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Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. We investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP). Genome-wide associations studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that we identified were used to map potential regulatory non-coding regions, which in turn were employed to predict transcription factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart-specific knockdown. Additionally, we showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, we showed that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, we documented correlations between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 and EGR2 in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans.

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia in humans, present in > 33 million people worldwide. Although AF is often developed secondary to cardiovascular diseases, endocrine disorders, or lifestyle factors, recent GWAS studies have identified >200 genetic variants that substantially contribute to AF risk. However, it is currently not known how these genetic predispositions contribute to the initiation and/or maintenance of AF-associated phenotypes. In this context, one major barrier to progress is the lack of experimental systems enabling to rapidly explore the function of large cohort of genes on rhythm parameters in models with human atrial relevance. To address these modeling challenges, we have developed a new multi-model platform enabling 1) high-throughput characterization of the role of AF-associated genes on action potential duration and rhythm parameters at the cellular level, using human iPSC-derived atrial-like cardiomyocytes (ACMs), and at the whole organ level, using the Drosophila heart model, and 2) validation of the physiological relevance of our experimental results using computational models of heterogenous human adult atrial myocytes (HAMs) and tissue. As proof of concept, we screened a cohort of 20 AF-associated genes and identified Phospholamban (PLN) loss of function as a top conserved hit that significantly shortens action potential duration in ACMs, HAMs and fly cardiomyocytes. Remarkably, while PLN knock-down (KD) was not sufficient to induce arrhythmia phenotypes, addition of environmental stressors (i.e fibroblasts, β-adrenergic stimulation) to the model systems, led to the robust generation of irregular beat to beat intervals, delayed after depolarizations, and triggered action potentials, as compared to controls. Finally, to delineate the mechanism underlying PLN KD-dependent arrhythmia, we used a logistic regression approach in HAM populations, and predicted that PLN functionally interacts with both NCX (loss of function) and L-type calcium channels (gain of function) to mediate these arrhythmic phenotypes. Consistent with our predictions, co-KD of PLN and NCX in ACMs and flies, led to increased arrhythmic events, while treatment of ACMs with L-type calcium channel inhibitor, verapamil, reverted these phenotypes. In summary, these results collectively demonstrate that our integrated multi-model system approach was successful in identifying and characterizing conserved roles (i.e regulation of Ca2+ homeostasis) for AF-associated genes and phenotypes, and thus paves the way for the discovery and molecular delineation of new gene regulatory networks controlling atrial rhythm with application to AF.

Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD). The pathogenesis of HLHS is poorly understood and due to the likely oligogenic complexity of the disease, definitive HLHS-causing genes have not yet been identified. Postulating that impaired cardiomyocyte proliferation as a likely important contributing mechanism to HLHS pathogenesis, and we conducted a genome-wide siRNA screen to identify genes affecting proliferation of human iPSC-derived cardiomyocytes (hPSC-CMs). This yielded ribosomal protein (RP) genes as the most prominent class of effectors of CM proliferation. In parallel, whole genome sequencing and rare variant filtering of a cohort of 25 HLHS proband-parent trios with poor clinical outcome revealed enrichment of rare variants of RP genes. In addition, in another familial CHD case of HLHS proband we identified a rare, predicted-damaging promoter variant affectingRPS15Athat was shared between the proband and a distant relative with CHD. Functional testing with an integrated multi-model system approach reinforced the idea that RP genes are major regulators of cardiac growth and proliferation, thus potentially contributing to hypoplastic phenotype observed in HLHS patients. Cardiac knockdown (KD) of RP genes with promoter or coding variants (RPS15A, RPS17, RPL26L1, RPL39, RPS15) reduced proliferation in generic hPSC-CMs and caused malformed hearts, heart-loss or even lethality inDrosophila. In zebrafish, diminishedrps15afunction caused reduced CM numbers, heart looping defects, or weakened contractility, while reducedrps17orrpl39function caused reduced ventricular size or systolic atrial dysfunction, respectively. Importantly, genetic interactions betweenRPS15Aand core cardiac transcription factorsTBX5in CMs,Drosocross, pannierandtinmanin flies, andtbx5andnkx2-7(nkx2-5paralog) in fish, support a specific role for RP genes in heart development. Furthermore,RPS15AKD-induced heart/CM proliferation defects were significantly attenuated byp53KD in both hPSC-CMs and zebrafish, and by Hippo activation (YAP/yorkieoverexpression) in developing fly hearts. Based on these findings, we conclude that RP genes play critical roles in cardiogenesis and constitute an emerging novel class of gene candidates likely involved in HLHS and other CHDs.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We therefore performed whole genome sequencing (WGS) on a large cohort of HLHS patients and their families to identify candidate genes that were then tested in Drosophila heart model for functional and structural requirements. Bioinformatic analysis of WGS data from an index family comprised of a HLHS proband born to consanguineous parents and postulated to have a homozygous recessive disease etiology, prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of the candidate HLHS gene homologs tested, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. Interestingly, these heart defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex’s role in maintaining cristae morphology and ETC complex assembly. Analysis of 183 genomes of HLHS patient-parent trios revealed five additional HLHS probands with rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes in these cases for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (E3 ubiquitin ligase), or SPTBN1 (scaffolding protein) caused synergistic heart defects, suggesting the potential involvement of a diverse set of pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.

Cardiomyocytes undergo significant structural and functional changes after birth, and these fundamental processes are essential for the heart to pump blood to the growing body. However, due to the challenges of isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of the mature phenotype remains poorly understood. Here we implement large-particle sorting and analyze single myocytes from neonatal to adult hearts. Early myocytes exhibit wide-ranging transcriptomic and size heterogeneity that is maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion reveals that peroxisome proliferator-activated receptor coactivator-1 signaling, which is active in vivo but inactive in pluripotent stem cell-derived cardiomyocytes, mediates the shift. This signaling simultaneously regulates key aspects of cardiomyocyte maturation through previously unrecognized proteins, including YAP1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and identifies a multifaceted regulator controlling cardiomyocyte maturation., Cardiomyocyte maturation and the acquisition of phenotypes is poorly understood at the single cell level. Here, the authors analyse the transcriptome of single cells from neonatal to adult heart and reveal that peroxisome proliferator-activated receptor coactivator-1 mediates the phenotypic shift.

Congenital Heart Disease (CHD) is driven by a strong genetic predisposition, yet only a small subset of patients (∼20%) are diagnosed with a precise genetic cause. Therefore, expanding the pool of genes associated with CHD and establishing the functional relationships between genes can assemble a more comprehensive genetic network to better understand cardiac development and pathogenesis. In our studies, we identified protein biogenesis cofactors Nascent polypeptide Associated Complex (NAC) and Signal Recognition Particle (SRP) that bind disparate subsets of emerging nascent polypeptides at the ribosome exit site to direct polypeptide fates, as novel regulators of cell differentiation and cardiac morphogenesis. Knockdown (KD) of the alpha-(Nacα)or beta- subunit (bicaudal, bic)of NAC in the developingDrosophilaheart led to disruption of cardiac remodeling during pupal stages resulting in an adult fly with no heart. Heart loss was rescued by combined KD ofNacαwith theHoxgeneAbd-B.Consistent with a central role for this interaction in the regulation of cardiogenesis, KD ofNacαin Cardiac Progenitors derived from human iPSCs impaired cardiac differentiation while co-KD with mammalianHoxgenesHOXC12 and HOXD12rescued this phenotype. The effect ofNacαKD on the fly heart was temporally regulated, in that KD in embryo or in pupae caused only a partial loss of the heart, whereas KD during both stages led to heart loss similar to continuous KD throughout life. This suggests thatNacαKD already in the embryo may reprogram cells leading to aberrant cardiac remodeling during pupal stages. Lastly, KD of several SRP subunits individually in the fly heart produced a range of cardiac phenotypes that targeted specific segments and cell types, indicating spatially regulated activities of SRP components in the heart. Together, these data suggest that despite NAC and SRP ubiquitous presence, they displayed spatially and temporally fine-tuned activities for proper cardiac morphogenesis.Nacα’sinteraction with cardiac-specificHoxgene functions builds upon the novel role of this pathway and expands our understanding of the complex genetic networks involved in cardiac development and pathogenesis.

Our understanding of the fundamental cellular and molecular factors leading to atrial fibrillation (AF) remains stagnant despite significant advancement in ablation and device technologies. Diagnosis and prevention strategies fall behind that of treatment, but expanding knowledge in AF genetics holds the potential to drive progress. We aim to review how an understanding of the genetic contributions to AF can guide an approach to individualized risk stratification and novel avenues in drug discovery. Rare familial forms of AF identified monogenic contributions to the development of AF. Genome-wide association studies (GWAS) further identified single-nucleotide polymorphisms (SNPs) suggesting polygenic and multiplex nature of this common disease. Polygenic risk scores accounting for the multitude of associated SNPs that each confer mildly elevated risk have been developed to translate genetic information into clinical practice, though shortcomings remain. Additionally, novel laboratory methods have been empowered by recent genetic findings to enhance drug discovery efforts. AF is increasingly recognized as a disease with a significant genetic component. With expanding sequencing technologies and accessibility, polygenic risk scores can help identify high risk individuals. Advancement in digital health tools, artificial intelligence and machine learning based on standard electrocardiograms, and genomic driven drug discovery may be integrated to deliver a sophisticated level of precision medicine in this modern era of emphasis on prevention. Randomized, prospective studies to demonstrate clinical benefits of these available tools are needed to validate this approach.

The comprehensive characterization of mechanisms safeguarding cell fate identity in differentiated cells is crucial for 1) our understanding of how differentiation is maintained in healthy tissues or misregulated in disease states and 2) to improve our ability to use direct reprogramming for regenerative purposes. To uncover novel fate-stabilizing regulators, we employed a genome-wide TF siRNA screen followed by a high-complexity combinatorial evaluation of top performing hits, in a cardiac reprogramming assay in mouse embryonic fibroblasts, and subsequently validated our findings in cardiac, neuronal and iPSCs reprogramming assays in primary human fibroblasts and adult endothelial cells. This approach identified a conserved set of 4 TFs (ATF7IP, JUNB, SP7, and ZNF207 [AJSZ]) that robustly opposes cell fate reprogramming, as demonstrated by up to 6-fold increases in efficiency upon AJSZ knockdown in both lineage- and cell type-independent manners. Mechanistically, ChIP-seq and single-cell ATAC-seq analyses, revealed that AJSZ bind to both open and closed chromatin in a genome-wide and regionalized fashion, thereby limiting reprogramming TFs access to target DNA and ability to remodel the chromatin. In parallel, integration of ChIP-seq and RNA-seq data followed by systematic functional gene testing, identified that AJSZ also promote cell fate stability by proximally down-regulating a conserved set of genes involved in the regulation of cell fate specification (MEF2C), proteome remodeling (TPP1, PPIC), ATP homeostasis (EFHD1), and inflammation signaling (IL7R), thereby limiting cells ability to undergo large-scale phenotypic changes. Finally, simultaneous knock-down of AJSZ in combination with cardiac reprogramming TFs overexpression improved heart function by 250% as compared to no treatment and 50% as compared to MGT, 1 month after myocardial infarction. In sum, this study uncovers a novel evolutionarily conserved mechanism mediating cell fate stability in differentiated cells and also identifies AJSZ as promising therapeutic targets for regenerative purposes in adult organs.Significance StatementDifferentiated cells can be converted from one cell type into another by overexpressing lineage-determining transcription factors. Direct lineage reprogramming represents a promising strategy for regenerative medicine, but current clinical applications remain limited by the low yield of the reprogramming process. Here, we present the identification and detailed mechanistic study of a novel mechanisms opposing reprogramming process and promoting cell fate stability. Using a highthroughput screening technique, we identified four transcription factors that act as blockades to cell type change. By tracking chromatin and gene expression changes, we reconstructed a conserved pathway mediating cell fate stability in differentiated cells.

Our understanding of the fundamental cellular and molecular factors leading to atrial fibrillation (AF) remains stagnant despite significant advancement in ablation and device technologies. Diagnosis and prevention strategies fall behind that of treatment, but expanding knowledge in AF genetics holds the potential to drive progress. We aim to review how an understanding of the genetic contributions to AF can guide an approach to individualized risk stratification and novel avenues in drug discovery.Rare familial forms of AF identified monogenic contributions to the development of AF. Genome-wide association studies (GWAS) further identified single-nucleotide polymorphisms (SNPs) suggesting polygenic and multiplex nature of this common disease. Polygenic risk scores accounting for the multitude of associated SNPs that each confer mildly elevated risk have been developed to translate genetic information into clinical practice, though shortcomings remain. Additionally, novel laboratory methods have been empowered by recent genetic findings to enhance drug discovery efforts. AF is increasingly recognized as a disease with a significant genetic component. With expanding sequencing technologies and accessibility, polygenic risk scores can help identify high risk individuals. Advancement in digital health tools, artificial intelligence and machine learning based on standard electrocardiograms, and genomic driven drug discovery may be integrated to deliver a sophisticated level of precision medicine in this modern era of emphasis on prevention. Randomized, prospective studies to demonstrate clinical benefits of these available tools are needed to validate this approach.

Background Deciphering the genetic architecture of cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. Drosophila has gained importance as a useful model to study heart development and function and allows the analysis of organismal traits in a physiologically relevant and accessible system. Our aim was to (i) identify in flies the loci associated to natural variations of cardiac performances among a natural population, (ii) decipher how these variants interact with each other and with the environment to impact cardiac traits, (iii) gain insights about the molecular and cellular processes affected, (iv) determine whether the genetic architecture of cardiac disorders is conserved with humans. Methods and Results We investigated the genetic architecture of natural variations of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP). Genome Wide Associations (GWA) for single markers and epistatic interactions identified genetic networks associated with natural variations of cardiac traits that were extensively validated in vivo. Non-coding variants were used to map potential regulatory non-coding regions which in turn were employed to predict Transcription Factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were validated by heart specific knockdown. We also analyzed natural variations of cardiac traits variance that revealed unique features of their micro-environmental plasticity. More importantly, correlations between genes associated with cardiac phenotypes both in flies and in humans support the conserved genetic architecture of cardiac functioning from arthropods to mammals. The characteristics of natural variations in cardiac function established in Drosophila may thus guide the analysis of cardiac disorders in humans. Using human iPSC-derived cardiomyocytes, we indeed characterized a conserved function for PAX9 and EGR2 in the regulation of the cardiac rhythm Conclusion In-depth analysis of the genetic architecture of natural variations of cardiac performance in flies combined with functional validations in vivo and in human iPSC-CM represents a major achievement in understanding the mechanisms underlying the genetic architecture of these complex traits and a valuable resource for the identification of genes and mechanisms involved in cardiac disorders in humans.

Mechanisms of myogenic commitment are deciphered through omics analysis of hiPSC-specific lineage specification measurements., Understanding the mechanisms of myogenesis in human induced pluripotent stem cells (hiPSCs) is a prerequisite to achieving patient-specific therapy for diseases of skeletal muscle. hiPSCs of different origin show distinctive kinetics and ability to differentiate into myocytes. To address the unique cellular and temporal context of hiPSC differentiation, we perform a longitudinal comparison of the transcriptomic profiles of three hiPSC lines that display differential myogenic specification, one robust and two blunted. We detail temporal differences in mechanisms that lead to robust myogenic specification. We show gene expression signatures of putative cell subpopulations and extracellular matrix components that may support myogenesis. Furthermore, we show that targeted knockdown of ZIC3 at the outset of differentiation leads to improved myogenic specification in blunted hiPSC lines. Our study suggests that β-catenin transcriptional cofactors mediate cross-talk between multiple cellular processes and exogenous cues to facilitate specification of hiPSCs to mesoderm lineage, leading to robust myogenesis.

Congenital heart diseases (CHDs), including hypoplastic left heart syndrome (HLHS), are genetically complex and poorly understood. Here, a multidisciplinary platform was established to functionally evaluate novel CHD gene candidates, based on whole-genome and iPSC RNA sequencing of a HLHS family-trio. Filtering for rare variants and altered expression in proband iPSCs prioritized 10 candidates. siRNA/RNAi-mediated knockdown in healthy human iPSC-derived cardiomyocytes (hiPSC-CM) and in developingDrosophilaand zebrafish hearts revealed that LDL receptor-related proteinLRP2is required for cardiomyocyte proliferation and differentiation. Consistent with hypoplastic heart defects, compared to parents the proband’s iPSC-CMs exhibited reduced proliferation. Interestingly, rare, predicted-damaging LRP2 variants were enriched in a HLHS cohort; however, understanding their contribution to HLHS requires further investigation. Collectively, we have established a multi-species high-throughput platform to rapidly evaluate candidate genes and their interactions during heart development, which are crucial first steps toward deciphering oligogenic underpinnings of CHDs, including hypoplastic left hearts.

Dietary emulsifiers are widely used in industrially processed foods, although the effects of these food additives on human gut microbiota are not well studied. Here, we investigated the effects of five different emulsifiers [glycerol monoacetate, glycerol monostearate, glycerol monooleate, propylene glycol monostearate, and sodium stearoyl lactylate (SSL)] on fecal microbiota in vitro. We found that 0.025% (w/v) of SSL reduced the relative abundance of the bacterial class Clostridia and others. The relative abundance of the families Clostridiaceae, Lachnospiraceae, and Ruminococcaceae was substantially reduced whereas that of Bacteroidaceae and Enterobacteriaceae was increased. Given the marked impact of SSL on Clostridia, we used genome reconstruction to predict community-wide production of short-chain fatty acids, which were experimentally assessed by GC-MS analysis. SSL significantly reduced concentrations of butyrate, and increased concentrations of propionate compared to control cultures. The presence of SSL increased lipopolysaccharide, LPS and flagellin in cultured communities, thereby enhancing the proinflammatory potential of SSL-selected bacterial communities.

ObjectivesPluripotent stem cell-derived cardiomyocytes are phenotypically immature, which limits their utility in downstream applications. Metabolism is dramatically reprogramed during cardiac maturationin vivoand presents a potential avenue to drivein vitromaturation. We aimed to identify and address metabolic bottlenecks in the generation of human pluripotent stem cell (hPSC)-derived cardiomyocytes.MethodshPSCs were differentiated into cardiomyocytes using an established, chemically-defined differentiation protocol. We applied 13C metabolic flux analysis (MFA) and targeted transcriptomics to characterize cardiomyocyte metabolism in during differentiation in the presence or absence of exogenous lipids.ResultshPSC-derived cardiomyocytes induced some cardiometabolic pathways (i.e. ketone body and branched-chain amino acid oxidation) but failed to effectively activate fatty acid oxidation. MFA studies indicated that lipid availability in cultures became limited during differentiation, suggesting potential issues with nutrient availability. Exogenous supplementation of lipids improved cardiomyocyte morphology, mitochondrial function, and promoted increased fatty acid oxidation in hPSC-derivatives.ConclusionhPSC-derived cardiomyocytes are dependent upon exogenous sources of lipids for metabolic maturation. Proper supplementation removes a potential roadblock in the generation of metabolically mature cardiomyocytes. These studies further highlight the importance of considering and exploiting metabolic phenotypes in thein vitroproduction and utilization of functional hPSC-derivatives.

The identification of genetic variants that predispose individuals to cardiovascular disease and a better understanding of their targets would be highly advantageous. Genome-wide association studies have identified variants that associate with QT-interval length (a measure of myocardial repolarization). Three of the strongest associating variants (single-nucleotide polymorphisms) are located in the putative promotor region of CNOT1, a gene encoding the central CNOT1 subunit of CCR4-NOT: a multifunctional, conserved complex regulating gene expression and mRNA stability and turnover. We isolated the minimum fragment of the CNOT1 promoter containing all three variants from individuals homozygous for the QT risk alleles and demonstrated that the haplotype associating with longer QT interval caused reduced reporter expression in a cardiac cell line, suggesting that reduced CNOT1 expression might contribute to abnormal QT intervals. Systematic siRNA-mediated knockdown of CCR4-NOT components in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) revealed that silencing CNOT1 and other CCR4-NOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, the cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes in vivo (CNOT1/Not1 and CNOT7/8/Pop2) was either lethal or resulted in dilated cardiomyopathy, reduced contractility or a propensity for arrhythmia. Silencing CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin also affected cardiac chamber size and contractility. Developmental studies suggested that CNOT1/Not1 and CNOT7/8/Pop2 are required during cardiac remodeling from larval to adult stages. To summarize, we have demonstrated how disease-associated genes identified by GWAS can be investigated by combining human cardiomyocyte cell-based and whole-organism in vivo heart models. Our results also suggest a potential link of CNOT1 and CNOT7/8 to QT alterations and further establish a crucial role of the CCR4-NOT complex in heart development and function. This article has an associated First Person interview with the first author of the paper., Summary: Genome-wide association studies combined with in vitro human cardiac cell assays and a model organism suitable for heart studies in vivo connect CNOT1, CNOT7 and overall the CCR4-NOT complex to human heart disease and morbidity.

Cardiomyocytes undergo significant levels of structural and functional changes after birth—fundamental processes essential for the heart to produce the volume and contractility to pump blood to the growing body. However, due to the challenges in isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of mature phenotypes remains poorly understood. Here we implemented large-particle sorting and analyzed single myocytes from neonatal to adult hearts. Early myocytes exhibited a wide-ranging transcriptomic and size heterogeneity, maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion revealed that peroxisome proliferator-activated receptor coactivator-1 signaling—activated in vivo but inactive in pluripotent stem cell-derived cardiomyocytes—mediates the shift. The signaling regulated key aspects of cardiomyocyte maturation simultaneously through previously unrecognized regulators, including Yap1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and unveils a multifaceted regulator controlling cardiomyocyte maturation.Significance StatementHow the individual single myocytes achieve full maturity remains a ‘black box’, largely due to the challenges with the isolation of single mature myocytes. Understanding this process is particularly important as the immaturity and early developmental arrest of pluripotent stem cell-derived myocytes has emerged a major concern in the field. Here we present the first study of high-quality single-cell transcriptomic analysis of cardiac muscle cells from neonatal to adult hearts. We identify a central transcription factor and its novel targets that control key aspects of myocyte maturation, including cellular hypertrophy, contractility, and mitochondrial activity.

Aims We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. Methods and results The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P

Congenital heart diseases (CHD), such as hypoplastic left heart syndrome (HLHS), are considered to have complex genetic underpinnings that are poorly understood. Here, an integrated multi-disciplinary approach was applied to identify novel genes and underlying mechanisms associated with HLHS. A family-based strategy was employed that coupled whole genome sequencing (WGS) with RNA sequencing of patient-derived induced pluripotent stem cells (iPSCs) from a sporadic HLHS proband-parent trio to identify, prioritize and functionally evaluate candidate genes in model systems. Consistent with the hypoplastic phenotype, the proband’s iPSCs had reduced proliferation capacity. Filtering WGS for rare de novo, recessive, and loss-of-function variants revealed 10 candidate genes with recessive variants and altered expression compared to the parents’ iPSCs. siRNA/RNAi-mediated knockdown in generic human iPSC-derived cardiac progenitors and in thein vivo Drosophilaheart model revealed that LDL receptor related proteinLRP2and apolipoproteinAPOBare required for robust hiPSC-derived cardiomyocyte proliferation and normal hear structure and function, possibly involving an oligogenic mechanism via growth-promoting WNT and SHH signaling.LRP2was further validated as a CHD gene in a zebrafish heart model and rare variant burden testing in an HLHS cohort. Collectively, this cross-functional genetic approach to complex congenital heart disease revealed LRP2 dysfunction as a likely novel genetic driver of HLHS, and hereby established a scalable approach to decipher the oligogenic underpinnings of maladaptive left heart development.One sentence summaryWhole genome sequencing and a multi-model system candidate gene validation - human iPSC-derived cardiomyocytes andDrosophilaand zebrafish hearts - identified lipoproteinLRP2as a new potential driver in congenital heart disease and suggests a deficit in proliferation as a hallmark of hypoplastic left heart syndrome.

Introduction Deciphering the genetic architecture of cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. Drosophila has gained importance as a useful model to study heart development and function and allows the analysis of organismal traits in a physiologically relevant and accessible system. Objectives Our aim was to (i) identify in flies the variants associated to natural variations of cardiac performances among a natural population, (ii) decipher how these variants interact with each other and with the environment to impact cardiac traits, (iii) gain insights about the molecular and cellular processes affected, (iv) determine whether the genetic architecture of cardiac disorders is conserved with humans. Methods We used the Drosophila Genetic Reference Panel, a community resource of sequenced inbred lines. Genome Wide Associations (GWA) for single markers and epistatic interactions were analyzed on cardiac traits related to rhythm and contractility. Results Genetic networks were unraveled and extensively validated in vivo. Non-coding variants were used to map potential regulatory non-coding regions and to predict Transcription Factors (TFs) binding sites. Cognate TFs were validated by heart specific knockdown. Natural variations of cardiac traits variance revealed unique features of the phenotypic plasticity of cardiac performance. Importantly, correlations between genes associated to GWAS for cardiac traits both in flies and humans supported conservation of the genetic architecture of cardiac disorders from arthropods to mammals. In addition, several conserved genes and pathways were validated in human iPSC-derived cardiomyocytes. Conclusion We provide an in-depth analysis of the genetic architecture of natural variations of cardiac performance in flies. Our data may guide the analysis of cardiac disorders in humans.

Summary Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through β-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.

Bipotent neuromesodermal progenitors (NMPs) residing in the caudal epiblast drive coordinated body axis extension by generating both posterior neuroectoderm and presomitic mesoderm. Retinoic acid (RA) is required for body axis extension, however the early molecular response to RA signaling is poorly defined, as is its relationship to NMP biology. As endogenous RA is first seen near the time when NMPs appear, we used WNT/FGF agonists to differentiate embryonic stem cells to NMPs which were then treated with a short 2-h pulse of 25 nM RA or 1 µM RA followed by RNA-seq transcriptome analysis. Differential expression analysis of this dataset indicated that treatment with 25 nM RA, but not 1 µM RA, provided physiologically relevant findings. The 25 nM RA dataset yielded a cohort of previously known caudal RA target genes including Fgf8 (repressed) and Sox2 (activated), plus novel early RA signaling targets with nearby conserved RA response elements. Importantly, validation of top-ranked genes in vivo using RA-deficient Raldh2−/− embryos identified novel examples of RA activation (Nkx1-2, Zfp503, Zfp703, Gbx2, Fgf15, Nt5e) or RA repression (Id1) of genes expressed in the NMP niche or progeny. These findings provide evidence for early instructive and permissive roles of RA in controlling differentiation of NMPs to neural and mesodermal lineages., Summary: The findings here demonstrate that the signaling molecule retinoic acid (RA) plays an early role in determining how embryonic progenitor cells decide to differentiate into either mesodermal or neural tissues.

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70–90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

The ability to produce unlimited numbers of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) harboring disease and patient-specific gene variants creates a new paradigm for modeling congenital heart diseases (CHDs) and predicting proarrhythmic liabilities of drug candidates. However, a major roadblock to implementing hiPSC-CM technology in drug discovery is that conventional methods for monitoring action potential (AP) kinetics and arrhythmia phenotypes in vitro have been too costly or technically challenging to execute in high throughput. Herein, we describe the first large-scale, fully automated and statistically robust analysis of AP kinetics and drug-induced proarrhythmia in hiPSC-CMs. The platform combines the optical recording of a small molecule fluorescent voltage sensing probe (VoltageFluor2.1.Cl), an automated high throughput microscope and automated image analysis to rapidly generate physiological measurements of cardiomyocytes (CMs). The technique can be readily adapted on any high content imager to study hiPSC-CM physiology and predict the proarrhythmic effects of drug candidates.

International audience; Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix–loop–helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation—Tcf3 and Foxa2—and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1–4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

Bipotent axial stem cells residing in the caudal epiblast during late gastrulation generate neuroectodermal and presomitic mesodermal progeny that coordinate somitogenesis with neural tube formation, but the mechanism that controls these two fates is not fully understood. Retinoic acid (RA) restricts the anterior extent of caudal fibroblast growth factor 8 (Fgf8) expression in both mesoderm and neural plate to control somitogenesis and neurogenesis, however it remains unclear where RA acts to control the spatial expression of caudal Fgf8. Here, we found that mouse Raldh2-/- embryos, lacking RA synthesis and displaying a consistent small somite defect, exhibited abnormal expression of key markers of axial stem cell progeny, with decreased Sox2+ and Sox1+ neuroectodermal progeny and increased Tbx6+ presomitic mesodermal progeny. The Raldh2-/- small somite defect was rescued by treatment with an FGF receptor antagonist. Rdh10 mutants, with a less severe RA synthesis defect, were found to exhibit a small somite defect and anterior expansion of caudal Fgf8 expression only for somites 1-6, with normal somite size and Fgf8 expression thereafter. Rdh10 mutants were found to lack RA activity during the early phase when somites are small, but at the 6-somite stage RA activity was detected in neural plate although not in presomitic mesoderm. Expression of a dominant-negative RA receptor in mesoderm eliminated RA activity in presomitic mesoderm but did not affect somitogenesis. Thus, RA activity in the neural plate is sufficient to prevent anterior expansion of caudal Fgf8 expression associated with a small somite defect. Our studies provide evidence that RA restriction of Fgf8 expression in undifferentiated neural progenitors stimulates neurogenesis while also restricting the anterior extent of the mesodermal Fgf8 mRNA gradient that controls somite size, providing new insight into the mechanism that coordinates somitogenesis with neurogenesis.

Tight control over the segregation of endoderm, mesoderm, and ectoderm is essential for normal embryonic development of all species, yet how neighboring embryonic blastomeres can contribute to different germ layers has never been fully explained. We postulated that microRNAs, which fine-tune many biological processes, might modulate the response of embryonic blastomeres to growth factors and other signals that govern germ layer fate. A systematic screen of a whole-genome microRNA library revealed that the let-7 and miR-18 families increase mesoderm at the expense of endoderm in mouse embryonic stem cells. Both families are expressed in ectoderm and mesoderm, but not endoderm, as these tissues become distinct during mouse and frog embryogenesis. Blocking let-7 function in vivo dramatically affected cell fate, diverting presumptive mesoderm and ectoderm into endoderm. siRNA knockdown of computationally predicted targets followed by mutational analyses revealed that let-7 and miR-18 down-regulate Acvr1b and Smad2, respectively, to attenuate Nodal responsiveness and bias blastomeres to ectoderm and mesoderm fates. These findings suggest a crucial role for the let-7 and miR-18 families in germ layer specification and reveal a remarkable conservation of function from amphibians to mammals.

Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon–exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS.