Human Embryonic Stem Cell-derived Cardiomyocytes Research Articles

Inhibition of Hmbox1 Promotes Cardiomyocyte Survival and Glucose Metabolism Through Gck Activation in Ischemia/Reperfusion Injury.

Exercise-induced physiological cardiac growth regulators may protect the heart from ischemia/reperfusion (I/R) injury. Homeobox-containing 1 (Hmbox1), a homeobox family member, has been identified as a putative transcriptional repressor and is downregulated in the exercised heart. However, its roles in exercise-induced physiological cardiac growth and its potential protective effects against cardiac I/R injury remain largely unexplored. We studied the function of Hmbox1 in exercise-induced physiological cardiac growth in mice after 4 weeks of swimming exercise. Hmbox1 expression was then evaluated in human heart samples from deceased patients with myocardial infarction and in the animal cardiac I/R injury model. Its role in cardiac I/R injury was examined in mice with adeno-associated virus 9 (AAV9) vector-mediated Hmbox1 knockdown and in those with cardiac myocyte-specific Hmbox1 ablation. We performed RNA sequencing, promoter prediction, and binding assays and identified glucokinase (Gck) as a downstream effector of Hmbox1. The effects of Hmbox1 together with Gck were examined in cardiomyocytes to evaluate their cell size, proliferation, apoptosis, mitochondrial respiration, and glycolysis. The function of upstream regulator of Hmbox1, ETS1, was investigated through ETS1 overexpression in cardiac I/R mice in vivo. We demonstrated that Hmbox1 downregulation was required for exercise-induced physiological cardiac growth. Inhibition of Hmbox1 increased cardiomyocyte size in isolated neonatal rat cardiomyocytes and human embryonic stem cell-derived cardiomyocytes but did not affect cardiomyocyte proliferation. Under pathological conditions, Hmbox1 was upregulated in both human and animal postinfarct cardiac tissues. Furthermore, both cardiac myocyte-specific Hmbox1 knockout and AAV9-mediated Hmbox1 knockdown protected against cardiac I/R injury and heart failure. Therapeutic effects were observed when sh-Hmbox1 AAV9 was administered after I/R injury. Inhibition of Hmbox1 activated the Akt/mTOR/P70S6K pathway and transcriptionally upregulated Gck, leading to reduced apoptosis and improved mitochondrial respiration and glycolysis in cardiomyocytes. ETS1 functioned as an upstream negative regulator of Hmbox1 transcription, and its overexpression was protective against cardiac I/R injury. Our studies unravel a new role for the transcriptional repressor Hmbox1 in exercise-induced physiological cardiac growth. They also highlight the therapeutic potential of targeting Hmbox1 to improve myocardial survival and glucose metabolism after I/R injury.

HLA-Ehigh /HLA-Ghigh /HLA-IIlow Human iPSC-Derived Cardiomyocytes Exhibit Low Immunogenicity for Heart Regeneration.

Although human pluripotent stem cells (hPSCs)-derived cardiomyocytes (hPSC-CMs) can remuscularize infarcted hearts and restore post-infarct cardiac function, post-transplant rejection resulting from human leukocyte antigen (HLA) mismatching is an enormous obstacle. It is crucial to identify hypoimmunogenic hPSCs for allogeneic cell therapy. This study is conducted to demonstrate the immune privilege of HLA-Ehigh /HLA-Ghigh /HLA-IIlow human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs). Ischemia-reperfusion surgery is done to create transmural myocardial infarction in rats. At post-infarct 4 days, hPSC-CMs (1.0×107 cells per kg), including human embryonic stem cell-derived cardiomyocytes (hESC-CMs), HLA-Elow/HLA-Glow/HLA-IIhigh hiPSC-CMs, and HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs, are injected into the infarcted myocardium. Under the treatment of very low dose cyclosporine A (CsA), only HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs survive in vivo and improved post-infarct cardiac function with infarct size reduction. HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs activate the SHP-1 signaling pathway of natural killer (NK) cells and cytotoxic T cells to evade attack by NK cells and cytotoxic T cells. Herein, it is demonstrated that using a clinically relevant CsA dose, HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs repair the infarcted myocardium and restore the post-infarct heart function. HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSCs are less immunogenic and may serve as platforms for regeneration medicine.

Myosin expression and contractile function are altered by replating stem cell-derived cardiomyocytes.

Myosin heavy chain (MyHC) is the main determinant of contractile function. Human ventricular cardiomyocytes (CMs) predominantly express the β-isoform. We previously demonstrated that ∼80% of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) express exclusively β-MyHC after long-term culture on laminin-coated glass coverslips. Here, we investigated the impact of enzymatically detaching hESC-CMs after long-term culture and subsequently replating them for characterization of cellular function. We observed that force-related kinetic parameters, as measured in a micromechanical setup, resembled α- rather than β-MyHC-expressing myofibrils, as well as changes in calcium transients. Single-cell immunofluorescence analysis revealed that replating hESC-CMs led to rapid upregulation of α-MyHC, as indicated by increases in exclusively α-MyHC- and in mixed α/β-MyHC-expressing hESC-CMs. A comparable increase in heterogeneity of MyHC isoform expression was also found among individual human induced pluripotent stem cell (hiPSC)-derived CMs after replating. Changes in MyHC isoform expression and cardiomyocyte function induced by replating were reversible in the course of the second week after replating. Gene enrichment analysis based on RNA-sequencing data revealed changes in the expression profile of mechanosensation/-transduction-related genes and pathways, especially integrin-associated signaling. Accordingly, the integrin downstream mediator focal adhesion kinase (FAK) promoted β-MyHC expression on a stiff matrix, further validating gene enrichment analysis. To conclude, detachment and replating induced substantial changes in gene expression, MyHC isoform composition, and function of long-term cultivated human stem cell-derived CMs, thus inducing alterations in mechanosensation/-transduction, that need to be considered, particularly for downstream in vitro assays.

Omentin-1 drives cardiomyocyte cell cycle arrest and metabolic maturation by interacting with BMP7.

Mammalian cardiomyocytes (CMs) undergo maturation during postnatal heart development to meet the increased demands of growth. Here, we found that omentin-1, an adipokine, facilitates CM cell cycle arrest and metabolic maturation. Deletion of omentin-1 causes mouse heart enlargement and dysfunction in adulthood and CM maturation retardation in juveniles, including delayed cell cycle arrest and reduced fatty acid oxidation. Through RNA sequencing, molecular docking analysis, and proximity ligation assays, we found that omentin-1 regulates CM maturation by interacting directly with bone morphogenetic protein 7 (BMP7). Omentin-1 prevents BMP7 from binding to activin type II receptor B (ActRIIB), subsequently decreasing the downstream pathways mothers against DPP homolog 1 (SMAD1)/Yes-associated protein (YAP) and p38 mitogen-activated protein kinase (p38 MAPK). In addition, omentin-1 is required and sufficient for the maturation of human embryonic stem cell-derived CMs. Together, our findings reveal that omentin-1 is a pro-maturation factor for CMs that is essential for postnatal heart development and cardiac function maintenance.

Expanding the apelin receptor pharmacological toolbox using novel fluorescent ligands.

The apelin receptor binds two distinct endogenous peptides, apelin and ELA, which act in an autocrine/paracrine manner to regulate the human cardiovascular system. As a class A GPCR, targeting the apelin receptor is an attractive therapeutic strategy. With improvements in imaging techniques, and the stability and brightness of dyes, fluorescent ligands are becoming increasingly useful in studying protein targets. Here, we describe the design and validation of four novel fluorescent ligands; two based on [Pyr1]apelin-13 (apelin488 and apelin647), and two based on ELA-14 (ELA488 and ELA647). Fluorescent ligands were pharmacologically assessed using radioligand and functional in vitro assays. Apelin647 was validated in high content imaging and internalisation studies, and in a clinically relevant human embryonic stem cell-derived cardiomyocyte model. Apelin488 and ELA488 were used to visualise apelin receptor binding in human renal tissue. All four fluorescent ligands retained the ability to bind and activate the apelin receptor and, crucially, triggered receptor internalisation. In high content imaging studies, apelin647 bound specifically to CHO-K1 cells stably expressing apelin receptor, providing proof-of-principle for a platform that could screen novel hits targeting this GPCR. The ligand also bound specifically to endogenous apelin receptor in stem cell-derived cardiomyocytes. Apelin488 and ELA488 bound specifically to apelin receptor, localising to blood vessels and tubules of the renal cortex. Our data indicate that the described novel fluorescent ligands expand the pharmacological toolbox for studying the apelin receptor across multiple platforms to facilitate drug discovery.

IGF-1 boosts mitochondrial function by a Ca2+ uptake-dependent mechanism in cultured human and rat cardiomyocytes.

A physiological increase in cardiac workload results in adaptive cardiac remodeling, characterized by increased oxidative metabolism and improvements in cardiac performance. Insulin-like growth factor-1 (IGF-1) has been identified as a critical regulator of physiological cardiac growth, but its precise role in cardiometabolic adaptations to physiological stress remains unresolved. Mitochondrial calcium (Ca2+) handling has been proposed to be required for sustaining key mitochondrial dehydrogenase activity and energy production during increased workload conditions, thus ensuring the adaptive cardiac response. We hypothesized that IGF-1 enhances mitochondrial energy production through a Ca2+-dependent mechanism to ensure adaptive cardiomyocyte growth. We found that stimulation with IGF-1 resulted in increased mitochondrial Ca2+ uptake in neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes, estimated by fluorescence microscopy and indirectly by a reduction in the pyruvate dehydrogenase phosphorylation. We showed that IGF-1 modulated the expression of mitochondrial Ca2+ uniporter (MCU) complex subunits and increased the mitochondrial membrane potential; consistent with higher MCU-mediated Ca2+ transport. Finally, we showed that IGF-1 improved mitochondrial respiration through a mechanism dependent on MCU-mediated Ca2+ transport. In conclusion, IGF-1-induced mitochondrial Ca2+ uptake is required to boost oxidative metabolism during cardiomyocyte adaptive growth.

Transcriptomics reveal stretched human pluripotent stem cell-derived cardiomyocytes as an advantageous hypertrophy model

Left ventricular hypertrophy, characterized by hypertrophy of individual cardiomyocytes, is an adaptive response to an increased cardiac workload that eventually leads to heart failure. Previous studies using neonatal rat ventricular myocytes (NRVMs) and animal models have revealed several genes and signaling pathways associated with hypertrophy and mechanical load. However, these models are not directly applicable to humans. Here, we studied the effect of cyclic mechanical stretch on gene expression of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using RNA sequencing. hiPSC-CMs showed distinct hypertrophic changes in gene expression at the level of individual genes and in biological processes. We also identified several differentially expressed genes that have not been previously associated with cardiomyocyte hypertrophy and thus serve as attractive targets for future studies. When compared to previously published data attained from stretched NRVMs and human embryonic stem cell-derived cardiomyocytes, hiPSC-CMs displayed a smaller number of changes in gene expression, but the differentially expressed genes revealed more pronounced enrichment of hypertrophy-related biological processes and pathways. Overall, these results establish hiPSC-CMs as a valuable in vitro model for studying human cardiomyocyte hypertrophy. • Mechanical load-associated genes in human cardiomyocytes are not fully understood. • Changes in response to mechanical stretch in hiPSC-CMs were investigated by RNAseq. • hiPSC-CMs showed distinct gene expression changes compared to NRVMs or hESC-CMs. • Differentially expressed genes were restricted to hypertrophy-related processes. • GAL, LINC00648 and SLC16A9 identified as new cardiac stretch-responsive genes.

RBM24 controls cardiac QT interval through CaMKIIδ splicing.

Calcium/calmodulin-dependent kinase II delta (CaMKIIδ) is the predominant cardiac isoform and it is alternatively spliced to generate multiple variants. Variable variants allow for distinct localization and potentially different functions in the heart. Dysregulation of CaMKIIδ splicing has been demonstrated to be involved in the pathogenesis of heart diseases, such as cardiac hypertrophy, arrhythmia, and diastolic dysfunction. However, the mechanisms that regulate CaMKIIδ are incompletely understood. Here, we show that RNA binding motif protein 24 (RBM24) is a key splicing regulator of CaMKIIδ. RBM24 ablation leads to the aberrant shift of CaMKIIδ towards the δ-C isoform, which is known to activate the L-type Ca current. In line with this, we found marked alteration in Ca2+ handling followed by prolongation of the ventricular cardiac action potential and QT interval in RBM24 knockout mice, and these changes could be attenuated by treatment with an inhibitor of CaMKIIδ. Importantly, knockdown of RBM24 in human embryonic stem cell-derived cardiomyocytes showed similar electrophysiological abnormalities, suggesting the important role of RBM24 in the human heart. Thus, our data suggest that RBM24 is a critical regulator of CaMKIIδ to control the cardiac QT interval, highlighting the key role of splicing regulation in cardiac rhythm.

Chick early amniotic fluid component improves heart function and protects against inflammation after myocardial infarction in mice.

Myocardial infarction (MI) is the major cause of mortality around the world. We recently demonstrated that chick early amniotic fluid (ceAF) can effectively rescue ischemic heart injury, indicating that it has a therapeutic function in MI. However, its functional components and the underlying mechanisms remain to be clarified. Here, we demonstrated that a fraction of ceAF, peak 8 (P8), had a protective effect on acute MI. P8 significantly decreased cardiomyocyte cross-sectional areas and cardiomyocyte apoptosis in MI mice. Using a human embryonic stem cell-derived cardiomyocyte model, which was subjected to hypoxia and reoxygenation, mimicking MI state, we found that P8 treatment reduced apoptosis and reversed myocardial contractility. Mechanistically, P8 improved cardiac function by inhibiting NF-κB signaling and downregulating inflammatory cytokine expression. Using mass spectrometry, we identified that guanosine and deoxynucleoside were the main functional components of P8 that suppressed the inflammatory response in human embryonic stem cell-derived cardiomyocytes. Collectively, our data suggest that specific components from ceAF are promising therapeutic agents for ischemic heart injury and could be a potential supplement to current medications for MI.

Abstract 10121: Hypoimmunogenic Human Induced Pluripotent Stem Cells Derived Cardiomyocytes for Cardiac Regeneration

Introduction: Post-transplant rejection resulting from human leukocyte antigen (HLA) mismatch is a challenge in allogeneic cell therapy. Human amniotic fluid stem cell-derived iPSCs (hAFSC-iPSCs) are proved to have immune privilege. Hypothesis: hAFSC-iPSC derived cardiomyocytes (hAFSC-iPSC-CMs) are less immunogenic. Methods and Results: hAFSC-iPSCs and hAFSC-iPSC-CMs expressed classical HLA-Ia (HLA-A, -B and -C) and non-classical HLA-Ib but did not have HLA-II expression, indicating that these cells may not be highly immunogenic. We did T Cell Assays to confirm low immunogenicity of hAFSC-iPSC-CMs. We did ischemia-reperfusion surgery of left anterior descending coronary artery to create large transmural myocardial infarction in rats. At post-infarct 4 days, we injected human pluripotent stem cell-derived cardiomyocytes (1.0х10 7 cells/kg), including human embryonic stem cell-derived cardiomyocytes (hESC-CMs), hiPSC-CMs and hAFSC-iPSC-CMs, into the infarcted myocardium. Using very low dose of cyclosporine A (CsA, 2.5 mg/kg/day), only hAFSC-iPSC-CMs transplantation could improve post-infarct cardiac function (left ventricular ejection fraction improvement in hAFSC-iPSC-CMs, hESC-CMs and hiPSC-CMs: 13.1 ± 1.8%, 0.6 ± 1.7% and -1.9 ± 1.6%, respectively). Compared with hESC-CMs and hiPSC-CMs, hAFSC-iPSC-CM transplantation remuscularized the infarcted myocardium with infarct size reduction. Using very low dose CsA, these engrafted hAFSC-iPSC-CMs resulted in a 3-month survival in vivo. hAFSC-iPSC-CMs expressing non-classical HLA-Ib evaded detection and attack by natural killer (NK) cells and cytotoxic T cells because these hAFSC-iPSC-CMs activated the protein tyrosine phosphatase SHP-1/SHP-2 signalling pathway of NK cells and cytotoxic T cells which downregulated signal transducer and activator of transcription 1 (STAT-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression. Conclusions: We demonstrated the immune privilege of hAFSC-iPSC-CM for cardiac regeneration. HLA-Ib-expressing hiPSCs with reduced HLA-II expression are less immunogenic and may serve as platforms for regenerative medicine.

Inhibition of HIPK2 protects stress-induced pathological cardiac remodeling.

SummaryBackgroundHomeodomain-Interacting Protein Kinase 2 (HIPK2) has been reported to maintain basal cardiac function, however, its role in pathological cardiac remodeling remains unclear.MethodsHIPK2 inhibitors (tBID and PKI1H) treated mice and two lines of HIPK2−/− mice were subjected to transverse aortic constriction (TAC). HIPK2 knockdown were performed in neonatal rat cardiomyocytes (NRCMs), neonatal rat cardiac fibroblasts (NRCFs), and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Microarray analysis was used to screen HIPK2 targets. Overexpression of early growth response 3 (EGR3) and C-type lectin receptor 4D (CLEC4D) were performed in NRCMs, while an activator of Smad3 was used in NRCFs, to rescue the effects of HIPK2 knockdown. Finally, the effects of EGR3 and CLEC4D knockdown by AAV9 in TAC were determined.FindingsHIPK2 was elevated in TAC mice model, as well as cardiomyocyte hypertrophy and NRCFs fibrosis model. Pharmacological and genetic inhibition of HIPK2 improved cardiac function and suppressed cardiac hypertrophy and fibrosis induced by TAC. In vitro, HIPK2 inhibition prevented cardiomyocyte hypertrophic growth and NRCFs proliferation and differentiation. At the mechanistic level, we identified EGR3 and CLEC4D as new targets of HIPK2, which were regulated by ERK1/2-CREB and mediated the protective function of HIPK2 inhibition in cardiomyocytes. Meanwhile, inhibition of phosphorylation of Smad3 was responsible for the suppression of cardiac fibroblasts proliferation and differentiation by HIPK2 inhibition. Finally, we found that inhibition of EGR3 or CLEC4D protected against TAC.InterpretationHIPK2 inhibition protects against pathological cardiac remodeling by reducing EGR3 and CLEC4D with ERK1/2-CREB inhibition in cardiomyocytes, and by suppressing the phosphorylation of Smad3 in cardiac fibroblasts.FundingThis work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to J.X.), National Natural Science Foundation of China (82020108002 and 81911540486 to J.X., 81400647 to MJ Xu), the grant from Science and Technology Commission of Shanghai Municipality (21XD1421300 and 20DZ2255400 to J.X.), the “Dawn” Program of Shanghai Education Commission (19SG34 to J.X.), and Shanghai Sailing Program (21YF1413200 to Q.Z.).

Single-nucleus transcriptomics reveals a gatekeeper role for FOXP1 in primate cardiac aging.

Aging poses a major risk factor for cardiovascular diseases, the leading cause of death in the aged population. However, the cell type-specific changes underlying cardiac aging are far from being clear. Here, we performed single-nucleus RNA-sequencing analysis of left ventricles from young and aged cynomolgus monkeys to define cell composition changes and transcriptomic alterations across different cell types associated with age. We found that aged cardiomyocytes underwent a dramatic loss in cell numbers and profound fluctuations in transcriptional profiles. Via transcription regulatory network analysis, we identified FOXP1, a core transcription factor in organ development, as a key downregulated factor in aged cardiomyocytes, concomitant with the dysregulation of FOXP1 target genes associated with heart function and cardiac diseases. Consistently, the deficiency of FOXP1 led to hypertrophic and senescent phenotypes in human embryonic stem cell-derived cardiomyocytes. Altogether, our findings depict the cellular and molecular landscape of ventricular aging at the single-cell resolution, and identify drivers for primate cardiac aging and potential targets for intervention against cardiac aging and associated diseases.

BNIP-2 Activation of Cellular Contractility Inactivates YAP for H9c2 Cardiomyoblast Differentiation.

Rho GTPases and Hippo kinases are key regulators of cardiomyoblast differentiation. However, how these signaling axes are coordinated spatiotemporally remains unclear. Here, the central and multifaceted roles of the BCH domain containing protein, BNIP-2, in orchestrating the expression of two key cardiac genes (cardiac troponin T [cTnT] and cardiac myosin light chain [Myl2]) in H9c2 and human embryonic stem cell-derived cardiomyocytes are delineated. This study shows that BNIP-2 mRNA and protein expression increase with the onset of cTnT and Myl2 and promote the alignment of H9c2 cardiomyocytes. Mechanistically, BNIP-2 is required for the inactivation of YAP through YAP phosphorylation and its cytosolic retention. Turbo-ID proximity labeling corroborated by super-resolution analyses and biochemical pulldown data reveals a scaffolding role of BNIP-2 for LATS1 to phosphorylate and inactivate YAP in a process that requires BNIP-2 activation of cellular contractility. The findings identify BNIP-2 as a pivotal signaling scaffold that spatiotemporally integrates RhoA/Myosin II and LATS1/YAP mechanotransduction signaling to drive cardiomyoblast differentiation, by switching the genetic programming from YAP-dependent growth to YAP-silenced differentiation. These findings offer insights into the importance of scaffolding proteins in bridging the gap between mechanical and biochemical signals in cell growth and differentiation and the prospects in translational applications.

Abstract P3121: Serological Mechanisms Driving Cardiotoxicity In Covid-19

Cardiac involvement is common in patients severely ill with COVID-19 and is associated with increased morbidity and mortality. The mechanism for myocardial injury in COVID-19 has been proposed to be at least in part due to a direct viral cytotoxic effect on cardiomyocytes. Preclinical data corroborate the notion that SARSCoV-2 can enter and replicate within hiPSC-derived cardiomyocytes. However, clinical evidence demonstrating direct viral effects on the heart remains limited. Alternatively, cardiac injury may result from excessive inflammatory cytokine production which is the main driver for the pathogenesis in COVID-19. Despite this, cardiotoxicity secondary to hyperinflammation constitutes a potentially amenable mechanism that remains largely unexplored. To investigate serological drivers of cardiotoxicity in COVID-19 we have established a bioassay that assessed the effects of serum from COVID-19 confirmed patients on human embryonic stem cell-derived cardiomyocytes (hESC-CMs). We demonstrate that serum from COVID-19 positive patients significantly reduced cardiomyocyte viability and contractility independent of viral transduction. Serum from patients with greater disease severity led to worse cardiomyocyte viability and this significantly correlated with levels of key inflammatory cytokines, including IL-6, TNF-α, IL1-β, IL-10, CRP and neutrophil to lymphocyte ratio with a specific reduction of CD4+ and CD8+ cells. Combinatorial blockade of IL-6 and TNF-α partially reversed the phenotype and preserved cardiomyocyte viability and function. Interestingly, treatment with serum from Systemic Inflammatory Response Syndrome (SIRS) patients recapitulates the effect observed with COVID-19 patients’ serum indicating that the phenotype observed is most likely not unique to COVID-19 disease. In conclusion, our results provide direct evidence that inflammatory cytokines are at least in part responsible for the cardiovascular damage seen in COVID-19 and indicate possible targets for therapeutic intervention.

Abstract P1032: Patterned Vessels In HIPSC-derived Engineered Myocardium Enhance Perfusion And Heart Regeneration In A Rat Myocardial Infarction Model

Globally, >6 million patients with heart failure (HF) have reduced ejection fraction due to prior myocardial infarction (MI). In the U.S., 50,000 become refractory to treatment each year and novel therapies are urgently needed to treat systolic dysfunction, as treatments to restore cardiac muscle are not available. Human embryonic or induced pluripotent stem cell-derived cardiomyocytes (hESC/hiPSC-CMs) have emerged as a leading candidate therapy for replacing CMs lost to MI and are being evaluated in preclinical models and clinical trials. Current delivery of CMs by intramuscular injection or engineered cardiac tissue is the first step in developing a new therapy but fails to address concerns of persistent ischemia in the host and implanted CMs. We hypothesize a dual remuscularization-revascularization therapy will promote vessel development to advance regeneration. We produce hiPSC-CMs and form human engineered myocardium (hEM) in custom molds by mixing CMs with 5% human primary cardiac fibroblasts in collagen. Sacrificial gelatin-alginate fibers coated with endothelial cells are embedded in the hEM to pattern patent channels 400 μm in diameter upon chelation and wash out of alginate. HEM is cultured with perfusion prior to implant in a rat model of ischemia/reperfusion MI to evaluate host-graft inosculation, perfused vascular bed morphology, graft size, and heart function at 2 weeks. Ex vivo optical coherence tomography reveals capillary-scale vessels in implants. Host vasculature and origin-tracking of inosculated vessels are quantitatively evaluated using micro-CT based 3D vascular reconstruction. Immunohistochemistry shows robust engraftment of hiPSC-CMs and chimeric vascular ingrowth in implants. Gene expression analysis from explanted hEM by bulk RNAseq is revealing a great benefit of vascularization on CM development of excitation-contraction coupling machinery and sarcomeres. In conclusion, remuscularization with revascularization therapy, featuring innovative approaches to pattern vessels, is enhancing multiple metrics of regeneration after hEM implantation. This study is the first of its kind and provides a new direction for development of regenerative therapies needed for the failing hearts of post-MI patients.

KCNQ1-deficient and KCNQ1-mutant human embryonic stem cell-derived cardiomyocytes for modeling QT prolongation

BackgroundThe slowly activated delayed rectifier potassium current (IKs) mediated by the KCNQ1 gene is one of the main currents involved in repolarization. KCNQ1 mutation can result in long-QT syndrome type 1 (LQT1). IKs does not participate in repolarization in mice; thus, no good model is currently available for research on the mechanism of and drug screening for LQT1. In this study, we established a KCNQ1-deficient human cardiomyocyte (CM) model and performed a series of microelectrode array (MEA) detection experiments on KCNQ1-mutant CMs constructed in other studies to explore the pathogenic mechanism of KCNQ1 deletion and mutation and perform drug screening.MethodKCNQ1 was knocked out in human embryonic stem cell (hESC) H9 line using the CRISPR/cas9 system. KCNQ1-deficient and KCNQ1-mutant hESCs were differentiated into CMs through a chemically defined differentiation protocol. Subsequently, high-throughput MEA analysis and drug intervention were performed to determine the electrophysiological characteristics of KCNQ1-deficient and KCNQ1-mutant CMs.ResultsDuring high-throughput MEA analysis, the electric field potential and action potential durations in KCNQ1-deficient CMs were significantly longer than those in wild-type CMs. KCNQ1-deficient CMs also showed an irregular rhythm. Furthermore, KCNQ1-deficient and KCNQ1-mutant CMs showed different responses to different drug treatments, which reflected the differences in their pathogenic mechanisms.ConclusionWe established a human CM model with KCNQ1 deficiency showing a prolonged QT interval and an irregular heart rhythm. Further, we used various drugs to treat KCNQ1-deficient and KCNQ1-mutant CMs, and the three models showed different responses to these drugs. These models can be used as important tools for studying the different pathogenic mechanisms of KCNQ1 mutation and the relationship between the genotype and phenotype of KCNQ1, thereby facilitating drug development.

Inducible apelin receptor knockdown reduces differentiation efficiency and contractility of hESC-derived cardiomyocytes.

The apelin receptor, a G protein-coupled receptor, has emerged as a key regulator of cardiovascular development, physiology, and disease. However, there is a lack of suitable human in vitro models to investigate the apelinergic system in cardiovascular cell types. For the first time we have used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and a novel inducible knockdown system to examine the role of the apelin receptor in both cardiomyocyte development and to determine the consequences of loss of apelin receptor function as a model of disease. Expression of the apelin receptor and its ligands in hESCs and hESC-CMs was determined. hESCs carrying a tetracycline-inducible short hairpin RNA targeting the apelin receptor were generated using the sOPTiKD system. Phenotypic assays characterized the consequences of either apelin receptor knockdown before hESC-CM differentiation (early knockdown) or in 3D engineered heart tissues as a disease model (late knockdown). hESC-CMs expressed the apelin signalling system at a similar level to the adult heart. Early apelin receptor knockdown decreased cardiomyocyte differentiation efficiency and prolonged voltage sensing, associated with asynchronous contraction. Late apelin receptor knockdown had detrimental consequences on 3D engineered heart tissue contractile properties, decreasing contractility and increasing stiffness. We have successfully knocked down the apelin receptor, using an inducible system, to demonstrate a key role in hESC-CM differentiation. Knockdown in 3D engineered heart tissues recapitulated the phenotype of apelin receptor down-regulation in a failing heart, providing a potential platform for modelling heart failure and testing novel therapeutic strategies.

Cpmer: A new conserved eEF1A2-binding partner that regulates Eomes translation and cardiomyocyte differentiation.

SummaryPrevious studies have shown that eukaryotic elongation factor 1A2 (eEF1A2) serves as an essential heart-specific translation elongation element and that its mutation or knockout delays heart development and causes congenital heart disease and death among species. However, the function and regulatory mechanisms of eEF1A2 in mammalian heart development remain largely unknown. Here we identified the long noncoding RNA (lncRNA) Cpmer (cytoplasmic mesoderm regulator), which interacted with eEF1A2 to co-regulate differentiation of mouse and human embryonic stem cell-derived cardiomyocytes. Mechanistically, Cpmer specifically recognized Eomes mRNA by RNA-RNA pairing and facilitated binding of eEF1A2 with Eomes mRNA, guaranteeing Eomes mRNA translation and cardiomyocyte differentiation. Our data reveal a novel functionally conserved lncRNA that can specifically regulate Eomes translation and cardiomyocyte differentiation, which broadens our understanding of the mechanism of lncRNA involvement in the subtle translational regulation of eEF1A2 during mammalian heart development.

Tomatidine-stimulated maturation of human embryonic stem cell-derived cardiomyocytes for modeling mitochondrial dysfunction.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have been reported to exhibit immature embryonic or fetal cardiomyocyte-like phenotypes. To enhance the maturation of hESC-CMs, we identified a natural steroidal alkaloid, tomatidine, as a new substance that stimulates the maturation of hESC-CMs. Treatment of human embryonic stem cells with tomatidine during cardiomyocyte differentiation stimulated the expression of several cardiomyocyte-specific markers and increased the density of T-tubules. Furthermore, tomatidine treatment augmented the number and size of mitochondria and enhanced the formation of mitochondrial lamellar cristae. Tomatidine treatment stimulated mitochondrial functions, including mitochondrial membrane potential, oxidative phosphorylation, and ATP production, in hESC-CMs. Tomatidine-treated hESC-CMs were more sensitive to doxorubicin-induced cardiotoxicity than the control cells. In conclusion, the present study suggests that tomatidine promotes the differentiation of stem cells to adult cardiomyocytes by accelerating mitochondrial biogenesis and maturation and that tomatidine-treated mature hESC-CMs can be used for cardiotoxicity screening and cardiac disease modeling.

Bisphosphonate of Zoledronate Has Antiapoptotic Effect on Hypoxia/Reoxygenation Injury in Human Embryonic Stem Cell-Derived Cardiomyocytes Through Trk Signaling Pathway.

In this work, we investigated the in vitro and in vivo functions of bisphosphonate of zoledronate (Zd) in hypoxia/reoxygenation (H/R) injured human embryonic stem cell-derived cardiomyocytes (hES-CMs). In the in vitro setting, the effects of Zd on hES-CM survival and differentiation were examined. We found that low and medium concentrations (<2 µm) of Zd did not induce cell death of hES-CMs. 0.5 µm Zd protected H/R-induced hES-CM apoptosis but did not affect key differentiation proteins, including hcTnl, PECM-1 Cnx43 and Pan-Cadherin. In addition, Zd-induced TrkA/B phosphorylation and promoted VEGF to counter the apoptotic effect of H/R injury. In the in vivo animal model of myocardial infarction, Zd treatment promoted the survival of hES-CMs by inducing PECAM1 and hcTnl. Thus, we concluded that Zd protected H/R-induced hES-CM apoptosis in vitro and promoted hES-CM survival in vivo. These data may facilitate the development of human embryonic stem cells into clinical applications for patients with ischemic heart disease.