Proliferation at the heart of preadolescence.

Cardiomyocytes, the cells of the heart muscle, lose nearly all of their proliferative capacity after birth, limiting the heart's ability to regenerate. Naqvi et al. now identify a transient burst of cardiomyocyte proliferation during preadolescence, driven by a thyroid hormone surge, with therapeutic implications for congenital and acquired heart diseases.

Inferring cell diversity in single cell data using consortium-scale epigenetic data as a biological anchor for cell identity.

Methods for cell clustering and gene expression from single-cell RNA sequencing (scRNA-seq) data are essential for biological interpretation of cell processes. Here, we present TRIAGE-Cluster which uses genome-wide epigenetic data from diverse bio-samples to identify genes demarcating cell diversity in scRNA-seq data. By integrating patterns of repressive chromatin deposited across diverse cell types with weighted density estimation, TRIAGE-Cluster determines cell type clusters in a 2D UMAP space. We then present TRIAGE-ParseR, a machine learning method which evaluates gene expression rank lists to define gene groups governing the identity and function of cell types. We demonstrate the utility of this two-step approach using atlases of in vivo and in vitro cell diversification and organogenesis. We also provide a web accessible dashboard for analysis and download of data and software. Collectively, genome-wide epigenetic repression provides a versatile strategy to define cell diversity and study gene regulation of scRNA-seq data.

Organization of gene programs revealed by unsupervised analysis of diverse gene-trait associations.

Genome wide association studies provide statistical measures of gene-trait associations that reveal how genetic variation influences phenotypes. This study develops an unsupervised dimensionality reduction method called UnTANGLeD (Unsupervised Trait Analysis of Networks from Gene Level Data) which organizes 16,849 genes into discrete gene programs by measuring the statistical association between genetic variants and 1,393 diverse complex traits. UnTANGLeD reveals 173 gene clusters enriched for protein-protein interactions and highly distinct biological processes governing development, signalling, disease, and homeostasis. We identify diverse gene networks with robust interactions but not associated with known biological processes. Analysis of independent disease traits shows that UnTANGLeD gene clusters are conserved across all complex traits, providing a simple and powerful framework to predict novel gene candidates and programs influencing orthogonal disease phenotypes. Collectively, this study demonstrates that gene programs co-ordinately orchestrating cell functions can be identified without reliance on prior knowledge, providing a method for use in functional annotation, hypothesis generation, machine learning and prediction algorithms, and the interpretation of diverse genomic data.

New Drug Targets and Preclinical Modelling Recommendations for Treating Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is the leading cause of morbidity and mortality worldwide and the primary underlying risk factor for heart failure. Despite decades of research and clinical trials, there are no drugs currently available to prevent organ damage from acute ischaemic injuries of the heart. In order to address the increasing global burden of heart failure, drug, gene, and cell-based regeneration technologies are advancing into clinical testing. In this review we highlight the burden of disease associated with AMI and the therapeutic landscape based on market analyses. New studies revealing the role of acid-sensitive cardiac ion channels and other proton-gated ion channels in cardiac ischaemia are providing renewed interest in pre- and post-conditioning agents with novel mechanisms of action that may also have implications for gene- and cell-based therapeutics. Furthermore, we present guidelines that couple new cell technologies and data resources with traditional animal modelling pipelines to help de-risk drug candidates aimed at treating AMI. We propose that improved preclinical pipelines and increased investment in drug target identification for AMI is critical to stem the increasing global health burden of heart failure.

Therapeutic Inhibition of Acid-Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

Single-cell RNA-seq of human induced pluripotent stem cells reveals cellular heterogeneity and cell state transitions between subpopulations.

Heterogeneity of cell states represented in pluripotent cultures has not been described at the transcriptional level. Since gene expression is highly heterogeneous between cells, single-cell RNA sequencing can be used to identify how individual pluripotent cells function. Here, we present results from the analysis of single-cell RNA sequencing data from 18,787 individual WTC-CRISPRi human induced pluripotent stem cells. We developed an unsupervised clustering method and, through this, identified four subpopulations distinguishable on the basis of their pluripotent state, including a core pluripotent population (48.3%), proliferative (47.8%), early primed for differentiation (2.8%), and late primed for differentiation (1.1%). For each subpopulation, we were able to identify the genes and pathways that define differences in pluripotent cell states. Our method identified four transcriptionally distinct predictor gene sets composed of 165 unique genes that denote the specific pluripotency states; using these sets, we developed a multigenic machine learning prediction method to accurately classify single cells into each of the subpopulations. Compared against a set of established pluripotency markers, our method increases prediction accuracy by 10%, specificity by 20%, and explains a substantially larger proportion of deviance (up to threefold) from the prediction model. Finally, we developed an innovative method to predict cells transitioning between subpopulations and support our conclusions with results from two orthogonal pseudotime trajectory methods.

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

Genetic Lineage Tracing of Sca-1+ Cells Reveals Endothelial but Not Myogenic Contribution to the Murine Heart.

BACKGROUND: The adult mammalian heart displays a cardiomyocyte turnover rate of ≈1%/y throughout postnatal life and after injuries such as myocardial infarction (MI), but the question of which cell types drive this low level of new cardiomyocyte formation remains contentious. Cardiac-resident stem cells marked by stem cell antigen-1 (Sca-1, gene name Ly6a) have been proposed as an important source of cardiomyocyte renewal. However, the in vivo contribution of endogenous Sca-1+ cells to the heart at baseline or after MI has not been investigated. METHODS: Here we generated Ly6a gene-targeted mice containing either a constitutive or an inducible Cre recombinase to perform genetic lineage tracing of Sca-1+ cells in vivo. RESULTS: We observed that the contribution of endogenous Sca-1+ cells to the cardiomyocyte population in the heart was <0.005% throughout all of cardiac development, with aging, or after MI. In contrast, Sca-1+ cells abundantly contributed to the cardiac vasculature in mice during physiological growth and in the post-MI heart during cardiac remodeling. Specifically, Sca-1 lineage-traced endothelial cells expanded postnatally in the mouse heart after birth and into adulthood. Moreover, pulse labeling of Sca-1+ cells with an inducible Ly6a-MerCreMer allele also revealed a preferential expansion of Sca-1 lineage-traced endothelial cells after MI injury in the mouse. CONCLUSIONS: Cardiac-resident Sca-1+ cells are not significant contributors to cardiomyocyte renewal in vivo. However, cardiac Sca-1+ cells represent a subset of vascular endothelial cells that expand postnatally with enhanced responsiveness to pathological stress in vivo.

Inhibition of ß-catenin signaling respecifies anterior-like endothelium into beating human cardiomyocytes.

During vertebrate development, mesodermal fate choices are regulated by interactions between morphogens such as activin/nodal, BMPs and Wnt/ß-catenin that define anterior-posterior patterning and specify downstream derivatives including cardiomyocyte, endothelial and hematopoietic cells. We used human embryonic stem cells to explore how these pathways control mesodermal fate choices in vitro. Varying doses of activin A and BMP4 to mimic cytokine gradient polarization in the anterior-posterior axis of the embryo led to differential activity of Wnt/ß-catenin signaling and specified distinct anterior-like (high activin/low BMP) and posterior-like (low activin/high BMP) mesodermal populations. Cardiogenic mesoderm was generated under conditions specifying anterior-like mesoderm, whereas blood-forming endothelium was generated from posterior-like mesoderm, and vessel-forming CD31(+) endothelial cells were generated from all mesoderm origins. Surprisingly, inhibition of ß-catenin signaling led to the highly efficient respecification of anterior-like endothelium into beating cardiomyocytes. Cardiac respecification was not observed in posterior-derived endothelial cells. Thus, activin/BMP gradients specify distinct mesodermal subpopulations that generate cell derivatives with unique angiogenic, hemogenic and cardiogenic properties that should be useful for understanding embryogenesis and developing therapeutics.

Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts.

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host­graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

The developmental origins and lineage contributions of endocardial endothelium.

Endocardial development involves a complex orchestration of cell fate decisions that coordinate with endoderm formation and other mesodermal cell lineages. Historically, investigations into the contribution of endocardium in the developing embryo was constrained to the heart where these cells give rise to the inner lining of the myocardium and are a major contributor to valve formation. In recent years, studies have continued to elucidate the complexities of endocardial fate commitment revealing a much broader scope of lineage potential from developing endocardium. These studies cover a wide range of species and model systems and show direct contribution or fate potential of endocardium giving rise to cardiac vasculature, blood, fibroblast, and cardiomyocyte lineages. This review focuses on the marked expansion of knowledge in the area of endocardial fate potential. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Transmembrane protein 88: a Wnt regulatory protein that specifies cardiomyocyte development.

Genetic regulation of the cell fate transition from lateral plate mesoderm to the specification of cardiomyocytes requires suppression of Wnt/ß-catenin signaling, but the mechanism for this is not well understood. By analyzing gene expression and chromatin dynamics during directed differentiation of human embryonic stem cells (hESCs), we identified a suppressor of Wnt/ß-catenin signaling, transmembrane protein 88 (TMEM88), as a potential regulator of cardiovascular progenitor cell (CVP) specification. During the transition from mesoderm to the CVP, TMEM88 has a chromatin signature of genes that mediate cell fate decisions, and its expression is highly upregulated in advance of key cardiac transcription factors in vitro and in vivo. In early zebrafish embryos, tmem88a is expressed broadly in the lateral plate mesoderm, including the bilateral heart fields. Short hairpin RNA targeting of TMEM88 during hESC cardiac differentiation increases Wnt/ß-catenin signaling, confirming its role as a suppressor of this pathway. TMEM88 knockdown has no effect on NKX2.5 or GATA4 expression, but 80% of genes most highly induced during CVP development have reduced expression, suggesting adoption of a new cell fate. In support of this, analysis of later stage cell differentiation showed that TMEM88 knockdown inhibits cardiomyocyte differentiation and promotes endothelial differentiation. Taken together, TMEM88 is crucial for heart development and acts downstream of GATA factors in the pre-cardiac mesoderm to specify lineage commitment of cardiomyocyte development through inhibition of Wnt/ß-catenin signaling.

High-content fluorescence bioassay investigates pore formation, ion channel modulation and cell membrane lysis induced by venoms.

Venoms comprise highly sophisticated bioactive molecules modulating ion channels, receptors, coagulation factors, and the cellular membranes. This array of targets and bioactivities requires advanced high-content bioassays to facilitate the development of novel envenomation treatments and biotechnological and pharmacological agents. In response to the existing gap in venom research, we developed a cutting-edge fluorescence-based high-throughput and high-content cellular assay. This assay enables the simultaneous identification of prevalent cellular activities induced by venoms such as membrane lysis, pore formation, and ion channel modulation. By integrating intracellular calcium with extracellular nucleic acid measurements, we have successfully distinguished these venom mechanisms within a single cellular assay. Our high-content bioassay was applied across three cell types exposed to venom components representing lytic, ion pore-forming or ion channel modulator toxins. Beyond unveiling distinct profiles for these action mechanisms, we found that the pore-forming latrotoxin α-Lt1a prefers human neuroblastoma to kidney cells and cardiomyocytes, while the lytic bee peptide melittin is not selective. Furthermore, evaluation of snake venoms showed that Elapid species induced rapid membrane lysis, while Viper species showed variable to no activity on neuroblastoma cells. These findings underscore the ability of our high-content bioassay to discriminate between clades and interspecific traits, aligning with clinical observations at venom level, beyond discriminating among ion pore-forming, membrane lysis and ion channel modulation. We hope our research will expedite the comprehension of venom biology and the diversity of toxins that elicit cytotoxic, cardiotoxic and neurotoxic effects, and assist in identifying venom components that hold the potential to benefit humankind.

Wnt dose escalation during the exit from pluripotency identifies tranilast as a regulator of cardiac mesoderm.

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.

HOPX-associated molecular programs control cardiomyocyte cell states underpinning cardiac structure and function.

Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function.

Cellular heterogeneity of pluripotent stem cell-derived cardiomyocyte grafts is mechanistically linked to treatable arrhythmias.

Preclinical data have confirmed that human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can remuscularize the injured or diseased heart, with several clinical trials now in planning or recruitment stages. However, because ventricular arrhythmias represent a complication following engraftment of intramyocardially injected PSC-CMs, it is necessary to provide treatment strategies to control or prevent engraftment arrhythmias (EAs). Here, we show in a porcine model of myocardial infarction and PSC-CM transplantation that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. Two unique surface marker signatures, signal regulatory protein α (SIRPA)+CD90-CD200+ and SIRPA+CD90-CD200-, identify arrhythmogenic and non-arrhythmogenic cardiomyocytes, respectively. Our data suggest that modifications to current PSC-CM-production and/or PSC-CM-selection protocols could potentially prevent EAs. We further show that pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias.