non-cardiomyocytes. An iPS cell line in which the dystrophin mutation was repaired by genome editing was used as a control. The cardiac organoids or EHTs were subjected to transient activation of fatty acid metabolism to promote the maturation of iPS-derived cardiomyocytes. Pathological conditions were then induced in these 3D-models by applying loads that mimicked the DMD-disease environment. Results The mature cardiac organoids displayed adult-like gene expression profiles in the components of dystrophin-associated-protein-complex and calcium handling. After continuous mechanical loading, cell death appeared in DMD-EHTs, although there was only a slight decrease in contractility and fibrosis. Elevated mitochondrial ROS production and DNA damage were observed as a part of the cell death mechanism. Furthermore, pathological stimuli in addition to mechanical loading reproduced progressive contractile dysfunction and further increase in fibrosis in DMD-EHTs. Conclusions From these observations, we concluded that our DMD-EHT model recapitulate the pathological development of DMD-cardiomyopathy. These results suggest that the key factors involved in the progression of DMD cardiomyopathy are (i) the fragility of cardiomyocytes to stress in early stages and (ii) the involvement of non-cardiomyocytes such as fibrosis. Our model provides insight into the molecular mechanisms underlying disease progression and novel therapeutic targets.

Phosphoproteomics of PLN R14del heart tissue reveals major myocyte degenerate phenotype, and is attenuated by PLN antisense treatment in vitro

Abstract Background/Purpose: Phospholamban (PLN) p.Arg14del (R14del, R14Δ/+) is a pathogenic variant that can cause cardiomyopathy, characterized by PLN protein aggregation, ultimately leading to heart failure (HF). The exact pathophysiology is unknown, we aim to uncover R14Δ/+ disease mechanisms and study potential treatment using PLN antisense oligonucleotides (PLN-ASOs). Methods Phosphoproteomics was performed on human heart tissue from end-stage R14Δ/+ patients (N=6) versus other etiologies of HF (N=10) as a control. CRISPR-Cas9 engineered R14Δ/+ and isogenic control (WT) induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were extensively characterized, including the phosphoproteome, calcium transients (FLUO 4-AM), mitochondrial respiration (Seahorse Mito Stress Test) and contractility in dynamic engineered heart tissues (dyn-EHTs) before and after PLN-ASO treatment, and the results were validated in R14Δ/+ iPSC-CMs derived from R14Δ/+ patients. Results Phosphoproteomics of heart tissue from R14Δ/+ vs. other etiologies of HF identified 138 differentially expressed proteins (DEPs) and 317 differentially expressed phosphoproteins (DEPPs), therewith establishing a disease-specific signature. Phosphoproteomics of WT vs. R14Δ/+ iPSC-CMs identified 451 DEPs and 1046 DEPPs. Gene ontology analysis of end-stage R14Δ/+ heart tissue and iPSC-CMs revealed a large overlap. Here, R14Δ/+ showed to have great impact on contraction (e.g. SYNPO2L, MYH10, TTN) and calcium (e.g. AHNAK, HRC, RYR2). In line with this, functional characterization revealed that R14Δ/+ iPSC-CMs have an increased Ca2+ systolic velocity and accelerated calcium transient (decreased T50 and T90), that associated with altered calcium-related DEPPs (e.g. RYR2). In dyn-EHTs, R14Δ/+ dyn-EHTs have a decreased contraction duration, time-to-peak and ability to pace, associated with altered contractility-related DEPPs (e.g. SYNPO2L, MYH7, TTN). PLN-ASOs dose-dependently altered PLN mRNA expression and protein levels in iPSC-CMs, and 62 DEPs and 372 DEPPs were identified. PLN-ASOs increased Ca2+ diastolic velocity, which associated with altered calcium-related DEPPs (e.g. PLN, AHNAK, HRC). PLN-ASOs increased the ability of dyn-EHTs to pace and therewith improved contractility, which associated with altered contractility-related DEPPs (e.g. SYNPO2L, MYH7, TTN). PLN-ASOs dose-dependently increased basal respiration and ATP production, which associated with altered mitophagy-related DEPs (GABARAPL2 and MAP1LC3). These functional characteristics were validated in R14Δ/+ patient iPSC-CMs. Discussion: R14Δ/+ cardiomyocytes have a degenerative phenotype characterized by altered calcium transients and reduced contractility and PLN-ASOs attenuate these characteristics and improve metabolism.

Identification of cell adhesion molecules for the maturation of engineered heart tissue

Abstract Background Engineered cardiac tissue (ECT) utilizing human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is anticipated to function as a translational in vitro model. However, hiPSC-CMs display phenotypes indicative of immaturity, and the structural organization of ECT composed of hiPSC-CMs is insuficient when compared to that of mature adult hearts. Although the mechanisms for hiPSC-CMs maturation have been reported, little is known about the mechanisms regulating the formation of in vitro tissue such as ECT. Cell adhesion molecules play important roles in heart formation in vivo through cell-cell interactions. However, specific cell adhesion molecules responsible for tissue structure formation in in vitro tissue models, such as ECTs, have not been identified. We hypothesize that cell adhesion molecules, which regulate cell-cell attachment, are crucial for in vitro tissue formation. Purpose This study aims to identify the key cell adhesion molecules involved in cell-cell interaction and assess the effects of their expression on hiPSC-CMs and EHT functions. Methods and Results We developed a cell attachment assay to evaluate the attachment ability of hiPCS-CMs and discovered that hiPSC-CMs produced via embryoid body method (EB-hiPSC-CMs) exhibited a higher attachment ability than those produced by monolayer method (Mono-hiPSC-CMs). Further analysis revealed no significant difference in anoikis sensitivity between EB-hiPSC-CMs and Mono-hiPSC-CMs, suggesting that the observed difference in attachment ability were due to differencies in cell adhesion efficiency between hiPSC-CMs generated by two methods. Moreover, RNA-seq analysis identified nine cell adhesion molecule genes that were highly expressed in EB-hiPSC-CMs. We identified the cell adhesion molecules responsible for the attachment ability of hiPSC-CMs by knockdown and over-expression experiments. Moreover, over-expression experiments using adeno-associated virus (AAV) demonstrate that hiPSC-CMs with overexpression of a cell adhesion molecule improved calcium handling and increased propagation speed, and ECTs with overexpression of a cell adhesion molecule showed enhanced sarcomere alignment and sarcomere length compared to controls. Conclusion(s) We observed differences in attachment ability between hiPSC-CMs generated by two differentiation methods. Transcriptome analysis of these hiPSC-CMs identified key cell adhesion molecules that regulate cell-cell attachment ability of hiPSC-CMs. Furthermore, increment of attachment ability of hiPSC-CMs by overexpression of a cell adhesion molecule enhanced hiPSC-CMs and ECT functions.

Investigating Inherited Heart Diseases Using Human Induced Pluripotent Stem Cell-Based Models

Inherited heart diseases (IHDs) are caused by genetic mutations that disrupt the physiological structure and function of the heart. Understanding the mechanisms behind these diseases is crucial for developing personalised interventions in cardiovascular medicine. Development of induced pluripotent stem cells, which can then be differentiated to any nucleated adult cell type, has enabled the creation of personalised single-cell and multicellular models, providing unprecedented insights into the pathophysiology of IHDs. This review provides a comprehensive overview of recent advancements in human iPSC models used to dissect the molecular and genetic underpinnings of common IHDs. We examine multicellular models and tissue engineering approaches, such as cardiac organoids, engineered heart tissue, and multicellular co-culture systems, which simulate complex intercellular interactions within heart tissue. Recent advancements in stem cell models offer a more physiologically relevant platform to study disease mechanisms, enabling researchers to observe cellular interactions, study disease progression, and identify therapeutic strategies. By leveraging these innovative models, we can gain deeper insights into the molecular and cellular mechanisms underlying IHDs, ultimately paving the way for more effective diagnostic and therapeutic strategies.

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.

The Effects Of In Vitro Exercise Of Engineered Heart Tissues On Cardiac Injury And Function

The Effects Of In Vitro Exercise Of Engineered Heart Tissues On Cardiac Injury And Function

Spaceflight-induced contractile and mitochondrial dysfunction in an automated heart-on-a-chip platform

With current plans for manned missions to Mars and beyond, the need to better understand, prevent, and counteract the harmful effects of long-duration spaceflight on the body is becoming increasingly important. In this study, an automated heart-on-a-chip platform was flown to the International Space Station on a 1-mo mission during which contractile cardiac function was monitored in real-time. Upon return to Earth, engineered human heart tissues (EHTs) were further analyzed with ultrastructural imaging and RNA sequencing to investigate the impact of prolonged microgravity on cardiomyocyte function and health. Spaceflight EHTs exhibited significantly reduced twitch forces, increased incidences of arrhythmias, and increased signs of sarcomere disruption and mitochondrial damage. Transcriptomic analyses showed an up-regulation of genes and pathways associated with metabolic disorders, heart failure, oxidative stress, and inflammation, while genes related to contractility and calcium signaling showed significant down-regulation. Finally, in silico modeling revealed a potential link between oxidative stress and mitochondrial dysfunction that corresponded with RNA sequencing results. This represents an in vitro model to faithfully reproduce the adverse effects of spaceflight on three-dimensional (3D)-engineered heart tissue.

Conductive, injectable, and self-healing collagen-hyaluronic acid hydrogels loaded with bacterial cellulose and gold nanoparticles for heart tissue engineering

The increasing demand for advanced biomaterials in nerve tissue engineering presents numerous challenges due to the complexity of nerve tissues and the need for materials that can accurately replicate their intricate structure and function. In response, this study introduces a novel injectable hydrogel that is thermosensitive, self-healing, and conductive, offering promising potential for heart and nerve tissue engineering applications. The hydrogel is based on collagen and hyaluronic acid functionalized with 3-aminopropyl-triethoxysilane (APTES)-grafted oxidized bacterial cellulose and gold nanoparticles (~50 nm). Rheological analysis reveals a substantial enhancement in the elastic modulus of the collagen-hyaluronic acid matrix with the incorporation of bacterial cellulose/gold nanoparticles, improving by an order of magnitude at 1 % strain. This improvement comes with a slight decrease in gelation temperature, from 36 °C to 32 °C. Besides thermo-sensitivity, the nanocomposite hydrogel exhibits a remarkable self-sealing response (about 80 % effectiveness) due to reversible physical crosslinking. Electrical spatial resistance measurements on human embryonic stem cell-derived cardiomyocytes-loaded hydrogels yield a value of ~0.1 S/m, which is suitable for electrical stimulation. In vitro extracellular field potential measurements also affirm the hydrogel's potential as an injectable scaffold for heart tissue engineering, i.e., the electrically stimulated human stem cells exhibit 47 beats per minute with a cell discharge (depletion) of 5.47 μv. A rapid gel formation in the physiological temperature (about 2 min) and high H9C2 cytotoxicity (viability of >90 % after 72 h incubation) is attainable. The developed collagen-based nanocomposite hydrogel offers an injectable, thermosensitive, and self-healing biomaterial platform for nerve or myocardium regeneration.

Simultaneous electromechanical monitoring in engineered heart tissues using a mesoscale framework.

Engineered heart tissues (EHTs) generated from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent powerful platforms for human cardiac research, especially in drug testing and disease modeling. Here, we report a flexible, three-dimensional electronic framework that enables real-time, spatiotemporal analysis of electrophysiologic and mechanical signals in EHTs under physiological loading conditions for dynamic, noninvasive, longer-term assessments. These electromechanically monitored EHTs support multisite measurements throughout the tissue under baseline conditions and in response to stimuli. Demonstrations include uses in tracking physiological responses to pharmacologically active agents and in capturing electrophysiological characteristics of reentrant arrhythmias. This platform facilitates precise analysis of signal location and conduction velocity in human cardiomyocyte tissues, as the basis for a broad range of advanced cardiovascular studies.

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.

P02-20 Development of an evaluation system for drug-induced cardiotoxicity via hepatic metabolism using co-culture of human cryopreserved hepatocytes and engineered heart tissue

P02-20 Development of an evaluation system for drug-induced cardiotoxicity via hepatic metabolism using co-culture of human cryopreserved hepatocytes and engineered heart tissue

Abstract We051: Chronic mavacamten treatment prevents development of contractile dysfunction in cMyBP-C null engineered heart tissue constructs, but not in constructs carrying the cMyBP-C W792R missense mutation

Hypercontractility, caused by mutations that increase the number of myosin heads in the disordered relaxed (DRX) state, is a prevalent cause of hypertrophic cardiomyopathy (HCM). A subgroup of HCM mutations cause primary hypocontractility, by stabilizing super-relaxed crossbridges (SRX). Mavacamten (mava) directly targets hypercontractility by reducing the number of DRX heads and improves outcomes in ~70% patients with obstructive HCM. We here assessed the efficacy of chronic mava treatment at preventing phenotype development in human iPSC engineered heart tissue (EHT) in a cMyBP-C null model (-/-), that increases the number of DRX heads, and EHT carrying the W792R (RR) HCM mutation. While the primary contractile defect caused by the W792R mutation is unknown, we postulated that it differed from the cMyBP-C -/- model, as RR mice die prior to weaning while -/- mice do not. Twitch force (TF) measurements performed on day 7 on control (+/+), -/- and RR EHT show that -/- EHT were hypercontractile while RR EHT were hypocontractile (Fig. 1). Following measurement, EHT from each genotype were either treated with vehicle or 100nM mava and cultured for 21 days. By day 28, TF production was similar between +/+ and -/- vehicle treated EHT while RR EHT remained hypocontractile (Fig. 2). Chronic mava treatment significantly reduced TF production in +/+ EHT but not in -/- or RR EHT (Fig. 2). Consistent with diastolic dysfunction, a hallmark of HCM in humans, day 28 -/- and RR EHT displayed slow relaxation, though the effect was more pronounced in RR EHT (Fig. 3). While chronic mava treatment accelerated relaxation in -/- EHT to levels that were similar to that observed in vehicle treated +/+ EHT, it failed to do so in RR EHT (Fig. 3). cMyBP-C -/- EHTs initially present with a hypercontractile phenotype, consistent with an increase in DRX crossbridges, while W792R RR EHT present with a hypocontractile phenotype. By 4 weeks in EHT culture, both genotypes present with impaired relaxation, that is more severe in RR EHT. While chronic mava treatment prevents development of slow relaxation in -/- EHT, it failed to do so in RR EHT. These findings suggest that the primary disease mechanism of the W792R mutation is not an increase in DRX crossbridges and that mavacamten may not be an effective treatment for patients carrying this mutation, further highlighting the need for studying the pathomechansims of HCM mutations and the development of novel therapies.

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.

Abstract We025: Human Engineered Heart Tissues Demonstrate Clinically-Relevant Disease indicators of Duchenne Muscular Dystrophy

Accurate in vitro modeling of healthy and disease conditions is crucial for assessing the safety and efficacy of novel therapeutics before clinical trials. For cardiac diseases, direct evaluation of muscle contraction is a reliable indicator of overall tissue function, however, this often necessitates expensive and low-throughput non-human in vivo models. Moreover, animal models may not accurately predict human responses to compound exposure. Here, we have developed a turnkey platform for facile fabrication of 3D engineered heart tissues (EHTs) that enables label-free, push-button measurements of contractility. The system allows for customized stimulation protocols to individual constructs, while simultaneously measuring contractility across 24 tissues in parallel with minimal user input. This approach enables stratification of muscle phenotypes and facilitates compound safety and efficacy screening. We present a 3D model of Duchenne muscular dystrophy that displays functional deficits consistent with in vivo disease indicators, including reduced contractile force and slowed relaxation kinetics, though beat rate remains unchanged. We show both acute and chronic effects of cardiotoxic drugs, as well as positive inotropic responses to external calcium and Isoproterenol. This highlights the utility of using EHTs to identify toxicity and derisk drug development and clinical testing outcomes. Spontaneously beating EHTs were also subjected to in situ stretching, leading to increased levels of cardiac troponin I (cTnI) in the tissue medium, indicating stretch induced injury. These data demonstrate a first-and-only commercial platform that integrates individual, fully-customizable, well-based control of electrical stimulation across a 24-well plate of tissues to pace muscle constructs, model force-frequency, and enable exercise regimens or damage protocols. Heart organoids were stretched under load presenting a challenge for therapeutics targeting injury prevention in the myocardium. Stimulation is coupled with automated assessment of 3D muscle contraction, providing an inclusive, medium-throughput platform for studying muscle-related disorders, advancing therapeutic discovery using a modality-agnostic biosystem.

Abstract Mo088: RBFOX2 haploinsufficiency impairs cardiomyocyte adhesion and contractility through faulty RNA metabolism

Background: Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease that is uniformly lethal if left untreated. Protein damaging mutations in one allele of RBFOX2, a highly conserved RNA binding protein, are enriched in HLHS patients, suggesting they are causal. However, it remains unclear how RBFOX2 haploinsufficiency contributes to disease pathogenesis as heterozygous animal models appear healthy. Methods: We generated and characterized RBFOX2 heterozygous and null human induced pluripotent stem cells (hiPSCs) and assessed their phenotypes in differentiated cardiomyocytes (iPSC-CMs) in 2D culture. To learn if altered function might be revealed in a more native microenvironment, we also generated 3D engineered heart tissues (EHTs) from RBFOX2 heterozygous hiPSC-CMs. To delineate the underlying molecular mechanisms, we performed bulk RNA-seq to reveal differential gene expression (DGE) and alternative splicing events (ASEs) between the different genetic cohorts and eCLIP-seq to detect RNAs directly bound by RBFOX2 in the CM lineage. Results: We observed dose-dependent reductions in cell adhesion, size, maturation, respiration, calcium handling, and action potential duration with no significant effect on proliferation in 2D culture. Although myofibril alignment and contractility were completely abolished in null hiPSC-CMs, these features were surprisingly preserved in heterozygous cells. However, in EHTs, we found that RBFOX2 is haploinsufficient for tissue maturation and contractility, suggesting that myocardial-intrinsic defects contribute to RBFOX2 -mediated HLHS. Integration of DGE, ASEs and eCLIP-seq datasets uncovered significant enrichment of DGE and/or ASEs of RBFOX2-bound and unbound transcripts involved in cell adhesion between the extracellular matrix (ECM) and the actin cytoskeleton/sarcomere network. Included in the direct targets with altered expression and alternative splicing was the ECM ligand FIBRONECTIN 1 ( FN1 ), which when downregulated was previously associated with HLHS. Conclusions: Our data suggest that RBFOX2 sits atop an expression and splicing hierarchy that promotes optimal CM cell growth, adhesion, and contractility that protects the heart from HLHS pathogenesis.