YAP induces a neonatal-like pro-renewal niche in the adult heart.

After myocardial infarction (MI), mammalian hearts do not regenerate, and the microenvironment is disrupted. Hippo signaling loss of function with activation of transcriptional co-factor YAP induces heart renewal and rebuilds the post-MI microenvironment. In this study, we investigated adult renewal-competent mouse hearts expressing an active version of YAP, called YAP5SA, in cardiomyocytes (CMs). Spatial transcriptomics and single-cell RNA sequencing revealed a conserved, renewal-competent CM cell state called adult (a)CM2 with high YAP activity. aCM2 co-localized with cardiac fibroblasts (CFs) expressing complement pathway component C3 and macrophages (MPs) expressing C3ar1 receptor to form a cellular triad in YAP5SA hearts and renewal-competent neonatal hearts. Although aCM2 was detected in adult mouse and human hearts, the cellular triad failed to co-localize in these non-renewing hearts. C3 and C3ar1 loss-of-function experiments indicated that C3a signaling between MPs and CFs was required to assemble the pro-renewal aCM2, C3+ CF and C3ar1+ MP cellular triad.

The Macrophage Landscape Across the Lifespan of a Human Cardiac Allograft.

BACKGROUND: Much of our knowledge of organ rejection after transplantation is derived from rodent models. METHODS: We used single-nucleus RNA sequencing to investigate the inflammatory myocardial microenvironment in human pediatric cardiac allografts at different stages after transplantation. We distinguished donor- from recipient-derived cells using naturally occurring genetic variants embedded in single-nucleus RNA sequencing data. RESULTS: Donor-derived tissue resident macrophages, which accompany the allograft into the recipient, are lost over time after transplantation. In contrast, monocyte-derived macrophages from the recipient populate the heart within days after transplantation and form 2 macrophage populations: recipient MP1 and recipient MP2. Recipient MP2s have cell signatures similar to donor-derived resident macrophages; however, they lack signatures of pro-reparative phagocytic activity typical of donor-derived resident macrophages and instead express profibrotic genes. In contrast, recipient MP1s express genes consistent with hallmarks of cellular rejection. Our data suggest that recipient MP1s activate a subset of natural killer cells, turning them into a cytotoxic cell population through feed-forward signaling between recipient MP1s and natural killer cells. CONCLUSIONS: Our findings reveal an imbalance of donor-derived and recipient-derived macrophages in the pediatric cardiac allograft that contributes to allograft failure.

Activated fibroblasts drive cellular interactions in end-stage pediatric hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a relatively rare but debilitating diagnosis in the pediatric population and patients with end-stage HCM require heart transplantation. In this study, we performed single-nucleus RNA sequencing on pediatric HCM and control myocardium. We identified distinct underling cellular processes in pediatric, end-stage HCM in cardiomyocytes, fibroblasts, endothelial cells, and myeloid cells, compared to controls. Pediatric HCM was enriched in cardiomyocytes exhibiting "stressed" myocardium gene signatures and underlying pathways associated with cardiac hypertrophy. Cardiac fibroblasts exhibited clear activation signatures and heightened downstream processes associated with fibrosis, more so than adult counterparts. There was notable depletion of tissue-resident macrophages, and increased vascular remodeling in endothelial cells. Our analysis provides the first single nuclei analysis focused on end-stage pediatric HCM.

scGIST: gene panel design for spatial transcriptomics with prioritized gene sets.

A critical challenge of single-cell spatial transcriptomics (sc-ST) technologies is their panel size. Being based on fluorescence in situ hybridization, they are typically limited to panels of about a thousand genes. This constrains researchers to build panels from only the marker genes of different cell types and forgo other genes of interest, e.g., genes encoding ligand-receptor complexes or those in specific pathways. We propose scGIST, a constrained feature selection tool that designs sc-ST panels prioritizing user-specified genes without compromising cell type detection accuracy. We demonstrate scGIST's efficacy in diverse use cases, highlighting it as a valuable addition to sc-ST's algorithmic toolbox.

Increased iron uptake by splenic hematopoietic stem cells promotes TET2-dependent erythroid regeneration.

Hematopoietic stem cells (HSCs) are capable of regenerating the blood system, but the instructive cues that direct HSCs to regenerate particular lineages lost to the injury remain elusive. Here, we show that iron is increasingly taken up by HSCs during anemia and induces erythroid gene expression and regeneration in a Tet2-dependent manner. Lineage tracing of HSCs reveals that HSCs respond to hemolytic anemia by increasing erythroid output. The number of HSCs in the spleen, but not bone marrow, increases upon anemia and these HSCs exhibit enhanced proliferation, erythroid differentiation, iron uptake, and TET2 protein expression. Increased iron in HSCs promotes DNA demethylation and expression of erythroid genes. Suppressing iron uptake or TET2 expression impairs erythroid genes expression and erythroid differentiation of HSCs; iron supplementation, however, augments these processes. These results establish that the physiological level of iron taken up by HSCs has an instructive role in promoting erythroid-biased differentiation of HSCs.

Endothelial cells adopt a pro-reparative immune responsive signature during cardiac injury.

Modulation of the heart's immune microenvironment is crucial for recovery after ischemic events such as myocardial infarction (MI). Endothelial cells (ECs) can have immune regulatory functions; however, interactions between ECs and the immune environment in the heart after MI remain poorly understood. We identified an EC-specific IFN responsive and immune regulatory gene signature in adult and pediatric heart failure (HF) tissues. Single-cell transcriptomic analysis of murine hearts subjected to MI uncovered an EC population (IFN-ECs) with immunologic gene signatures similar to those in human HF. IFN-ECs were enriched in regenerative-stage mouse hearts and expressed genes encoding immune responsive transcription factors (Irf7, Batf2, and Stat1). Single-cell chromatin accessibility studies revealed an enrichment of these TF motifs at IFN-EC signature genes. Expression of immune regulatory ligand genes by IFN-ECs suggests bidirectional signaling between IFN-ECs and macrophages in regenerative-stage hearts. Our data suggest that ECs may adopt immune regulatory signatures after cardiac injury to accompany the reparative response. The presence of these signatures in human HF and murine MI models suggests a potential role for EC-mediated immune regulation in responding to stress induced by acute injury in MI and chronic adverse remodeling in HF.

In vivo expansion of gene-targeted hepatocytes through transient inhibition of an essential gene.

Homology Directed Repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested "Repair Drive", a novel method for improving targeted gene insertion in the liver by selectively expanding correctly repaired hepatocytes in vivo. Our system consists of transient conditioning of the liver by knocking down an essential gene, and delivery of an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive dramatically increases the percentage of correctly targeted hepatocytes, up to 25%. This resulted in a five-fold increased expression of a therapeutic transgene. Repair Drive was well-tolerated and did not induce toxicity or tumorigenesis in long term follow up. This approach will broaden the range of liver diseases that can be treated with somatic genome editing.

Regrowing the heart, one TREE at a time.

While many animals can completely repair injured tissues, the mammalian heart possesses limited regenerative capabilities. Yan and Cigliola et al. show that AAV-mediated, zebrafish-derived tissue regeneration enhancer elements (TREEs) can direct pro-regenerative gene expression in injured cardiac tissue of mice and pigs that turn off following repair.

Yap and Taz promote osteogenesis and prevent chondrogenesis in neural crest cells in vitro and in vivo.

Neural crest cells (NCCs) are multipotent stem cells that can differentiate into multiple cell types, including the osteoblasts and chondrocytes, and constitute most of the craniofacial skeleton. Here, we show through in vitro and in vivo studies that the transcriptional regulators Yap and Taz have redundant functions as key determinants of the specification and differentiation of NCCs into osteoblasts or chondrocytes. Primary and cultured NCCs deficient in Yap and Taz switched from osteogenesis to chondrogenesis, and NCC-specific deficiency for Yap and Taz resulted in bone loss and ectopic cartilage in mice. Yap bound to the regulatory elements of key genes that govern osteogenesis and chondrogenesis in NCCs and directly regulated the expression of these genes, some of which also contained binding sites for the TCF/LEF transcription factors that interact with the Wnt effector ß-catenin. During differentiation of NCCs in vitro and NCC-derived osteogenesis in vivo, Yap and Taz promoted the expression of osteogenic genes such as Runx2 and Sp7 but repressed the expression of chondrogenic genes such as Sox9 and Col2a1. Furthermore, Yap and Taz interacted with ß-catenin in NCCs to coordinately promote osteoblast differentiation and repress chondrogenesis. Together, our data indicate that Yap and Taz promote osteogenesis in NCCs and prevent chondrogenesis, partly through interactions with the Wnt-ß-catenin pathway.

LATS1/2 control TGFB-directed epithelial-to-mesenchymal transition in the murine dorsal cranial neuroepithelium through YAP regulation.

Hippo signaling, an evolutionarily conserved kinase cascade involved in organ size control, plays key roles in various tissue developmental processes, but its role in craniofacial development remains poorly understood. Using the transgenic Wnt1-Cre2 driver, we inactivated the Hippo signaling components Lats1 and Lats2 in the cranial neuroepithelium of mouse embryos and found that the double conditional knockout (DCKO) of Lats1/2 resulted in neural tube and craniofacial defects. Lats1/2 DCKO mutant embryos had microcephaly with delayed and defective neural tube closure. Furthermore, neuroepithelial cell shape and architecture were disrupted within the cranial neural tube in Lats1/2 DCKO mutants. RNA sequencing of embryonic neural tubes revealed increased TGFB signaling in Lats1/2 DCKO mutants. Moreover, markers of epithelial-to-mesenchymal transition (EMT) were upregulated in the cranial neural tube. Inactivation of Hippo signaling downstream effectors, Yap and Taz, suppressed neuroepithelial defects, aberrant EMT and TGFB upregulation in Lats1/2 DCKO embryos, indicating that LATS1/2 function via YAP and TAZ. Our findings reveal important roles for Hippo signaling in modulating TGFB signaling during neural crest EMT.

Integrated multi-omic characterization of congenital heart disease.

The heart, the first organ to develop in the embryo, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of patients with CHD survive into adulthood, but many suffer premature death from heart failure and non-cardiac causes1. Here, to gain insight into this disease progression, we performed single-nucleus RNA sequencing on 157,273 nuclei from control hearts and hearts from patients with CHD, including those with hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot, two common forms of cyanotic CHD lesions, as well as dilated and hypertrophic cardiomyopathies. We observed CHD-specific cell states in cardiomyocytes, which showed evidence of insulin resistance and increased expression of genes associated with FOXO signalling and CRIM1. Cardiac fibroblasts in HLHS were enriched in a low-Hippo and high-YAP cell state characteristic of activated cardiac fibroblasts. Imaging mass cytometry uncovered a spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD, in agreement with the predilection in CHD to infection and cancer2. Our comprehensive phenotyping of CHD provides a roadmap towards future personalized treatments for CHD.

EPIXplorer: A web server for prediction, analysis and visualization of enhancer-promoter interactions.

Long distance enhancers can physically interact with promoters to regulate gene expression through formation of enhancer-promoter (E-P) interactions. Identification of E-P interactions is also important for profound understanding of normal developmental and disease-associated risk variants. Although the state-of-art predictive computation methods facilitate the identification of E-P interactions to a certain extent, currently there is no efficient method that can meet various requirements of usage. Here we developed EPIXplorer, a user-friendly web server for efficient prediction, analysis and visualization of E-P interactions. EPIXplorer integrates 9 robust predictive algorithms, supports multiple types of 3D contact data and multi-omics data as input. The output from EPIXplorer is scored, fully annotated by regulatory elements and risk single-nucleotide polymorphisms (SNPs). In addition, the Visualization and Downstream module provide further functional analysis, all the output files and high-quality images are available for download. Together, EPIXplorer provides a user-friendly interface to predict the E-P interactions in an acceptable time, as well as understand how the genome-wide association study (GWAS) variants influence disease pathology by altering DNA looping between enhancers and the target gene promoters. EPIXplorer is available at https://www.csuligroup.com/EPIXplorer.

Hippo signaling in cardiac fibroblasts during development, tissue repair, and fibrosis.

The evolutionarily conserved Hippo signaling pathway plays key roles in regulating the balance between cell proliferation and apoptosis, cell differentiation, organ size control, tissue repair, and regeneration. Recently, the Hippo pathway has been shown to regulate heart fibrosis, defined as excess extracellular matrix (ECM) deposition and increased tissue stiffness. Cardiac fibroblasts (CFs) are the primary cell type that produces, degrades, and remodels the ECM during homeostasis, aging, inflammation, and tissue repair and regeneration. Here, we review the available evidence from the current literature regarding how the Hippo pathway regulates the formation and function of CFs during heart development and tissue repair.

The cell-autonomous and non-cell-autonomous roles of the Hippo pathway in heart regeneration.

Cardiomyocytes are differentiated heart muscle cells with minimal self-renewal ability. Thus, loss of cardiomyocytes from cardiovascular disease and injury cannot be effectively replenished. Recent studies in animal models have indicated that induction of endogenous cardiomyocyte proliferation is essential for cardiac renewal and that inhibiting the Hippo signaling pathway can stimulate cardiomyocyte proliferation and heart regeneration. Increasing evidence has suggested that cardiomyocyte proliferation requires a permissive microenvironment that consists of multiple cell types. In this review, we summarize recent studies that highlight how the Hippo pathway regulates heart regeneration through cell-autonomous and non-cell-autonomous mechanisms. We also discuss recent translational studies in large animal models that demonstrate the therapeutic potential of targeting the Hippo pathway in the treatment of heart disease.

Decoding the PITX2-controlled genetic network in atrial fibrillation.

Atrial fibrillation (AF), the most common sustained cardiac arrhythmia and a major risk factor for stroke, often arises through ectopic electrical impulses derived from the pulmonary veins (PVs). Sequence variants in enhancers controlling expression of the transcription factor PITX2, which is expressed in the cardiomyocytes (CMs) of the PV and left atrium (LA), have been implicated in AF predisposition. Single nuclei multiomic profiling of RNA and analysis of chromatin accessibility combined with spectral clustering uncovered distinct PV- and LA-enriched CM cell states. Pitx2-mutant PV and LA CMs exhibited gene expression changes consistent with cardiac dysfunction through cell type-distinct, PITX2-directed, cis-regulatory grammars controlling target gene expression. The perturbed network targets in each CM were enriched in distinct human AF predisposition genes, suggesting combinatorial risk for AF genesis. Our data further reveal that PV and LA Pitx2-mutant CMs signal to endothelial and endocardial cells through BMP10 signaling with pathogenic potential. This work provides a multiomic framework for interrogating the basis of AF predisposition in the PVs of humans.

RNA splicing to cytoskeleton: A new path to cardiomyocyte ploidy and division?

Mammalian cardiomyocytes (CMs) undergo polyploidization after birth, accompanied by the loss of CM proliferation and regenerative capacity, although why this occurs is still poorly understood. In this issue of Developmental Cell, Gan et al. show that premature CM polyploidization, through defective RNA splicing, is detrimental to ventricular wall growth.