Stem Cell-derived Cardiomyocytes Research Articles

Enhancing Maturation and Translatability of Human Pluripotent Stem Cell-Derived Cardiomyocytes through a Novel Medium Containing Acetyl-CoA Carboxylase 2 Inhibitor.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) constitute an appealing tool for drug discovery, disease modeling, and cardiotoxicity screening. However, their physiological immaturity, resembling CMs in the late fetal stage, limits their utility. Herein, we have developed a novel, scalable cell culture medium designed to enhance the maturation of hPSC-CMs. This medium facilitates a metabolic shift towards fatty acid utilization and augments mitochondrial function by targeting Acetyl-CoA carboxylase 2 (ACC2) with a specific small molecule inhibitor. Our findings demonstrate that this maturation protocol significantly advances the metabolic, structural, molecular and functional maturity of hPSC-CMs at various stages of differentiation. Furthermore, it enables the creation of cardiac microtissues with superior structural integrity and contractile properties. Notably, hPSC-CMs cultured in this optimized maturation medium display increased accuracy in modeling a hypertrophic cardiac phenotype following acute endothelin-1 induction and show a strong correlation between in vitro and in vivo target engagement in drug screening efforts. This approach holds promise for improving the utility and translatability of hPSC-CMs in cardiac disease modeling and drug discovery.

Therapeutic Efficacy of Mexiletine for Long QT Syndrome Type 2: Evidence From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, Transgenic Rabbits, and Patients.

Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.

Abstract We014: Creating Cell-specific Computational Models of Stem Cell-derived Cardiomyocytes Using Optical Experiments

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have gained traction as a powerful model in cardiac disease and therapeutics research, since iPSCs are self-renewing and can be derived from healthy and diseased patients without invasive surgery. However, current iPSC-CM differentiation methods produce cardiomyocytes with immature, fetal-like electrophysiological phenotypes, and the variety of maturation protocols in the literature results in phenotypic differences between labs. Heterogeneity of iPSC donor genetic backgrounds contributes to additional phenotypic variability. Several mathematical models of iPSC-CM electrophysiology have been developed to help understand the ionic underpinnings of, and to simulate, various cell responses, but these models individually do not capture the phenotypic variability observed in iPSC-CMs. Here, we tackle these limitations by developing a computational pipeline to calibrate cell preparation-specific iPSC-CM electrophysiological parameters. We used the genetic algorithm (GA), a heuristic parameter calibration method, to tune ion channel parameters in a mathematical model of iPSC-CM physiology. To systematically optimize an experimental protocol that generates sufficient data for parameter calibration, we created simulated datasets by applying various protocols to a population of in silico cells with known conductance variations, and we fitted to those datasets. We found that calibrating models to voltage and calcium transient data under 3 varied experimental conditions, including electrical pacing combined with ion channel blockade and changing buffer ion concentrations, improved model parameter estimates and model predictions of unseen channel block responses. This observation held regardless of whether the fitted data were normalized, suggesting that normalized fluorescence recordings, which are more accessible and higher throughput than patch clamp recordings, could sufficiently inform conductance parameters. Therefore, this computational pipeline can be applied to different iPSC-CM preparations to determine cell line-specific ion channel properties and understand the mechanisms behind variability in perturbation responses.

Abstract Tu092: Role of Z-disc as a Driver of Hypertrophy and Sarcomere Assembly in Hypertrophic Cardiomyopathy

The Z-disc is an electron-dense structure demarcating the lateral boundaries of the sarcomere and postulated to play a key role in both cell signaling and sarcomere assembly. We performed EM on both HCM patient septal myectomies and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with 3 different HCM MYH7 mutations, finding marked Z-disc widening along with a hallmark feature of HCM, myofibrillar disarray. Mutations in MYH7 that cause HCM do so by increasing force at the sarcomere level, leading to the proposal that the Z-disc may act as a mechanosensor that converts myosin-generated force into the intracellular signaling that drives hypertrophy. We focused on understanding the mechanisms of Z-disc mechanotransduction of both hypertrophy and sarcomere disarray. RNA-seq from HCM myectomies demonstrated 52 Z-disc differentially expressed genes (DEGs). scRNA-seq from HCM hiPSC-CMs identified 24 Z-disc DEGs. 14 Z-disc DEGs were shared across both datasets, most notably 5 upstream regulators of calcineurin signalling. To correlate changes in gene expression with altered force we transfected hiPSC-CMs with a FRET-based tension sensor in the Z-disc protein vinculin allowing the direct measurement of force experienced by the Z-disc. Live-cell confocal imaging of hiPSC-CMs expressing EGFP-α-actinin-2 showed that indistinct α-actinin-2 foci coalesced into sarcomeres over the course of 24-48 h after replating. Imaging at nm scale using cryo-electron tomography revealed marked disorder in individual thick filaments in HCM hiPSC-CMs, distinct from the classical disarray at the level of whole myofibrils and which would not be visible on conventional EM. Our data suggest that early alterations in sarcomeric micro-structure and signaling induced by altered myosin force may precede hypertrophy and could occur decades before clinical signs (hypertrophy on ECG or echo) are present, with potential implications for early preventive intervention.

Abstract We035: PTMA-MBD3 axis is a core heart regenerative driver in mammals

Introduction: The adult mammalian heart has an extremely limited ability for regeneration after injury, whereas the fetal heart can steadily regenerate. Uncovering the molecular underpinnings of proliferative cardiomyocytes and manipulating the core genes activate cardiomyocyte proliferation may provide new insight into adult heart regrowth and functional repair post-injury. Methods and Results: We performed single-cell RNA-sequencing of mouse embryonic hearts from various developmental stages (E8.5-E17.5), and identified a cardiomyocyte population in proliferative phase with 120 highly expressed feature genes. Combining with our bulk RNA-seq data on isolated cardiomyocytes from different heart developmental periods (E14.5, P1-P56), we screened and demonstrated that Ptma is the core conservative factor in promoting mouse, rat and human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) proliferation. Cardiac specifically knocking out Ptma blocked cardiomyocyte proliferation and neonatal mouse heart regeneration after apical resection injury. AAV9-delivered Ptma overexpression extended neonatal heart regenerative time window and showed therapeutic benefit in driving adult cardiomyocyte proliferation and cardiac repair. As PTMA is a polypeptide, we further validated that PTMA derived conservative peptide Tα1 displayed more effective in treating adult cardiac injury compared to Decapeptide. PTMA is an unstructured, intrinsically disordered nuclear protein that functions by binding to target proteins. Using Co-IP, IP and Native PAGE, we found PTMA promoted cardiomycyte proliferaton by binding to MBD3, which decreases the formation of MBD3-HDAC1 NuRD complex . We further decreased MBD3-HDAC1 NuRD complex formation by inhibiting MBD3, boosting both rat and mouse cardiomyocyte proliferation. Conclusions: We identified the PTMA-MBD3 axis as an effective target for promoting mammalian heart regeneration and provided a new insight into cardiomyocyte proliferation through blocking MBD3-HDAC1 NuRD complex.

Abstract We030: Microtubule Organizing Centers in Cardiomyocyte Proliferation and Maturation

Mammalian cardiomyocytes undergo a loss of proliferative capacity due to cell division failure, leading to polyploidy (genome duplication) during postnatal maturation. Centrosomes, primary microtubule organizing centers (MTOCs) in animal cells comprised of centrioles and pericentriolar matrix (PCM), play a crucial role in cell division. In differentiated cells, non-centrosomal (nc) MTOCs can reorganize microtubules, with cellular localization varying between cell types to mediate specialized functions. The specific role of ncMTOCs in cardiomyocyte division and maturation remains unknown. This study investigates the formation and function of ncMTOCs in cardiomyocytes, aiming to elucidate the regulating mechanism. Our results demonstrate that during the maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), centrosomes disassemble, and their PCM translocates to the nuclear envelope, establishing perinuclear ncMTOCs. Furthermore, maturing CMs with ncMTOCs often exhibit polyploidy, indicating incomplete cell division. Microtubule dynamic experiments reveal that ncMTOCs serve as organizing centers for microtubules, promoting their assembly around the nucleus in maturing cardiomyocytes. To understand the mechanism regulating the transition from centrosomes to ncMTOCs in cardiomyocytes, we identified a significant downregulation of the nuclear lamina protein Lamin B2 (LMNB2) during CM maturation. Our results indicate that LMNB2 is essential for mitotic spindle formation in CMs but not in stem cells, suggesting a distinct role for LMNB2 in CMs. Our results also suggest an increased centrosome to ncMTOC transition in LMNB2-depleted CMs at an early differentiation stage. Ongoing research aims to investigate the impact of perinuclear ncMTOCs on cardiomyocyte proliferation through pericentriolar matrix knockdown in hiPSC-CMs. Additionally, the study seeks to further elucidate the role of Lamin B2 in the transition of centrosome proteins and its underlying mechanism.

Abstract Mo053: Acacetin's Antiarrhythmic Potential in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Harboring Hypertrophic and Dilated Cardiomyopathy-Related Mutations

Background: Hypertrophic cardiomyopathy (HCM) is often caused by mutations such as p.R943X in the Myosin Binding Protein C3 (MYBPC3), leading to early and delayed afterdepolarizations, myofibrillar disarray, and calcium dysfunction, which may induce lethal arrhythmias. Dilated cardiomyopathy (DCM) can also result from mutations, such as the p.R14del mutation in the phospholamban (PLN) protein, which is crucial for calcium signaling regulation, leading to arrhythmogenesis when provoked. This study explores the arrhythmogenic potential in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the HCM MYBPC3 p.R943X mutation and the DCM PLN p.R14del mutation. Methods: We utilized a 384-well optical physiological system to record action potentials (APs) in hiPSC-CMs carrying the MYBPC3 p.R943X mutation and the DCM PLN p.R14del mutation, including their isogenic controls. Action potential metrics were measured under spontaneous and electrically paced conditions. The effects of Acacetin, NS-5806, nifedipine, and GS-967 on the recorded AP types were analyzed to understand the underlying channelopathies and arrhythmogenic potential. Special attention was given to the variability between the HCM MYBPC3 p.R943X and DCM PLN p.R14del cell lines' susceptibility to arrhythmogenesis and their isogenic controls. Results: The HCM MYBPC3 p.R943X hiPSC-CMs exhibited a significant increase in early afterdepolarizations, non-sustained and sustained ventricular tachycardia, and AP notches compared to their isogenic control. AP notches and arrhythmogenic events were notably provoked by NS-5806 in both the HCM MYBPC3 p.R943X and DCM PLN p.R14del hiPSC-CMs. However, these were significantly ameliorated (p&lt;0.05) by Acacetin administration (5 µM), highlighting its potential as an effective antiarrhythmic in managing both HCM- and DCM-induced arrhythmias. GS-967 also showed efficacy in reducing early afterdepolarizations (EADs) in the HCM model. Conclusions: This study provides novel insights into the arrhythmogenic potential of hiPSC-CMs carrying MYBPC3 p.R943X and PLN p.R14del mutations, underscoring the significant role of Acacetin in attenuating these effects. It paves the way for developing targeted therapies that address the complex molecular mechanisms underlying arrhythmogenesis in cardiomyopathies. The findings suggest that Acacetin could be a promising candidate for the pharmacologic treatment of cardiomyopathies characterized by arrhythmogenic risks.

Exosomal Preconditioning of Human iPSC-Derived Cardiomyocytes Beneficially Alters Cardiac Electrophysiology and Micro RNA Expression.

Experimental evidence, both in vitro and in vivo, has indicated cardioprotective effects of extracellular vesicles (EVs) derived from various cell types, including induced pluripotent stem cell-derived cardiomyocytes. The biological effects of EV secretion, particularly in the context of ischemia and cardiac electrophysiology, remain to be fully explored. Therefore, the goal of this study was to unveil the effects of exosome (EXO)-mediated cell-cell signaling during hypoxia by employing a simulated preconditioning approach on human-induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs). Electrophysiological activity of hIPSC-CMs was measured using a multielectrode array (MEA) system. A total of 16 h of hypoxic stress drastically increased the beat period. Moreover, hIPSC-CMs preconditioned with EXOs displayed significantly longer beat periods compared with non-treated cells after 16 h of hypoxia (+15.7%, p < 0.05). Furthermore, preconditioning with hypoxic EXOs resulted in faster excitation-contraction (EC) coupling compared with non-treated hIPSC-CMs after 16 h of hypoxia (-25.3%, p < 0.05). Additionally, microRNA (miR) sequencing and gene target prediction analysis of the non-treated and pre-conditioned hIPSC-CMs identified 10 differentially regulated miRs and 44 gene targets. These results shed light on the intricate involvement of miRs, emphasizing gene targets associated with cell survival, contraction, apoptosis, reactive oxygen species (ROS) regulation, and ion channel modulation. Overall, this study demonstrates that EXOs secreted by hIPSC-CM during hypoxia beneficially alter electrophysiological properties in recipient cells exposed to hypoxic stress, which could play a crucial role in the development of targeted interventions to improve outcomes in ischemic heart conditions.

Abstract Tu069: Development of a Novel Biallelic, Haploinsufficient Mouse Model of MYBPC3 Related Hypertrophic Cardiomyopathy

Background: No current animal models re-capitulate MyBP-c haploinsufficiency and hypertrophic cardiomyopathy (HCM) observed in patients with heterozygous MYBPC3 truncating variants, the most common genetic cause of HCM. We recently identified a hypomorphic (partial) loss of function MYBPC3 variant that increases disease severity in HCM patients when combined with an MYBPC3 truncating variant. Hypothesis: Additive effects from a hypomorphic loss of function MYBPC3 variant observed in human HCM will result in MyBP-c protein haploinsufficiency and HCM in a biallelic murine model. Methods: To test MYBPC3 dose effects, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were first derived from an HCM patient with biallelic hypomorphic and complete loss of function variants in MYBPC3 . We then generated Mybpc3 knock out (Mybpc3 KO ) and hypomorphic knock in (Mybpc3 KI ) alleles in mice. Control (Mybpc3 +/+ ), monoallelic, and biallelic mice and iPSC-CMs were characterized with morphometric measurements, echocardiography, mass spectroscopy, and RNA sequencing. Results: Biallelic iPSC-CMs demonstrated haploinsufficiency not present in heterozygous models through dose reduction in MYBPC3 protein (Fig 1A). Similarly, biallelic Mybpc3 KI/KO mice had a reduction in Mybpc3 transcripts (62±3%, p&lt;0.001) with 43±11%% reduction in MyBP-c protein (Fig 1B) and cardiac hypertrophy (10±5% increase in heart mass, p=0.002, Fig 1C). Conclusions: Graded reduction in Mybpc3 transcripts in a biallelic mouse model results in MyBP-c haploinsufficiency similar to that observed in HCM patients. Corresponding hypertrophy establishes this approach as a clinically relevant, novel model of MYBPC3 HCM.

Abstract Mo092: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as an In Vitro Model to Evaluate the Efficacy of a Gene Replacement Therapy for Plakophilin-2- Associated Arrhythmogenic Right Ventricular Cardiomyopathy

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disorder characterized by severe arrhythmias and heart dysfunction. Mutations in desmosome gene Plakophilin-2 ( PKP2 ) account for 40% of ARVC cases, with current palliative therapies failing to address the genetic cause. Herein, we generated an in vitro ARVC model using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) harboring a pathogenic PKP2 variant (c.2146G&gt;C), as a platform to test a PKP2 -gene replacement approach developed at Tenaya Therapeutics for the treatment of PKP2 -associated ARVC patients. Methods: Three isogenic human iPSC lines (wild-type, heterozygous and homozygous PKP2 mutant) were differentiated to iPSC-CM; and monolayers and engineered heart tissues (EHTs) were generated. Gene and protein expression were assessed by RNA-Seq/RT-qPCR, and immunocytochemistry, respectively. Contractility, electrophysiology and calcium handling were measured. A proprietary adeno-associated virus gene therapy (AAV9: PKP2 ) was used to transduce human iPSC-CM, and changes in gene and protein expression, and contractile function were evaluated. Results: A human iPSC-CM beating monolayer was achieved for all three lines. PKP2 expression was depleted in a genotype-dependent manner, and desmosome structure was disrupted in the mutant lines. Functional assessment of human iPSC-CM monolayers showed impaired contractile properties and abnormal electrophysiological and calcium transients in the mutant lines, such as decreased contraction amplitude and prolonged field and action potential duration. Transcriptional analysis revealed changes in desmosome, gap junctions, sarcomere, ion channels, metabolic and apoptosis gene expression. Characterization of EHTs displayed a deficit in contractility, slower action and field potential kinetics and irregular calcium homeostasis in the mutant lines. The administration of AAV9: PKP2 to the mutant lines restored ion channel and desmosome gene and protein expression, and contractile function. Conclusions: Our PKP2 human iPSC-CM disease model recapitulated the main hallmarks of ARVC phenotype. Administration of AAV9: PKP2 restored desmosome protein expression and contractility. This model lays a foundation for the understanding of the underlying molecular pathophysiological mechanisms of PKP2 -ARVC, and the potential for AAV9: PKP2 as a one-time dose to correct the genetic cause of disease in individuals with PKP2 -associated ARVC.

Abstract We046: Building Mass or Strength: Stage-specific Effects of Insulin and Wnt on Cell Fate Decisions in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background: Reactivation of the so-called fetal gene programs and pathways have been reported in failing hearts, however, it remains unclear whether this phenomenon is beneficial or detrimental to the remaining functional myocardium. During cardiac development, the orchestration of these embryonic growth pathways, facilitate temporal and stage-specific cell fate decisions such as required for specification and growth of the various myocardial compartments. Previously, we demonstrated that molecular activation of the Wnt pathway resulted in massive expansion of immature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Yet, it is unknown what exact cues guide the cell fate decisions of immature hiPSC-CMs to either proliferate or terminally differentiate into more mature cardiomyocytes. Purpose: This work uncovers the embryonic growth pathways that control the cues for proliferation versus terminal differentiation in functional immature hiPSC-CMs. Methods: We utilised spontaneously beating hiPSC-CMs at day 11 of differentiation and performed a combinatory screen for various dosages of CHIR99021/Wnt and Insulin/Akt pathway. Proliferation was determined by quantification of cell number and the proportion of Ki67 positive hiPSC-CMs. Terminal differentiation was evaluated by presence of more mature sarcomere markers and cell cycle exit. Results: Our combinatory screen for Insulin/Akt and CHIR99021/Wnt demonstrates synergistic effects on proliferation of immature hiPSC-CMs, whereas the absence of these pathway activators leads to rapid cell cycle exit and terminal differentiation of hiPSC-CMs. Detailed characterization reveals that CHIR99021/Wnt also importantly regulates sarcomere homeostasis and architecture in hiPSC-CMs. Moreover, we identify a novel temporal interplay between CHIR99021/Wnt via TCF and Insulin/Akt via FOXO as regulators of cell-fate decision in immature hiPSC-CMs. Conclusions: Molecular targeting of the embryonic growth pathways in acute and chronic forms of heart failure requires a detailed understanding of the specific effects on cardiomyocyte function and sarcomere architecture. This work provides a starting point to develop future strategies to target acute and chronic forms of heart failure via modulation of embryonic growth pathways.

Abstract Mo080: Clinically Variable Penetrant MYH7 G256E Mutation Shows Gene-Dose-Dependent HCM Disease Phenotype on a Transcriptomic, Proteomic, and Functional Level

Introduction: Hypertrophic cardiomyopathy (HCM) is a monogenic disorder caused by heterozygous (HET) mutations in sarcomere genes such as MYH7 . Recently, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying HET HCM mutations have shed new insights into the molecular mechanisms of HCM. However, the cause of phenotype variability and the role of allelic imbalance in phenotype penetrance of HET missense mutations such as MYH7 G256E remain unclear. Hypothesis: We hypothesize that hiPSC CMs carrying clinically variable penetrant HCM missense mutations such as MYH7 G256E show a gene dose-dependent disease phenotype indicating their susceptibility for allelic imbalance as a cause for variable disease phenotype penetrance. Methods: We generated HET and homozygous (HOM) hiPSC-lines for variable penetrant HCM mutation MYH7 G256E. We performed multiplexed single-cell RNA sequencing and quantitative proteomics analyses on wildtype (WT), HET, and HOM iPSC-CMs at day 30 of cardiac differentiation. Additional Digital Droplet PCR studies were performed to investigate the MYH7 mutant allele expression. The functional contractile phenotype was assessed with Sony SI8000 Cell motion imaging system. Results: We detected a large clone-to-clone and batch-to-batch variability in HET iPSC-CMs on a transcriptomic and proteomic level that obscured the identification of a consistent mutant phenotype in HET MYH7 G256E vs WT CMs. These phenotypes correlated with a 1:1 bulk ratio of mutant vs WT mRNA and protein expression, while overall MYH7 gene and protein expression remained comparable. Transcriptomic and proteomic analyses of WT, HET, and HOM CMs show a gene dose-dependent regulation of known hypertrophy response markers such as NPPB (mRNA: HET vs WT: +1.76x p .adj=7.0*10 -7 , HOM vs WT: +5.42x p.adj=1.5*10 -48 ; protein: HET vs WT: +1.15x, 95% CI [0.86, 1.58]; HOM vs WT: +1.51x, 95% CI [1.12, 2.01]) which functionally correlated with enhanced contractility (contraction velocity in μm/s: WT: 5.84 +/-2.24, HET: 6.04 +/-2.89, HOM: 7.77 +/-3.352, p=0.3*10 -2 ANOVA) and the more frequent occurrence of arrhythmias (WT: 1/46, HET: 0/55, HOM: 5/41). Conclusions: Hypertrophic disease phenotype in MYH7 G256E mutation iPSC-CMs shows a gene dose-dependent penetrance on a transcriptomic, proteomic, and functional level. This indicates susceptibility to allelic imbalance as a potential cause for variable disease penetrance.

Abstract We050: Elevated Cell-Free RNA in Maternal Circulation during Single Ventricle Heart Defect Pregnancies Dysregulate Human Cardiomyocyte Proliferation

Congenital single-ventricle heart defects (SVHDs) are life-threatening conditions in newborns, where one of the heart's ventricular chambers is severely underdeveloped. While medical advancements have improved survival rates, many SVHD patients still suffer lifelong cardiac complications and comorbidities. Clinical diagnosis of fetal SVHD occurs at around 20 weeks of gestation through echocardiogram. In this study, we aimed to identify novel maternal blood biomarkers that could signal SVHD development in fetuses. The study's premise is based on the limited regenerative capacity of adult human cardiomyocytes, like those in pregnant mothers, compared to the extensive proliferation seen in fetal cardiomyocytes that form the functional heart. We hypothesized small signaling molecules secreted by proliferating fetal cardiomyocytes enter the maternal circulatory system and could indicate abnormal ventricular growth in SVHD-affected fetuses. Both clinical and in vitro approaches were employed to identify potential cell-free miRNAs released by the developing fetal heart into maternal circulation. Blood plasma samples from pregnant women carrying healthy and SVHD-affected fetuses were screened for differentially expressed cell-free miRNAs and were compared with those from the supernatants of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) grown from healthy and SVHD donors. This comparative approach led to the identification of an miRNA panel (miR-487b, miR-433, mi-134, and miR-889) that were elevated in pregnant women carrying SVHD-affected fetuses and in proliferating iPSC-CMs from SVHD patients compared to healthy controls (p.adj&lt;0.05). From this panel, forced-expression of miR-487b and mir-134 modulated the proliferation profiles of human cardiomyocytes in vitro . Results from this study establish these circulating cell-free miRNAs in maternal blood as potential non-invasive biomarkers for predicting abnormal fetal cardiac development leading to SVHDs. These findings could potentially revolutionize SVHD screening as an alternative diagnostic tool to echocardiogram.

Abstract Mo093: Rescue of Desmin Insufficiency Restores Contractile Function in Cardiomyocytes with MYH7 E848G Dilated Cardiomyopathy Variant

Dilated cardiomyopathy (DCM), characterized by systolic dysfunction, is a significant cause of heart failure. Myosin heavy chain 7 ( MYH7 ) pathogenic variants are common causes of DCM; however, no specific disease-modifying therapy for MYH7 DCM exists. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing the pathogenic MYH7 E848G DCM variant, we confirmed hypocontractility at the single cell level as assessed with traction force microscopy. hiPSC-CMs were cultured on polyacrylamide hydrogels with physiological stiffness in 15x100 µm patterned Matrigel-coated rectangles, each containing one cell. Upon 1 Hz electrical stimulation, contracting cells displaced fluorescent beads embedded within the gel, which were measured to estimate twitch force. MYH7 E848G/+ hiPSC-CMs had reduced maximum twitch force (97 ± 11 nN, n=46 cells) compared to MYH7 +/+ hiPSC-CMs (179 ± 18 nN, n=49 cells), recapitulating the disease phenotype. RNAseq analysis of MYH7 E848G/+ hiPSC-CMs compared to isogenic control MYH7 +/+ hiPSC-CMs found significant downregulation of desmin ( DES) (76.4 ± 8.2% reduction, n=4 bio reps), which encodes a muscle-specific type III intermediate filament. Desmin protein expression in MYH7 E848G/+ hiPSC-CMs was significantly attenuated relative to MYH7 +/+ hiPSC-CMs (90.9 ± 1.8% reduction, n=4 bio reps), matching gene expression data. Gene expression of the transcription factor MEF2C , a known transactivator of DES, was also reduced (40.4 ± 7.0% reduction, n=4 bio reps), suggesting MYH7 E848G/+ suppresses the MEF2C - DES signaling axis. We hypothesized restoring desmin protein levels may improve contractility in MYH7 E848G/+ hiPSC-CMs. To test this, we created an adeno-associated virus (AAV9) packaged with full-length desmin cDNA fused with a T2A self-cleaving peptide and mApple fluorescent reporter. hiPSC-CMs were transfected with this mApple-T2A-DES AAV at 1 MOI and contractility was assessed via traction force microscopy. mApple + MYH7 E848G/+ hiPSC-CMs had increased maximum twitch force (141 ± 11 nN, n=75 cells) relative to mApple - MYH7 E848G/+ hiPSC-CMs (65 ± 20 nN, n=21 cells), approaching the maximum twitch force of mApple + MYH7 +/+ hiPSC-CMs (165 ± 10 nN, n=87 cells). In conclusion, suppression of the MEF2C - DES signaling axis is a potential novel mechanism by which pathogenic MYH7 variants cause DCM. By leveraging this mechanistic insight, we found that rescuing desmin insufficiency can partially restore contractile function.

Abstract Mo096: High-Throughput Functional Determination of TNNI3 Variant Pathogenicity

Introduction: Mutations in the thin filament protein TNNI3 (cardiac troponin I) can perturb calcium cycling and cause hypertrophic, restrictive, and dilated cardiomyopathies. However, the majority of TNNI3 variants observed in patients have unknown functional significance and uncertain contribution to disease. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful system for studying the effect of genetic variants on human cardiomyocyte function. TNNI3 has been challenging to study in these cells as they predominantly express TNNI1. In this study, we combined a novel scalable in vitro gene replacement method with simultaneous measurement of contractility and calcium cycling to determine the functional effect of TNNI3 variants. Hypothesis: Using our gene replacement platform, we expect that TNNI3 variants causing Hypertrophic Cardiomyopathy (HCM) will increase force generation and calcium sensitivity relative to wild-type, whereas TNNI3 variants causing Dilated Cardiomyopathy (DCM) will decrease force generation and calcium sensitivity relative to wild-type. Methods: We generated double knockout TNNI1 -/- TNNI3 -/- hiPSCs. Cardiomyocytes differentiated from these hiPSCs were transduced with lentivirus carrying either wild-type or mutant TNNI3 during replating onto polyacrylamide gels with fluorescent beads. After a week of incubation, the cells were stained with a fluorescent calcium probe, and contractility, diastolic tension, and calcium sensitivity were measured by our high-throughput physiologic imaging and analysis platform. Results: hiPSC-CMs expressing the HCM-associated variant R192H show increased peak force, diastolic tension, and calcium sensitivity relative to wild-type TNNI3, whereas hiPSC-CMs expressing the DCM-associated variant K36Q show decreased peak force and calcium sensitivity relative to wild-type TNNI3. Conclusion: We validated our TNNI3 in vitro gene replacement and high-throughput physiologic imaging and analysis platform by comparing reference HCM and DCM variants to wild-type TNNI3. Ongoing scaled studies will evaluate the naturally occurring variants in ClinVar to characterize their functional effect on cardiac physiology.

Abstract We023: P53 Activation Promotes Maturational Characteristics of Pluripotent Stem Cell-derived Cardiomyocytes in 3D Suspension Culture via FOXO-FOXM1 Regulation

Background: Current protocols can generate highly-pure populations of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate key characteristics of mature in vivo cardiomyocytes. Yet, there exists a risk of arrhythmias when hiPSC-CMs are injected into large animal models. This prompts further investigation into the mechanisms of hiPSC-CM maturation to facilitate clinical translation. Hypothesis: The forkhead box (FOX) family of transcription factors can regulate maturation in neonatal cardiomyocytes through a balance between FOXO and FOXM1. We also previously found that p53 activation could enhance hiPSC-CM maturation. Therefore, we hypothesized that p53 activation increases FOXO and decreases FOXM1 to promote hiPSC-CM maturation in three-dimensional (3D) suspension culture. Methodology: 3D cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator), LOM612 (FOXO activator), AS1842856 (FOXO inhibitor), or RCM-1 (FOXM1 inhibitor), starting 2 days after onset of beating. The hiPSC-CMs were assessed for maturation in metabolic, contractile, and electrophysiological properties, by Seahorse mito stress test, Multi electrode array, and VALA kinetic image cytometer respectively. Results: P53 activation promoted FOXO upregulation and FOXM1 downregulation in hiPSC-CMs, measured by RT-qPCR and immunostaining. Alongside this, p53 activation also promoted hiPSC-CM metabolic and contractile maturational characteristics, seen by increase in oxygen consumption and beat amplitude respectively. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2 and eliminated spontaneous beating. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics of hiPSC-CMs such as increase in contractility, oxygen consumption, and voltage peak maximum upstroke velocity. Further, by single-cell RNAseq, FOXO activated groups showed increase in cardiac maturational pathways compared against DMSO control groups. Conclusions: These results show that p53 activation promotes FOXO and suppresses FOXM1, which modulate hiPSC-CM maturation in 3D suspension. Our study expands current understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3D culture system.

Abstract We048: Exon-skipping therapy can restore functional dystrophin attenuating calcium overload for DMD cardiomyopathy with mutations in actin-binding domain 1

Antisense oligonucleotide (AO)-mediated exon-skipping is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), which is caused by loss-of-function mutations in the DMD gene encoding Dp427m, leading to progressive cardiomyopathy. In-frame deletion of exons 3–9 (Δ3–9) manifesting asymptomatic or very mild clinical phenotype is an ideal goal for exon-skipping by targeting actin-binding domain 1 (ABD1); however, the efficacy of this therapy for DMD cardiomyopathy remains unclear. We compared three isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing Δ3–9, frameshifting Δ3–7, and intact DMD. RNA-seq revealed high similarity between Δ3–9 and wild-type hiPSC-CMs. Furthermore, we observed electrophysiological alterations with accelerated CaMKII activation in Δ3–7 hiPSC-CMs compared to Δ3–9 and wild-type hiPSC-CMs, while Δ3–9 Dp427m was stably expressed, maintaining substantial binding to F-actin and retention of desmin. AOs targeting exon 8 efficiently induced exons 8–9 skipping to restore functional Dp427m and electrophysiological parameters in Δ3–7 hiPSC-CMs. Exon-skipping therapy converting the reading frame to Δ3–9 might be promising for DMD cardiomyopathy.

Abstract We031: Optimizing Immunosuppressive Strategies for Enhanced Survival of Allogeneic Pluripotent Stem Cell-Derived Cardiomyocytes in Non-Human Primate Transplantation Model

Introduction: Accumulating evidence suggests that the transplantation of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) regenerates injured hearts in animal studies. Currently, several clinical studies are underway, transplanting allogeneic human PSC-CMs. In these clinical studies, patients have been treated with the same combination of immunosuppressants used after heart transplantation, including steroids, calcineurin inhibitors (CNIs), and mycophenolate mofetil (MMF). However, the optimal treatment following PSC-CM transplantation has yet to be established. The purpose of this study is to determine the optimal treatment for grafted PSC-CM survival by regulating the immune response using an allogeneic non-human primate transplantation model. Methods and Results: We prepared 9 male cynomolgus monkeys as recipient animals. Donor cardiomyocytes were generated from induced pluripotent stem (iPS) cells derived from another monkey, with MHC class I and class II types differing from those in the recipients. The recipient animals underwent ischemia/reperfusion injury of the left anterior descending artery two weeks before iPSC-CMs transplantation. Initially, animals were treated with the same combination of immunosuppressants (steroid/CNIs/MMF: N=3), and grafted PSC-CMs survived without apparent immune rejection. Subsequently, we conducted experiments aiming to reduce immunosuppressants. The animals were treated with the same combination, but steroids were terminated at 4 weeks post-transplantation (N=4). Histological analysis at 8 or 12 weeks post-transplantation revealed that grafted cardiomyocytes were rejected, with a significant infiltration of CD3-positive lymphocytes. When recipients were treated with another combination of immunosuppressants (steroid/abatacept/MMF), and steroids were terminated at 4 weeks post-transplantation, grafted cardiomyocytes survived without apparent infiltration of CD3-positive lymphocytes at 8 weeks post-transplantation (N=2). Conclusion: A different combination of immunosuppressants from heart transplantation allows transplanted allogeneic PSC-CMs to survive without the long-term use of steroids.

Abstract Mo018: Using a mitochondria-rich hiPSC-CM model to investigate doxorubicin-induced cardiotoxicity

Doxorubicin (DOX) is commonly used to treat cancer but can induce severe cardiotoxicity by damaging the mitochondria. Dexrazoxane is clinically approved to protect against DOX-induced cardiotoxicity (DCT) but has been shown to reduce the anti-cancer efficacy of DOX and cause secondary neoplasm. Human induced pluripotent stem cell derived-cardiomyocyte (hiPSC-CMs) models have been used to study DOX-induced cardiotoxicity (DCT), but some do not correctly recapitulate patient response to dexrazoxane. This was previously attributed to the low mitochondrial abundance of these cells and is a hurdle against the identification of new therapeutics against DCT. Our aim is to use our patient-derived, mitochondria-rich hiPSC-CM model to identify compounds which can protect against DCT. We first checked that our hiPSC-CMs recapitulate the cardioprotective effects of dexrazoxane. Using this validated model, we showed that compound I1 suppressed both the mitochondrial and non-mitochondrial damage induced by DOX, at levels that are comparable to that of dexrazoxane. However, unlike dexrazoxane and contrary to its effects in CMs, compound I1 was cytotoxic to a panel of cancer cell lines. Mechanistic investigations indicated that the divergent effects of CMs vs cancer cells is due the ability of compound I1 to inhibit two distinct molecular targets in the two cell types. Lastly, we validated that compound I1 can protect the heart in an in vivo mouse model of DCT. In summary, compound I1 offers the dual benefits of cardioprotection and tumour suppression and is potentially superior to conventional treatment. Our results provide important proof of principle that a single compound can be used to simultaneously target both the heart and cancer via different targets/pathways to improve clinical outcome.

Effect and mechanism of T lymphocytes on human induced pluripotent stem cell-derived cardiomyocytes via Proteomics

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