Pluripotent Stem Cell-derived Cardiac Myocytes Research Articles

Induced pluripotent stem cell-derived cardiac myocytes to assess anthracycline cardiotoxicity

Abstract Background Current oncological treatments improved survival but also increased awareness of cardiovascular side-effects following cancer therapy. Anthracyclines are a class of highly effective chemotherapeutics, yet their use is regularly complicated by anthracycline-induced cardiotoxicity (AIC). The advent of induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) offers the potential for disease modeling and prediction of genetic cardiomyopathies. Purpose We aim to use iPSC-CMs to assess susceptibility to cardiotoxicity induced by anti-cancer treatments on an individual patient level. Furthermore, the assay could be exploited to unravel cellular processes and pathways involved in AIC. Methods Five heart transplant patients following AIC and five anthracycline treated patients without AIC were included in our study. We reprogrammed patient-derived lymphocytes from both groups toward iPSCs using Sendai Virus; afterwards, patient derived iPSCs were differentiated into CM-like cells. Cytotoxicity following chemotherapy exposure was assessed using flow cytometry (FC), in which iPSC-CM-specific cells were first selected based on the expression of signal-regulatory protein alpha (SIRPA) surface marker; thereafter the percentage of apoptotic versus viable cells following doxorubicin exposure was quantitatively assessed. Ultrastructural changes were evaluated using electron microscopy (EM) images, comparing doxorubicin treated and untreated iPSC-CMs. Reactive Oxygen Species (ROS) generation following doxorubicin exposure was assessed using a luciferin precursor-based plate reader assay (ROS-GLO) and FC. Furthermore, whole genome sequencing (WGS) was performed and variants were filtered based on allele frequency, pathogenicity using HumVar trained variant effect predictors and presence in patients versus controls. Results The cytotoxic effect of increasing concentrations of doxorubicin was quantifiable using FC on SIRPA positive iPSC-CMs (Figure 1 panel A). On the ultrastructural level, doxorubicin treated iPSC-CMs manifested darker and more condensed mitochondria as seen with EM (Figure 1 panel B). Increased ROS production was observed following doxorubicin exposure as assessed using ROS-GLO plate reader assay and FC (Figure 1 panel C). We used iPSC-CMs of transplant patients and matched controls to assess inter-individual susceptibility to anthracycline-induced cardiotoxicity. Included patient profiles can be found in Figure 2 panel A. There was a higher rate of cell death at any concentration of doxorubicin in patients with proven AIC as opposed to controls (Figure 2 panel B). WGS analysis showed 2 pathogenic variants in the PRAMEF6 gene present in cases and not in controls (Figure 3 panel C). Conclusion iPSC-CMs represent a robust disease model to study cancer therapy-induced cardiotoxicity. Furthermore, iPSC-CMs allow to study inter-individual differences in susceptibility to cancer therapy-induced cardiotoxicity.Mechanism of AICSusceptibility to AIC

Abstract 11679: The Crucial Role of Estrogen-Related Receptor in Human Cardiomyocyte Maturation

During the developmental transition from fetal to adult, the heart undergoes dramatic mitochondrial and sarcomeric maturation. This process is partially reversed during development of heart failure. We have identified the nuclear receptors, estrogen-related receptor (ERR) α and γ, as critical transcriptional regulators of energy metabolic and sarcomeric genes during postnatal maturation of mouse heart. Here, we aimed to define the transcriptional mechanisms involved in ERR-mediated maturation in human cardiomyocytes. Genomic interrogation with published datasets revealed that approximately 50% of super-enhancer regions (SEs) overlapped with ERRγ peaks in human-induced pluripotent stem cell-derived cardiac myocytes. Intersection of the ERRγ cistrome with a published H3K27ac Hi-ChIP dataset from human hearts identified over 3,000 ERR-containing enhancer-promoter loops, which are associated with genes related to cardiac contractile function. These results highlighted the essential role of ERR in cardiomyocyte enhancer function. Among the SEs defined by mediator complex deposition, we found that both ERRα and γ occupied the enhancer region flanking the MYH6/MYH7 cluster, a well-characterized locus for fetal to adult developmental switching. Targeting ERR recruitment on the enhancer using catalytically-dead Cas9-KRAB or proteolysis targeting chimeric (PROTAC) strategies resulted in the reversal of MYH6/7 switching. RNA-sequencing analysis following PROTAC treatment revealed that deactivation of ERRα resulted in downregulation of a wide array of genes encoding cardiac structural proteins, particularly adult-type ventricular sarcomeric proteins. Additional cistrome analyses demonstrated that PROTAC treatment resulted in decreased ERRα peak signals on cardiac structural genes, including enhancer regions directing MYH6/7 transcription consistent with the observed gene expression changes. In summary, ERR signaling is essential for the transcriptional program of structural maturation in human cardiomyocytes. This process likely involves establishing proper cardiac chromatin architecture. These results support a novel therapeutic strategy of utilizing ERR activators to reverse fetal shifts in failing hearts.

Activation of the integrated stress response rewires cardiac metabolism in Barth syndrome

Barth Syndrome (BTHS) is an inherited cardiomyopathy caused by defects in the mitochondrial transacylase TAFAZZIN (Taz), required for the synthesis of the phospholipid cardiolipin. BTHS is characterized by heart failure, increased propensity for arrhythmias and a blunted inotropic reserve. Defects in Ca2+-induced Krebs cycle activation contribute to these functional defects, but despite oxidation of pyridine nucleotides, no oxidative stress developed in the heart. Here, we investigated how retrograde signaling pathways orchestrate metabolic rewiring to compensate for mitochondrial defects. In mice with an inducible knockdown (KD) of TAFAZZIN, and in induced pluripotent stem cell-derived cardiac myocytes, mitochondrial uptake and oxidation of fatty acids was strongly decreased, while glucose uptake was increased. Unbiased transcriptomic analyses revealed that the activation of the eIF2α/ATF4 axis of the integrated stress response upregulates one-carbon metabolism, which diverts glycolytic intermediates towards the biosynthesis of serine and fuels the biosynthesis of glutathione. In addition, strong upregulation of the glutamate/cystine antiporter xCT increases cardiac cystine import required for glutathione synthesis. Increased glutamate uptake facilitates anaplerotic replenishment of the Krebs cycle, sustaining energy production and antioxidative pathways. These data indicate that ATF4-driven rewiring of metabolism compensates for defects in mitochondrial uptake of fatty acids to sustain energy production and antioxidation.

Abstract P2175: Induced Pluripotent Stem Cell-derived Cardiac Myocytes To Assess Anthracycline Cardiotoxicity

Background: Anthracyclines are a class of highly effective chemotherapeutics, yet their use is regularly complicated by anthracycline-induced cardiotoxicity (AIC). The advent of induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) offers the potential for disease modeling and prediction of genetic cardiomyopathies. Purpose: We aim to use iPSC-CMs to assess susceptibility to AIC. Methods: Five AIC heart transplant patients and five anthracycline treated patients without AIC were included in our study. We reprogrammed lymphocytes from both groups toward iPSCs using Sendai Virus, followed by differentiation into CM-like cells. Cytotoxicity following chemotherapy exposure was assessed using flow cytometry (FC), electron microscopy (EM) and reactive oxygen species generation (ROS). Furthermore, whole genome sequencing (WGS) was performed and variants were filtered based on allele frequency, pathogenicity using HumVar trained variant effect predictors and presence in patients versus controls. Results: The cytotoxic effect of increasing concentrations of doxorubicin was quantifiable using FC on SIRPA positive iPSC-CMs ( A ). There was a higher rate of cell death at any concentration of doxorubicin in patients with AIC as opposed to controls ( B ). On the ultrastructural level, doxorubicin treated iPSC-CMs manifested darker and more condensed mitochondria as seen with EM ( C ). Increased ROS production was observed following doxorubicin exposure ( D ). WGS analysis showed 2 pathogenic variants in the PRAMEF6 gene present in cases and not in controls ( E ). Conclusion: iPSC-CMs represent a robust disease model to study mechanisms of and susceptibility to AIC.

Human induced pluripotent stem cell-derived cardiac myocytes and sympathetic neurons in disease modelling.

Human induced pluripotent stem cells (hiPSC) offer an unprecedented opportunity to generate model systems that facilitate a mechanistic understanding of human disease. Current differentiation protocols are capable of generating cardiac myocytes (hiPSC-CM) and sympathetic neurons (hiPSC-SN). However, the ability of hiPSC-derived neurocardiac co-culture systems to replicate the human phenotype in disease modelling is still in its infancy. Here, we adapted current methods for efficient and replicable induction of hiPSC-CM and hiPSC-SN. Expression of cell-type-specific proteins were confirmed by flow cytometry and immunofluorescence staining. The utility of healthy hiPSC-CM was tested with pressor agents to develop a model of cardiac hypertrophy. Treatment with angiotensin II (AngII) resulted in: (i) cell and nuclear enlargement, (ii) enhanced fetal gene expression, and (iii) FRET-activated cAMP responses to adrenergic stimulation. AngII or KCl increased intracellular calcium transients in hiPSC-SN. Immunostaining in neurocardiac co-cultures demonstrated anatomical innervation to myocytes, where myocyte cytosolic cAMP responses were enhanced by forskolin compared with monocultures. In conclusion, human iPSC-derived cardiac myocytes and sympathetic neurons replicated many features of the anatomy and (patho)physiology of these cells, where co-culture preparations behaved in a manner that mimicked key physiological responses seen in other mammalian systems. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Proteasomal Degradation of TRAF2 Mediates Mitochondrial Dysfunction in Doxorubicin-Cardiomyopathy.

Cytokines such as tumor necrosis factor-α (TNFα) have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). Although TNFα can elicit different cellular responses, including survival or death, the mechanisms underlying these divergent outcomes in the heart remain cryptic. The E3 ubiquitin ligase TRAF2 (TNF receptor associated factor 2) provides a critical signaling platform for K63-linked polyubiquitination of RIPK1 (receptor interacting protein 1), crucial for nuclear factor-κB (NF-κB) activation by TNFα and survival. Here, we investigate alterations in TNFα-TRAF2-NF-κB signaling in the pathogenesis of DOX cardiotoxicity. Using a combination of in vivo (4 weekly injections of DOX 5 mg·kg-1·wk-1) in C57/BL6J mice and in vitro approaches (rat, mouse, and human inducible pluripotent stem cell-derived cardiac myocytes), we monitored TNFα levels, lactate dehydrogenase, cardiac ultrastructure and function, mitochondrial bioenergetics, and cardiac cell viability. In contrast to vehicle-treated mice, ultrastructural defects, including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFα levels, were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFα in DOX cardiotoxicity, we discovered that NF-κB was readily activated by TNFα. However, TNFα-mediated NF-κB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked polyubiquitination of RIPK1 from the proteasomal degradation of TRAF2. Furthermore, TRAF2 protein abundance was markedly reduced in hearts of patients with cancer treated with DOX. We further established that the reciprocal actions of the ubiquitinating and deubiquitinating enzymes cellular inhibitors of apoptosis 1 and USP19 (ubiquitin-specific peptidase 19), respectively, regulated the proteasomal degradation of TRAF2 in DOX-treated cardiac myocytes. An E3-ligase mutant of cellular inhibitors of apoptosis 1 (H588A) or gain of function of USP19 prevented proteasomal degradation of TRAF2 and DOX-induced cell death. Furthermore, wild-type TRAF2, but not a RING finger mutant defective for K63-linked polyubiquitination of RIPK1, restored NF-κB signaling and suppressed DOX-induced cardiac cell death. Last, cardiomyocyte-restricted expression of TRAF2 (cardiac troponin T-adeno-associated virus 9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX. Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX sensitizes cardiac myocytes to TNFα-mediated necrotic cell death and DOX cardiotoxicity.

Transcriptional Effects of Candidate COVID-19 Treatments on Cardiac Myocytes.

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) has emerged as a major cause of morbidity and mortality worldwide, placing unprecedented pressure on healthcare. Cardiomyopathy is described in patients with severe COVID-19 and increasing evidence suggests that cardiovascular involvement portends a high mortality. To facilitate fast development of antiviral interventions, drugs initially developed to treat other diseases are currently being repurposed as COVID-19 treatments. While it has been shown that SARS-CoV-2 invades cells through the angiotensin-converting enzyme 2 receptor (ACE2), the effect of drugs currently repurposed to treat COVID-19 on the heart requires further investigation.MethodsHuman induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) were treated with five repurposed drugs (remdesivir, lopinavir/ritonavir, lopinavir/ritonavir/interferon beta (INF-β), hydroxychloroquine, and chloroquine) and compared with DMSO controls. Transcriptional profiling was performed to identify global changes in gene expression programs.ResultsRNA sequencing of hiPSC-CMs revealed significant changes in gene programs related to calcium handling and the endoplasmic reticulum stress response, most prominently for lopinavir/ritonavir and lopinavir/ritonavir/interferon-beta. The results of the differential gene expression analysis are available for interactive access at https://covid19drugs.jakobilab.org.ConclusionTranscriptional profiling in hiPSC-CMs treated with COVID-19 drugs identified unfavorable changes with lopinavir/ritonavir and lopinavir/ritonavir/INF-β in key cardiac gene programs that may negatively affect heart function.

MicroRNA-365 regulates human cardiac action potential duration

Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human cardiac action potential and ask whether their manipulation allows for therapeutic modulation of action potential abnormalities. Quantitative analysis of the microRNA targetomes in human cardiac myocytes identifies miR-365 as a primary microRNA to regulate repolarizing ion channels. Action potential recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes show that elevation of miR-365 significantly prolongs action potential duration in myocytes derived from a Short-QT syndrome patient, whereas specific inhibition of miR-365 normalizes pathologically prolonged action potential in Long-QT syndrome myocytes. Transcriptome analyses in these cells at bulk and single-cell level corroborate the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional action potential-regulating genes by this microRNA. Whole-cell patch-clamp experiments confirm miR-365-dependent regulation of repolarizing ionic current Iks. Finally, refractory period measurements in human myocardial slices substantiate the regulatory effect of miR-365 on action potential in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac action potential duration by targeting key factors of cardiac repolarization.

Quantifying Propagation Velocity from Engineered Cardiac Tissues with High-Speed Fluorescence Microscopy and Automated Analysis Software.

Many acquired or inherited forms of heart disease as well as drugs are known to increase the susceptibility of patients to arrhythmias. To predict arrhythmogenic events and discover new therapeutic strategies to mitigate them, approaches to efficiently quantify the velocity of propagation in engineered cardiac tissues are important research tools. In this chapter, we describe how to collect videos of propagating calcium waves in engineered cardiac tissues with a high-speed camera mounted on an inverted fluorescence microscope. We also provide instructions for downloading and using our software package to analyze these videos and calculate propagation velocity. These techniques should be compatible with a variety of voltage- or calcium-sensitive fluorescent dyes or genetically encoded sensors. Although these approaches were originally developed for engineered neonatal rat cardiac tissues, the same procedures can likely be used with human-induced pluripotent stem cell-derived cardiac myocytes, paving the way for patient-specific analysis of propagation due to features such as tissue architecture, substrate rigidity, genetic mutations, or drug treatments.

Human BIN1 isoforms grow, maintain, and regenerate excitation-contraction couplons in adult rat and human stem cell-derived cardiomyocytes.

In ventricular myocytes, transverse-tubules (T-tubules) are instrumental for excitation-contraction (EC)coupling and their disarray is a hallmark of cardiac diseases. BIN1 is a key contributor to their biogenesis. Our study set out to investigate the role of human BIN1 splice variants in the maintenance and regeneration of EC-coupling in rat adult ventricular myocytes and human-induced pluripotent stem cell-derived cardiac myocytes (hiPS-CMs). In heart samples from healthy human donors expression patterns of five BIN1 splice variants were identified. Following viral transduction of human BIN1 splice variants in cellular models of T-tubular disarray, we employed high-speed confocal calcium imaging and CaCLEAN analysis to identify functional EC-coupling sites (couplons) and T-tubular architecture. Adult rat ventricular myocytes were used to investigate the regeneration after loss and maintenance of EC-coupling while we studied the enhancement of EC-coupling in hiPS-CMs. All five human BIN1 splice variants induced de-novo generation of T-tubules in both cell types. Isoforms with the phosphoinositide-binding motif (PI) were most potent in maintenance and regeneration of T-tubules and functional EC-coupling in adult rat myocytes. In hiPSC-CMs, BIN1 variants with PI-motif-induced de novo generation of T-tubules, functional couplons and enhanced calcium handling. BIN1 is essential for the maintenance, regeneration, and de novo generation of functional T-tubules. Isoforms with PI-motifs appeared as particulalrly potent. These T-tubules trigger the development of functional couplons resulting in enhanced calcium handling.

Structural and Functional Characterization of a Nav1.5-Mitochondrial Couplon.

The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined. To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria. Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A. We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.

Co-expression of calcium and hERG potassium channels reduces the incidence of proarrhythmic events.

Cardiac electrical activity is extraordinarily robust. However, when it goes wrong it can have fatal consequences. Electrical activity in the heart is controlled by the carefully orchestrated activity of more than a dozen different ion conductances. While there is considerable variability in cardiac ion channel expression levels between individuals, studies in rodents have indicated that there are modules of ion channels whose expression co-vary. The aim of this study was to investigate whether meta-analytic co-expression analysis of large-scale gene expression datasets could identify modules of co-expressed cardiac ion channel genes in human hearts that are of functional importance. Meta-analysis of 3653 public human RNA-seq datasets identified a strong correlation between expression of CACNA1C (L-type calcium current, ICaL) and KCNH2 (rapid delayed rectifier K+ current, IKr), which was also observed in human adult heart tissue samples. In silico modelling suggested that co-expression of CACNA1C and KCNH2 would limit the variability in action potential duration seen with variations in expression of ion channel genes and reduce susceptibility to early afterdepolarizations, a surrogate marker for proarrhythmia. We also found that levels of KCNH2 and CACNA1C expression are correlated in human-induced pluripotent stem cell-derived cardiac myocytes and the levels of CACNA1C and KCNH2 expression were inversely correlated with the magnitude of changes in repolarization duration following inhibition of IKr. Meta-analytic approaches of multiple independent human gene expression datasets can be used to identify gene modules that are important for regulating heart function. Specifically, we have verified that there is co-expression of CACNA1C and KCNH2 ion channel genes in human heart tissue, and in silico analyses suggest that CACNA1C-KCNH2 co-expression increases the robustness of cardiac electrical activity.

Systematic analysis of different pluripotent stem cell-derived cardiac myocytes as potential testing model for cardiocytoprotection

IntroductionStem cell-derived cardiac myocytes are potential sources for testing cardiocytoprotective molecules against ischemia/reperfusion injury in vitro. Materials and methodsHere we performed a systematic analysis of two different induced pluripotent stem cell lines (iPSC 3.4 and 4.1) and an embryonic stem cell (ESC) line-derived cardiac myocytes at two different developmental stages. Cell viability in simulated ischemia/reperfusion (SI/R)-induced injury and a known cardiocytoprotective NO-donor, S-nitroso-n-acetylpenicillamine (SNAP) was tested. ResultsAfter analysis of full embryoid bodies (EBs) and cardiac marker (VCAM and cardiac troponin I) positive cells of three lines at 6 conditions (32 different conditions altogether), we found significant SI/R injury-induced cell death in both full EBs and VCAM+ cardiac cells at later stage of their differentiation. Moreover, full EBs of the iPS 4.1 cell line after oxidative stress induction by SNAP was protected at day-8 samples. ConclusionWe have shown that 4.1 iPS-derived cardiomyocyte line could serve as a testing platform for cardiocytoprotection.

Abstract 222: The Estrogen-related Receptor (ERR) Drives Cardiac Myocyte Maturation in Cooperation With GATA4

During transition from fetal to adult developmental stages, the heart undergoes robust mitochondrial biogenesis in parallel with growth and structural maturation. Recently we identified a role for ERRα/γ in this broad program of postnatal cardiac maturation. We sought to delineate the mechanisms whereby ERRs coordinate transcriptional regulation of both metabolic and structural genes in the maturation of human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs). CRISPR-based gene deletion studies demonstrated that ERRα/γ is necessary for activation of genes involved in mitochondrial and structural maturation during hiPSC-CM differentiation including mitochondrial function, ion transport, Ca 2+ handling, and the adult sarcomere. Whole-genome RNA-sequencing and ChIP-sequencing (ChIP-seq) studies indicated that ERRγ directly regulates both metabolic and structural genes by remodeling H3K27ac depositions in related enhancer regions. Integration of the ERRγ ChIP-seq data with published hiPSC-CM datasets demonstrated that approximately 50% of super-enhancer regions overlapped ERRγ peaks, suggesting ERR is involved in the maintenance and function of cardiac super-enhancer regions. Corresponding motif analyses and intersection analyses with published GATA4 ChIP-seq datasets suggested that ERRγ cooperates with GATA4 on many structural, but not metabolic targets. Specifically, binding sites for both factors are often co-localized around cardiac contractile, ion channel, and Ca 2+ handling genes, including TNNI3, MYBPC3, MYH7, TTN, KCNQ1, and RYR2 . In contrast, ERRγ sites on target genes involved in mitochondrial maturation such as ATP5B, COX4I1, CPT1B , and FABP3 lacked GATA4 sites. Experiments using luciferase reporters demonstrated functional cooperation between ERRγ and GATA4 that depends on the known ERR coactivator, peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1α). We conclude that the ERR cooperates with GATA4 to drive cardiac structural gene transcription during cardiac differentiation, whereas ERR regulates genes involved in energy metabolism independent of GATA4. Activation of ERR signaling could prove to be an effective tool to drive hiPSC-CM maturation.

Deep Characterization of Circular RNAs from Human Cardiovascular Cell Models and Cardiac Tissue.

For decades, cardiovascular disease (CVD) has been the leading cause of death throughout most developed countries. Several studies relate RNA splicing, and more recently also circular RNAs (circRNAs), to CVD. CircRNAs originate from linear transcripts and have been shown to exhibit tissue-specific expression profiles. Here, we present an in-depth analysis of sequence, structure, modification, and cardiac circRNA interactions. We used human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs), human healthy and diseased (ischemic cardiomyopathy, dilated cardiomyopathy) cardiac tissue, and human umbilical vein endothelial cells (HUVECs) to profile circRNAs. We identified shared circRNAs across all samples, as well as model-specific circRNA signatures. Based on these circRNAs, we identified 63 positionally conserved and expressed circRNAs in human, pig, and mouse hearts. Furthermore, we found that the sequence of circRNAs can deviate from the sequence derived from the genome sequence, an important factor in assessing potential functions. Integration of additional data yielded evidence for m6A-methylation of circRNAs, potentially linked to translation, as well as, circRNAs overlapping with potential Argonaute 2 binding sites, indicating potential association with the RISC complex. Moreover, we describe, for the first time in cardiac model systems, a sub class of circRNAs containing the start codon of their primary transcript (AUG circRNAs) and observe an enrichment for m6A-methylation for AUG circRNAs.

A Critical Role for Estrogen-Related Receptor Signaling in Cardiac Maturation.

The heart undergoes dramatic developmental changes during the prenatal to postnatal transition, including maturation of cardiac myocyte energy metabolic and contractile machinery. Delineation of the mechanisms involved in cardiac postnatal development could provide new insight into the fetal shifts that occur in the diseased heart and unveil strategies for driving maturation of stem cell-derived cardiac myocytes. To delineate transcriptional drivers of cardiac maturation. We hypothesized that ERR (estrogen-related receptor) α and γ, known transcriptional regulators of postnatal mitochondrial biogenesis and function, serve a role in the broader cardiac maturation program. We devised a strategy to knockdown the expression of ERRα and γ in heart after birth (pn-csERRα/γ [postnatal cardiac-specific ERRα/γ]) in mice. With high levels of knockdown, pn-csERRα/γ knockdown mice exhibited cardiomyopathy with an arrest in mitochondrial maturation. RNA sequence analysis of pn-csERRα/γ knockdown hearts at 5 weeks of age combined with chromatin immunoprecipitation with deep sequencing and functional characterization conducted in human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CM) demonstrated that ERRγ activates transcription of genes involved in virtually all aspects of postnatal developmental maturation, including mitochondrial energy transduction, contractile function, and ion transport. In addition, ERRγ was found to suppress genes involved in fibroblast activation in hearts of pn-csERRα/γ knockdown mice. Disruption of Esrra and Esrrg in mice during fetal development resulted in perinatal lethality associated with structural and genomic evidence of an arrest in cardiac maturation, including persistent expression of early developmental and noncardiac lineage gene markers including cardiac fibroblast signatures. Lastly, targeted deletion of ESRRA and ESRRG in hiPSC-CM derepressed expression of early (transcription factor 21 or TCF21) and mature (periostin, collagen type III) fibroblast gene signatures. ERRα and γ are critical regulators of cardiac myocyte maturation, serving as transcriptional activators of adult cardiac metabolic and structural genes, an.d suppressors of noncardiac lineages including fibroblast determination.

Upregulation of the MAGUK Sap97 Enhances Protein Expression in Stem Cell Derived Myocytes

Human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) hold promise for personalized disease modeling and therapy development, however use of hiPSC-CMs is limited by problems of cell immaturity. Studies show that hiPSC-CMs have inadequate expression of proteins necessary for normal structure and function. Membrane-associated guanylate kinases (MAGUKs) are important for protein targeting/clustering needed for signal transduction. Synapse associated protein 97, the archetypical MAGUK, interacts with cell adhesion molecules, receptors/ion channels, and other MAGUKs, which necessary for cell differentiation and maturation. Preliminary evidence shows that Sap97 protein levels are marginal or undetectable in hiPSC-CMs. We tested the hypothesis that upregulation of Sap97 will increase protein expression and thereby enhance hiPSC-CMs maturation. Methods and Results: hiPSC-CMs were infected with control adenoviral constructs (Adv-GFP) or with Sap97 constructs (Adv-Sap97), and 6-well plates of cells were collected after 72 hours for biochemical analyses. Control hiPSC-CMs had marginal to undetectable levels of Sap97 expression. Infection with Adv-Sap97 increased Sap97 expression by 0.35+/- 0.01 (10 MOI; n=4), and by 0.70 +/- 0.01 (50 MOI; n=4). Furthermore, Adv-Sap97 (50 MOI) enhanced expression of N-cadherin and myosin VI, respectively, from 0.48+/- 0.01 to 0.78 +/- 0.01 (p=0.0003; n=5), and from 0.48 +/-0.4 to 1.04 +/-0.1 p=0.0027; n=5). Moreover, Adv-Sap97 (50 MOI) increased expression of HCN4 and CX43 proteins, respectively, from 1.05+/- 0.01 to 2.5 +/- 0.01 p= 0.0425; n=4) and from 0.43+/- 0.01 to 0.9 +/- 0.01 p=0.0001; n=5). In optically measured action potentials, Adv-Sap97 treated hiPSC-CM monolayers had slower spontaneous depolarization rate (1.1±0.22 Hz, n=7) than control (Adv-GFP, 1.68±0.12 Hz, n=6). Conclusions: Upregulation of MAGUK protein, Sap97, enhances expression of proteins that are important for structural and electrical maturation of hiPSC-CMs.

Comparative effects of parent and heated cinnamaldehyde on the function of human iPSC-derived cardiac myocytes

Many e-cigarette products contain cinnamaldehyde as a primary constituent of cinnamon flavorings. When used as a food additive, cinnamaldehyde is generally regarded as safe for ingestion. However, little is known about the effects of cinnamaldehyde or its degradation products, generated after heating and inhalation, which may lead to elevated circulatory exposure to the heart. Hence, in this study, we tested the in vitro cardiac toxicity of cinnamaldehyde and its thermal degradation products generated by heating at low (200 ± 50 °C) and high temperatures (700 ± 50 °C) on the contractility, rhythmicity and electrical signaling properties of human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs). Cellular impedance measurements on spontaneously beating hiPSC-CMs revealed that cinnamaldehyde significantly alters contraction-dependent signal amplitude, beating rate, and cell morphology. These effects were attenuated after cinnamaldehyde was subjected to heating at low or high temperatures. Current clamp analysis of hiPSC-CM action potentials (APs) showed only modest effects of acute application of 1–100 μM cinnamaldehyde on resting membrane potential, while prolonged (~20 min) application of 100 μM cinnamaldehyde resulted in progressive depolarization and loss of rhythmic AP spiking activity. Collectively, these results suggest that micromolar levels of cinnamaldehyde could alter cardiac excitability, in part by impairing the processes that regulate membrane potential and depolarization. Our results further suggest that heating cinnamaldehyde by itself does not directly lead to the formation of products with greater cardiotoxicity in vitro.

Abstract 151: Organized 3D Neo-Angiogenic and Neo-Lymphangiogenic Vascular Networks for Cardiac Regeneration

Introduction: Cardiovascular tissue engineering has been an area of intense investigation. The major challenge to these approaches has been the inability to vascularize and perfuse the in vitro engineered tissue constructs. Engineering a tissue of clinically relevant magnitude requires the formation of extensive and stable microvascular networks within the tissue. Hypothesis: Functional vascularized cardiac graft can be generated by the interaction of multipotent human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-eCMs) in a 3D collagen cell carrier (CCC) scaffold. Methods and Results: To achieve the above aim, we have developed an in vitro 3D functional vascularized cardiac muscle construct using hiPSC-eCMs and hMSCs. Initially, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured on CCCs for 7 days under vasculogenic culture conditions. hCMVECs/hMSCs underwent maturation and differentiation characteristic of microvessel morphogenesis and formed extensive plexuses of capillary networks. Next, the hiPSC-eCMs and hMSCs were co-cultured onto these generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. hiPSC-eCMS/hMSCs underwent maturation and differentiation, and demonstrated spontaneous rhythmic beating. The vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular and functional levels. Expression analyses of the differentiated cells revealed neo-angiogenesis and/or neo-lymphangiogenesis as well as neo-cardiomyogenesis. Conclusions: Our unique 3D co-culture system provides us the apt in vitro functional prevascularized 3D cardiac graft that can be utilized for myocardial repair and/or regeneration.

N-cadherin overexpression enhances the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes in infarcted mouse hearts.

In regenerative medicine, cellular cardiomyoplasty is one of the promising options for treating myocardial infarction (MI); however, the efficacy of such treatment has shown to be limited due to poor survival and/or functional integration of implanted cells. Within the heart, the adhesion between cardiac myocytes (CMs) is mediated by N-cadherin (CDH2) and is critical for the heart to function as an electromechanical syncytium. In this study, we have investigated whether the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) can be enhanced through CDH2 overexpression. CDH2-hiPSC-CMs and control wild-type (WT)-hiPSC-CMs were cultured in myogenic differentiation medium for 28 days. Using a mouse MI model, the cell survival/engraftment rate, infarct size, and cardiac functions were evaluated post-MI, at Day 7 or Day 28. In vitro, conduction velocities were significantly greater in CDH2-hiPSC-CMs than in WT-hiPSC-CMs. While, in vivo, measurements of cardiac functions: left ventricular (LV) ejection fraction, reduction in infarct size, and the cell engraftment rate were significantly higher in CDH2-hiPSC-CMs treated MI group than in WT-hiPSC-CMs treated MI group. Mechanistically, paracrine activation of ERK signal transduction pathway by CDH2-hiPSC-CMs, significantly induced neo-vasculogenesis, resulting in a higher survival of implanted cells. Collectively, these data suggest that CDH2 overexpression enhances not only the survival/engraftment of cultured CDH2-hiPSC-CMs, but also the functional integration of these cells, consequently, the augmentation of the reparative properties of implanted CDH2-hiPSC-CMs in the failing hearts.