Induced Pluripotent Stem Cell-derived Cardiomyocytes Research Articles

Myeloperoxidase is a critical mediator of anthracycline-induced cardiomyopathy

Cardiotoxicity is a major complication of anthracycline therapy that negatively impacts prognosis. Effective pharmacotherapies for prevention of anthracycline-induced cardiomyopathy (AICM) are currently lacking. Increased plasma levels of the neutrophil-derived enzyme myeloperoxidase (MPO) predict occurrence of AICM in humans. We hypothesized that MPO release causally contributes to AICM. Mice intravenously injected with the anthracycline doxorubicin (DOX) exhibited higher neutrophil counts and MPO levels in the circulation and cardiac tissue compared to saline (NaCl)-treated controls. Neutrophil-like HL-60 cells exhibited increased MPO release upon exposition to DOX. DOX induced extensive nitrosative stress in cardiac tissue alongside with increased carbonylation of sarcomeric proteins in wildtype but not in Mpo−/− mice. Accordingly, co-treatment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with DOX and MPO aggravated loss of hiPSC-CM-contractility compared to DOX treatment alone. DOX-treated animals exhibited pronounced cardiac apoptosis and inflammation, which was attenuated in MPO-deficient animals. Finally, genetic MPO deficiency and pharmacological MPO inhibition protected mice from the development of AICM. The anticancer efficacy of DOX was unaffected by MPO deficiency. Herein we identify MPO as a critical mediator of AICM. We demonstrate that DOX induces cardiac neutrophil infiltration and release of MPO, which directly impairs cardiac contractility through promoting oxidation of sarcomeric proteins, cardiac inflammation and cardiomyocyte apoptosis. MPO thus emerges as a promising pharmacological target for prevention of AICM.

Empagliflozin Suppresses the Differentiation/Maturation of Human Epicardial Preadipocytes and Improves Paracrine Secretome Profile

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce epicardial adipose tissue (EAT) in humans, enhancing cardioprotective effects on heart failure and atrial fibrillation. We investigated the direct effect of the SGLT2 inhibitor empagliflozin on human primary epicardial adipocytes and preadipocytes. SGLT2 is primarily expressed in human preadipocytes in the EAT. The expression levels of SGLT2 significantly diminished when the preadipocytes were terminally differentiated. Adipogenesis of preadipocytes was attenuated by empagliflozin treatment without affecting cell proliferation. The messenger RNA levels and secreted protein levels of interleukin 6 and monocyte chemoattractant protein 1 were significantly decreased in empagliflozin-treated adipocytes. Coculture of human induced pluripotent stem cell-derived atrial cardiomyocytes and adipocytes pretreated with or without empagliflozin revealed that empagliflozin significantly suppressed reactive oxygen species. IL6 messenger RNA expression in human EAT showed significant clinically relevant associations. Empagliflozin suppresses human epicardial preadipocyte differentiation/maturation, likely inhibiting epicardial adipogenesis and improving the paracrine secretome profile of EAT, particularly by regulating IL6 expression.

Abstract P1042: Stem Cell Models Reveal Sarcomere Inhibition As A Potential Treatment For Pediatric Restrictive Cardiomyopathy

Introduction: Pediatric Restrictive Cardiomyopathy (RCM) is a genetic cardiomyopathy resulting from profoundly increased stiffness of the wall of the cardiac ventricle with normal or low normal contractility. Treatment options are limited, with most patients necessitating heart transplant. Therapies that can improve diastolic function are needed for this severe disease. Recently, direct inhibition of the sarcomere with mavacamten, a cardiac myosin antagonist, has been shown to be effective in treating a related disease, Hypertrophic Cardiomyopathy (HCM), in adults. However, whether it would be useful for the diastolic dysfunction seen in RCM is unknown. Hypothesis: Treatment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) derived from RCM patients with mavacamten will rescue diastolic deficits. Methods: Cardiomyocytes were differentiated from induced pluripotent stem cells (iPSCs) originally derived from a pediatric patient with RCM, as well as a paired isogenic CRISPR-corrected line. Cells were treated with increasing doses of mavacamten and contractility and resting tension were measured using a novel traction force microscopy platform. Results: At baseline, RCM cells showed elevated contractility, resting tension, and calcium sensitivity compared to their isogenic control. Mavacamten treatment decreased both contractility and resting tension of RCM cardiomyocytes. Conclusions: Mavacamten improves both systolic and diastolic derangements in an hiPSC model of RCM.

Abstract P1150: Integrative Transcriptomics And Cell Systems Analyses Reveal Protective Pathways Controlled By Igfbp-3 In Anthracycline-induced Cardiotoxicity

Rationale: Anthracyclines such as doxorubicin (Dox) are effective chemotherapeutic agents. Their use however is hindered by the risk of anthracycline-induced cardiotoxicity (AIC). Our understanding of protective pathways activated by cardiomyocytes following AIC remains incomplete. Insulin-like growth factor binding protein (Igfbp)-3, the most abundant Igfbp family member in the circulation, is associated with effects on the metabolism, proliferation, and survival of various cell types. Whereas Igfbp-3 is significantly induced by Dox in the heart, its role in AIC remains ill-defined. Methods and Results: We investigated molecular mechanisms as well as systems-level transcriptomic consequences of manipulating Igfbp-3 in AIC using neonatal rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our findings reveal that Dox induces the nuclear enrichment of Igfbp-3 in cardiomyocytes. Furthermore, Igfbp-3 overexpression leads to a significant reduction of γ-H2AX, a marker of DNA damage (56% reduction vs. control, n=3, P<0.05), and impedes topoisomerase (Top)-2β expression which forms a Top2β-Dox-DNA cleavage complex leading to DNA double-strand breaks (44% reduction vs. control, n=3, P<0.01). Next generation sequencing and gene network analyses of iPSC-CMs treated with Dox reveal that Igfbp-3 perturbation modulates genes implicated in microtubule function and cardiomyocyte contraction. Stimulated Emission Depletion (STED) microscopy and immunoblotting further indicate that Igfbp-3 knockdown exacerbates detyrosinated microtubule accumulation (STED, 21% increase vs. control, n=16-19, P<0.05; immunoblotting, 62% increase vs. control, n=6, P<0.01) – a hallmark of increased cardiomyocyte stiffness and heart failure – and favorably affects contractility following Dox treatment. Conclusion: Our delineation of the protective role of Igfbp-3 in AIC provides novel insight into pathways induced by cardiomyocytes to mitigate DNA damage and apoptosis, and improve contractile function.

Abstract P3048: Ppp1r1b-lncRNA Regulates Cardiomyocytes Differentiation Via Epigenetic Modulation Of Tbx5 Promoter

Introduction: Myogenic differentiation is essential to cardiac development as well as regeneration and repair in response to genetic lesions or injury in the context of cardiomyopathy and congenital heart defects (CHDs). We have previously shown that mouse Ppp1r1b-lncRNA modulates epigenetic features of myogenic master regulators through targeted interaction with histone-modifying complex and other co-factors. However, the molecular mechanisms that underlie human PPP1R1B-lncRNA -mediated regulation of cardiomyocyte differentiation remain unknown. Objectives: To elucidate the mechanisms by which human PPP1R1B-lncRNA regulates cardiomyocyte differentiation. Methods/Results: PPP1R1B-lncRNA possessed conserved function in promoting myogenic differentiation in human cardiac and skeletal myocytes and its expression was dysregulated in CHDs. Mechanistically, by silencing PPP1R1B-lncRNA , myogenic differentiation was impaired in PPP1R1B-lncRNA -deficient human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The expressions of myogenic transcription factors, including GATA4 and TBX5 , as well as sarcomere proteins, including TNNT2 and MYOZ2, were significantly suppressed in PPP1R1B-lncRNA deficient hiPSC-CMs. Epigenetic analysis revealed increased H3K27 tri-methylation at TBX5 promoter in the PPP1R1B-lncRNA knockdown cardiac progenitors. PPP1R1B-lncRNA was found to bind to EZH2, the enzymatic core of the polycomb repressive complex (PRC2). Furthermore, chromatin isolation by RNA purification (ChIRP)-seq revealed enriched interaction of PPP1R1B-lncRNA with TBX5 , MEF2C, and GATA4 promoters. Meanwhile, chromatin immunoprecipitation (ChIP)-PCR demonstrated increased enrichments of EZH2 at TBX5 promoter in the PPP1R1B-lncRNA-depleted cells, resembling what was observed in the pre-differentiation state. Conclusions: Human PPP1R1B-lncRNA promotes cardiomyocyte differentiation via interactions with promoters of key cardiac transcription factors. PPP1R1B-lncRNA enhances chromatin accessibility at TBX5 promoter by interfering with PRC2 binding. Understanding PPP1R1B-lncRNA function in cardiomyocyte differentiation may lead to novel therapies for cardiopathies and CHDs.

Abstract P1079: Regeneration Of Primate Hearts With Human IPS Cell-derived Cardiac Spheroids

Background: Clinical application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for cardiac repair started with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of hiPSC-CMs into the cardiac muscle layer, which was shown to induce electrical integration, was unclear because of concerns regarding poor engraftment of dispersed cardiomyocytes (CMs) and post-transplant arrhythmias. Thus, in this study, we prepared clinical-grade purified hiPSC-CM aggregates, known as cardiac spheroids (CSs), and confirmed whether the direct injection of CSs could regenerate infarcted non-human primate hearts. Methods: We performed two separate experiments to explore the appropriate number of CMs derived from human induced pluripotent stem cells (hiPSCs). In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks pre-transplantation and were designated as recipients of CSs containing 2х10 7 CMs or the vehicle. The animals were killed 12 weeks post-transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6х10 7 CMs). Results: Recipients of CSs containing 2х10 7 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle, with a low incidence of post-transplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits. The incidence of post-transplant arrhythmia slightly increased in recipients of CSs containing 6х10 7 CMs. Conclusions: We demonstrated that direct injection of hiPSC-derived CSs, prepared as clinical-grade cell products, restores the contractile functions of injured primate hearts with an acceptable risk of post-transplant arrhythmia. These findings provide strong rationale for conducting clinical trials using the equivalent CM products.

Abstract P1019: High-throughput Generation And Functional Characterization Of Myosin Heavy Chain 7 Variants In Genome-edited Cardiomyocytes

Hypertrophic cardiomyopathy (HCM), an inheritable cardiac condition characterized by idiopathic left ventricular hypertrophy, can cause sudden cardiac death or heart failure. Autosomal-dominant missense mutations in MYH7 (encoding the sarcomeric βMHC protein) account for 33% of genotype-positive HCM cases. While pathogenic MYH7 variants are clinically-actionable, 75% of MYH7 missense variants in ClinVar are variants of unknown significance (VUS). Gene-editing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can help determine the function of MYH7 VUS, however, the discovery of new variants by genetic testing outpaces the ability to gene-edit hiPSCs. Thus, we developed a novel method called CR ISPR a On- T arget E diting R etrieval (CRaTER) which increases the success of gene-editing by 25-fold compared to current methods. We leveraged CRaTER to enrich for hiPSCs edited with a library of MYH7 missense variants and recovered 113 different heterozygous variants. As deleterious mutations can reduce protein stability, we flow sorted MYH7 variant hiPSC-CMs into bins based on differing βMHC abundance followed by next generation sequencing of each bin to calculate variant abundance scores. This assay identified 31 MYH7 missense variants as functionally abnormal (lower abundance scores than synonymous controls), including all known pathogenic variants in the library. This βMHC depletion phenotype was validated in clonal hiPSC-CMs and supported by western blotting of myocardium from a patient with a pathogenic MYH7 missense mutation. Furthermore, we rescued βMHC protein levels by genetically ablating the gene encoding for Muscle RING-finger protein-1, an E3 protein ligase that targets βMHC for degradation, and found that the cardiomyocyte hypertrophy induced by the pathogenic MYH7 E848G HCM variant was significantly blunted. In summary, CRaTER enables the generation of gene-edited hiPSC lines at unprecedented scale. Functional analysis of MYH7 variant hiPSC-CMs reveals a novel correlation between loss of βMHC protein and clinical pathogenicity that can be leveraged for clinical variant interpretation. Interventions that prevent the loss of βMHC may potentially be therapeutic for MYH7 -associated HCM.

Abstract P1066: Engineering Of Ferritin Overexpressing Human Induced Pluripotent Stem Cells For In Vivo Tracking Of Grafts Using Magnetic Resonance Imaging

Introduction: Transplantation of human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) emerges as a promising approach for the treatment of heart diseases. However, it is an unmet challenge to precisely localize the hiPSC-CMs upon transplantation and quantify the graft size in the live large animals. To address this issue, we established a new approach to track grafted CMs in large animals using magnetic resonance imaging detectable reporter molecules, which allows the assessment of graft size in live animals at different time points. Methods: We employed a CRISPR/Cas9 system as a virus-free approach to overexpress ferritin in hiPSCs. Results: hiPSC cells were transfected with ferritin heavy chain CRISPR activation plasmid for 24 hours. Before antibiotic selection, we established a kill curve to determine the appropriate antibiotic concentration. Based on the kill curve, we conducted antibiotic selection using puromycin for 7 days and isolated transfected colonies for further culture. Next, we treated various concentrations of iron chloride (0-100 μM) to the FHC transfected hiPSC for 4 hours to examine a proper concentration of iron loading for MRI detection. There was no significant difference in cell viability between 0 and 25 μM of iron chloride. In addition, we confirmed transfection efficiency with higher ferritin expression in transfected cells compared to controls (non-transfected wild-type hiPSC). Following, we plan to differentiate ferritin overexpressing hiPSC into cardiomyocytes, inject the cells into porcine hearts, and conduct magnetic resonance imaging to detect the engrafted transgenic cells in live animals. Conclusion: The ferritin-expressing hiPSC line has the potential to provide a reliable measurement of the graft size in large animals.

Abstract P3192: Prdm16 Regulates Cardiac Metabolic Genes Through Perm1

The heart is a metabolically demanding organ that requires significant amounts of energy to function properly, and even minor metabolic deficiencies during development can lead to heart failure. Cardiomyopathies are a group of conditions that affect the heart's contractile function, and PRDM16 deficiency is a primary cause of cardiomyopathies associated with 1p36 deletion syndrome in humans. Conditional deletion of Prmd16 in the heart causes a spectrum of cardiac phenotypes including left ventricular non-compaction, late-onset heart failure, and dilated cardiomyopathy. Still, the mechanisms underlying these cardiac phenotypes with loss of Prdm16 are not fully understood. We and others demonstrated that lack of Prdm16 in the heart causes alterations in fatty acid (FA) oxidation and oxidative phosphorylation (OXPHOS) gene expression. Further, we now show that Prdm16 cardiac deletion reduces the expression of key regulators of FA oxidation and OXPHOS genes including Perm1. Loss and gain of Prdm16 function in vitro confirmed a cell-autonomous regulation of Perm1 by Prdm16 in H9c2 rat cardiomyoblasts and neonatal rat ventricular myocytes (NRVMs). Prdm16 ChIP-sequencing analysis of embryonic hearts revealed direct binding of Prdm16 to the promoter of Perm1. Prdm16 binding to Perm1 promoter was further confirmed by ChIP-qPCR in H9c2 cells. Knockdown of Prdm16 in NRVMs reduced maximal mitochondrial respiration, which was rescued by over-expressing Perm1. Further, PERM1 mRNA was diminished in human induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) derived from a patient with a PRDM16 mutation (Q187X). These results are evidence that PRDM16 is an upstream regulator of PERM1 in the heart.

Extracellular Vesicle-Encapsulated AAVs for Therapeutic Gene Delivery to the Heart.

Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use is preexisting neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction, and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance. We developed a 2-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. We compared the gene delivery and therapeutic efficacy of EV-AAVs with an equal titer of free AAVs in the presence of NAbs, both in vitro and in vivo. In addition, we investigated the mechanism of EV-AAV uptake in human left ventricular and human induced pluripotent stem cell cardiomyocytes in vitro and mouse models in vivo using a combination of biochemical techniques, flow cytometry, and immunofluorescence imaging. Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAbs, both to human left ventricular and human induced pluripotent stem cell cardiomyocytes in vitro and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9- sarcoplasmic reticulum calcium ATPase 2a to infarcted hearts in preimmunized mice significantly improved ejection fraction and fractional shortening compared with AAV9-sarcoplasmic reticulum calcium ATPase 2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Trafficking studies using human induced pluripotent stem cell-derived cells in vitro and mouse hearts in vivo showed significantly higher expression of EV-AAV6/9-delivered genes in cardiomyocytes compared with noncardiomyocytes, even with comparable cellular uptake. Using cellular subfraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake. Together, using 5 different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared with free AAVs in the presence of NAbs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure.

Materials and Design Approaches for a Fully Bioresorbable, Electrically Conductive and Mechanically Compliant Cardiac Patch Technology.

Myocardial infarction (MI) is one of the leading causes of death and disability. Recently developed cardiac patches provide mechanical support and additional conductive paths to promote electrical signal propagation in the MI area to synchronize cardiac excitation and contraction. Cardiac patches based on conductive polymers offer attractive features; however, the modest levels of elasticity and high impedance interfaces limit their mechanical and electrical performance. These structures also operate as permanent implants, even in cases where their utility is limited to the healing period of tissue damaged by the MI. The work presented here introduces a highly conductive cardiac patch that combines bioresorbable metals and polymers together in a hybrid material structure configured in a thin serpentine geometry that yields elastic mechanical properties. Finite element analysis guides optimized choices of layouts in these systems. Regular and synchronous contraction of human induced pluripotent stem cell-derived cardiomyocytes on the cardiac patch and ex vivo studies offer insights into the essential properties and the bio-interface. These results provide additional options in the design of cardiac patches to treat MI and other cardiac disorders.

Comparing the signaling and transcriptome profiling landscapes of human iPSC-derived and primary rat neonatal cardiomyocytes

The inaccessibility of human cardiomyocytes significantly hindered years of cardiovascular research efforts. To overcome these limitations, non-human cell sources were used as proxies to study heart function and associated diseases. Rodent models became increasingly acceptable surrogates to model the human heart either in vivo or through in vitro cultures. More recently, due to concerns regarding animal to human translation, including cross-species differences, the use of human iPSC-derived cardiomyocytes presented a renewed opportunity. Here, we conducted a comparative study, assessing cellular signaling through cardiac G protein-coupled receptors (GPCRs) in rat neonatal cardiomyocytes (RNCMs) and human induced pluripotent stem cell-derived cardiomyocytes. Genetically encoded biosensors were used to explore GPCR-mediated nuclear protein kinase A (PKA) and extracellular signal-regulated kinase 1/ 2 (ERK1/2) activities in both cardiomyocyte populations. To increase data granularity, a single-cell analytical approach was conducted. Using automated high content microscopy, our analyses of nuclear PKA and ERK1/2 signaling revealed distinct response clusters in rat and human cardiomyocytes. In line with this, bulk RNA-seq revealed key differences in the expression patterns of GPCRs, G proteins and downstream effector expression levels. Our study demonstrates that human stem cell-derived models of the cardiomyocyte offer distinct advantages for understanding cellular signaling in the heart.

Activation of MG53 Enhances Cell Survival and Engraftment of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Injured Hearts

Background and ObjectiveOur previous studies demonstrated that MG53 protein can protect the myocardium, but its use as a therapeutic is challenging due to its short half-life in blood circulation. This study aimed to investigate the cardioprotective role of MG53 on human induced pluripotent stem cell-derived cardiomyocytes (HiPSC-CMs) in the context of myocardial ischemia/reperfusion (I/R).MethodsIn vitro: HiPSC-CMs were transfected with adenoviral MG53 (HiPSC-CMsMG53), in which the expression of MG53 can be controlled by doxycycline (Dox), and the cells were then exposed to H2O2 to mimic ischemia/reperfusion injury. In vivo: HiPSC-CMsMG53 were transplanted into the peri-infarct region in NSG™ mice after I/R. After surgery, mice were treated with Dox (+ Dox) to activate MG53 expression (sucrose as a control of -Dox) and then assessed by echocardiography and immunohistochemistry.ResultsMG53 can be expressed in HiPSC-CMMG53 and released into the culture medium after adding Dox. The cell survival rate of HiPSC-CMMG53 was improved by Dox under the H2O2 condition. After 14 and 28 days of ischemia/reperfusion (I/R), transplanted HiPSC-CMsMG53 + Dox significantly improved heart function, including ejection fraction (EF) and fractional shortening (FS) in mice, compared to HiPSC-CMsMG53-Dox, and reduced the size of the infarction. Additionally, HiPSC-CMMG53 + Dox mice demonstrated significant engraftment in the myocardium as shown by staining human nuclei-positive cells. In addition, the cell survival-related AKT signaling was found to be more active in HiPSC-CMMG53 + Dox transplanted mice’s myocardium compared to the HiPSC-CMMG53-Dox group. Notably, the Dox treatment did not cause harm to other organs.Conclusion Inducible MG53 expression is a promising approach to enhance cell survival and engraftment of HiPSC-CMs for cardiac repair.Graphical

Nuclear translocation of mitochondrial dehydrogenases as an adaptive cardioprotective mechanism

Chemotherapy-induced cardiac damage remains a leading cause of death amongst cancer survivors. Anthracycline-induced cardiotoxicity is mediated by severe mitochondrial injury, but little is known about the mechanisms by which cardiomyocytes adaptively respond to the injury. We observed the translocation of selected mitochondrial tricarboxylic acid (TCA) cycle dehydrogenases to the nucleus as an adaptive stress response to anthracycline-cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes and in vivo. The expression of nuclear-targeted mitochondrial dehydrogenases shifts the nuclear metabolic milieu to maintain their function both in vitro and in vivo. This protective effect is mediated by two parallel pathways: metabolite-induced chromatin accessibility and AMP-kinase (AMPK) signaling. The extent of chemotherapy-induced cardiac damage thus reflects a balance between mitochondrial injury and the protective response initiated by the nuclear pool of mitochondrial dehydrogenases. Our study identifies nuclear translocation of mitochondrial dehydrogenases as an endogenous adaptive mechanism that can be leveraged to attenuate cardiomyocyte injury.

Evaluation of Porcine Psoas Major as a Scaffold Material for Engineered Heart Tissues.

Decellularized porcine myocardium is commonly used as scaffolding for engineered heart tissues (EHTs). However, structural and mechanical heterogeneity in the myocardium complicate production of mechanically consistent tissues. In this study, we evaluate the porcine psoas major muscle (tenderloin) as an alternative scaffold material. Head-to-head comparison of decellularized tenderloin and ventricular scaffolds showed only minor differences in mean biomechanical characteristics, but tenderloin scaffolds were less variable and less dependent on the region of origin than ventricular samples. The active contractile behavior of EHTs made by seeding tenderloin versus ventricular scaffolds with human-induced pluripotent stem cell-derived cardiomyocytes was also comparable, with only minor differences observed. Collectively, the data reveal that the behavior of EHTs produced from decellularized porcine psoas muscle is almost identical to those made from porcine left ventricular myocardium, with the advantages of being more homogeneous, biomechanically consistent, and readily obtainable.

In vitro effect of hydroxychloroquine on pluripotent stem cells and their cardiomyocytes derivatives.

Introduction: Hydroxychloroquine (HDQ) is an antimalarial drug that has also shown its effectiveness in autoimmune diseases. Despite having side effects such as retinopathy, neuromyopathy and controversial cardiac toxicity, HDQ has been presented and now intensively studied for the treatment and prevention of coronavirus disease 2019 (COVID-19). Recent works revealed both beneficial and toxic effects during HDQ treatment. The cardiotoxic profile of HDQ remains unclear and identifying risk factors is challenging. Methods: Here, we used well-established cell-cultured to study the cytotoxic effect of HDQ, mouse induced pluripotent stem cells (miPSC) and their cardiomyocytes (CMs) derivatives were exposed to different concentrations of HDQ. Cell colony morphology was assessed by microscopy whereas cell viability was measured by flow cytometry and impedance-based methods. The effect of HDQ on beating activity of mouse and human induced pluripotent stem cell-derived CMs (miPSC-CMs and hiPSC-CMs, respectively) and mouse embryonic stem cell-derived CMs (mESC-CMs) were captured by the xCELLigence RTCA and microelectrode array (MEA) systems. Results and discussion: Our results revealed that 20µM of HDQ promotes proliferation of stem cells used suggesting that if appropriately monitored, HDQ may have a cardioprotective effect and may also represent a possible candidate for tissue repair. In addition, the field potential signals revealed that higher doses of this medication caused bradycardia that could be reversed with a higher concentration of ß-adrenergic agonist, Isoproterenol (Iso). On the contrary, HDQ caused an increase in the beating rate of hiPSC-CMs, which was further helped upon application of Isoproterenol (Iso) suggesting that HDQ and Iso may also work synergistically. These results indicate that HDQ is potentially toxic at high concentrations and can modulate the beating activity of cardiomyocytes. Moreover, HDQ could have a synergistic inotropic effect with isoproterenol on cardiac cells.

Topological butterfly wings for human induced pluripotent stem cell-derived cardiomyocyte maturation and myocardial infarction treatment

Myocardial infarction (MI) causes irreversible myocardial cell loss; however, current treatments are limited to slowing disease progression rather than restoring lost myocardium. Cardiac tissue engineering, based on inexhaustible human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), becomes an ideal treatment method for supplying exogenous cardiomyocytes (CMs) to help contractile function recovery. However, hiPSC-CMs currently used are usually immature, which might trigger ventricular arrhythmias in MI treatments and limit their application in myocardial tissue engineering. The topological structure, as one of the most effective cues in controlling cellular functions, may benefit hiPSC-CM maturation. Here, the natural topological Morpho Menelaus butterfly wings (BFW) were chosen to regulate hiPSC-CM maturation and form the grafts for MI treatment. Immunofluorescence and calcium imaging tests confirmed that the parallel nanoridges on the surface of BFW oriented cellular growth and enhanced gap junction formation and improved the calcium handling ability of hiPSC-CMs, which demonstrated the maturation of hiPSC-CMs. BFW-matured hiPSC-CMs were transplanted as a patch for MI treatment evaluation, and twenty-eight days after engraftment, the histological destruction and contractile function of MI mice were improved obviously. This work provides an effective method for promoting hiPSC-CM maturation and potentially expands the application of natural materials in myocardial tissue engineering.

Supramolecular Assemblies of Glycopeptides Enhance Gap Junction Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes via Inducing Spheroids Formation to Optimize Cardiac Repair.

Stem cell-based therapies have demonstrated significant potential for use in heart regeneration. An effective paradigm for heart repair in rodent and large animal models is the transplantation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Despite this, the functional and phenotypical immaturity of two-dimensional (2D)-cultured hiPSC-CMs, particularly their low electrical integration, poses a caveat for clinical translation. In this study, a supramolecular assembly of a glycopeptide containing a cell adhesion motif-RGD and saccharide-glucose (Bio-Gluc-RGD) was designed to enable the three-dimensional (3D) spheroid formation of hiPSC-CMs, promoting cell-cell and cell-matrix interactions that occur during spontaneous morphogenesis. HiPSC-CMs in spheroids were prone to be phenotypically mature and developed robust gap junctions via activation of the integrin/ILK/p-AKT/Gata4 pathway. Monodispersed hiPSC-CMs encapsulated in the Bio-Gluc-RGD hydrogel were more likely to form aggregates and, therefore, survive in the infarcted myocardium of mice, accompanied by more robust gap junction formation in the transplanted cells, and hiPSC-CMs delivered with the hydrogels also displayed angiogenic effect and anti-apoptosis capacity in the peri-infarct area, enhancing their overall therapeutic efficacy in myocardial infarction. Collectively, our findings illustrate a novel concept for modulating hiPSC-CM maturation by spheroid induction, which has the potential for post-MI heart regeneration. This article is protected by copyright. All rights reserved.

Autoimmune Atrial Fibrillation.

Atrial fibrillation (AF) is by far the most common cardiac arrhythmia. In about 3% of individuals, AF develops as a primary disorder without any identifiable trigger (idiopathic or historically termed lone AF). In line with the emerging field of autoantibody-related cardiac arrhythmias, the objective of this study was to explore whether autoantibodies targeting cardiac ion channels can underlie unexplained AF. Peptide microarray was used to screen patient samples for autoantibodies. We compared patients with unexplained AF (n=37 pre-existent AF; n=14 incident AF on follow-up) to age- and sex-matched controls (n=37). Electrophysiological properties of the identified autoantibody were then tested in vitro with the patch clamp technique and in vivo with an experimental mouse model of immunization. A common autoantibody response against Kir3.4 protein was detected in patients with AF and even before the development of clinically apparent AF. Kir3.4 protein forms a heterotetramer that underlies the cardiac acetylcholine-activated inwardly rectifying K+ current, IKACh. Functional studies on human induced pluripotent stem cell-derived atrial cardiomyocytes showed that anti-Kir3.4 IgG purified from patients with AF shortened action potentials and enhanced the constitutive form of IKACh, both key mediators of AF. To establish a causal relationship, we developed a mouse model of Kir3.4 autoimmunity. Electrophysiological study in Kir3.4-immunized mice showed that Kir3.4 autoantibodies significantly reduced atrial effective refractory period and predisposed animals to a 2.8-fold increased susceptibility to AF. To our knowledge, this is the first report of an autoimmune pathogenesis of AF with direct evidence of Kir3.4 autoantibody-mediated AF.

Analysis of Fatty Acid Metabolism in Fetal and Failing Hearts by Single-Cell RNA Sequencing Revealed SLC27A6 as a Critical Gene in Heart Maturation.

Heart failure is associated with shifts in substrate preferences and energy insufficiency. Although cardiac metabolism has been explored at the organ level, the metabolic changes at the individual cell level remain unclear. This study employed single-cell ribonucleic acid (RNA) sequencing to investigate the cell-type-specific characteristics of gene expression related to fatty acid metabolism. Single-cell RNA sequencing data from fetal hearts were processed to analyze gene expression patterns related to fatty acid metabolism. Immunofluorescence staining and Western blotting techniques were employed to validate the expression of specific proteins. Additionally, calcium recording and contractility measurements were performed to assess the functional implications of fatty acid metabolism in cardiomyocytes. Based on single-cell RNA sequencing data analysis, we found that a decrease in overall energy requirements underlies the downregulation of fatty acid oxidation-related genes in the later period of heart maturation and the compensatory increase of fatty acid metabolism in individual cardiomyocytes during heart failure. Furthermore, we found that solute carrier family 27 member 6 (SLC27A6), a fatty acid transport protein, is involved in cardiac maturation. SLC27A6 knockdown in human induced pluripotent stem cell-derived cardiomyocytes resulted in an immature cardiomyocyte transcriptional profile, abnormal morphology, impaired Ca2+ handling activity, and contractility. Overall, our study offers a novel perspective for exploring cardiac fatty acid metabolism in fetal and failing hearts along with new insights into the cellular mechanism underlying fatty acid metabolic alterations in individual cardiac cells. It thus facilitates further exploration of cardiac physiology and pathology.