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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.

Background: The intestine occupies the critical interface between cholesterol absorption and excretion. Surprisingly little is known about the role of de novo cholesterol synthesis in this organ, and its relationship to whole body cholesterol homeostasis. Here, we investigate the physiological importance of this pathway through genetic deletion of the rate-limiting enzyme. Methods: Mice lacking 3-hydroxy-3-methylglutaryl-coenzyme A reductase ( Hmgcr ) in intestinal villus and crypt epithelial cells were generated using a Villin -Cre transgene. Plasma lipids, intestinal morphology, mevalonate pathway metabolites, and gene expression were analyzed. Results: Mice with intestine-specific loss of Hmgcr were markedly smaller at birth, but gain weight at a rate similar to wild-type littermates, and are viable and fertile into adulthood. Intestine lengths and weights were greater relative to body weight in both male and female Hmgcr intestinal knockout mice. Male intestinal knockout had decreased plasma cholesterol levels, whereas fasting triglycerides were lower in both sexes. Lipidomics revealed substantial reductions in numerous nonsterol isoprenoids and sterol intermediates within the epithelial layer, but cholesterol levels were preserved. Hmgcr intestinal knockout mice also showed robust activation of SREBP-2 (sterol-regulatory element binding protein-2) target genes in the epithelium, including the LDLR (low-density lipoprotein receptor). At the cellular level, loss of Hmgcr is compensated for quickly after birth through a dramatic expansion of the stem cell compartment, which persists into adulthood. Conclusions: Loss of Hmgcr in the intestine is compatible with life through compensatory increases in intestinal absorptive surface area, LDLR expression, and expansion of the resident stem cell compartment.

PDF file - 1574K, YAP1 and SOX9 expression in EC cell lines.

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PDF file - 1015K, YAP1 Induces SOX9 promoter activity in 293T cells and primer design for SOX9 ChIP-PCR.

PDF file - 127K, SOX9 is elevated in mouse liver tumors with inactivating Hippo pathway mutations.

Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies., Combined methylmalonic acidemia (MMA) and hyperhomocysteinemias are inborn errors of vitamin B12 metabolism, and mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) underlie some forms of these disorders. Here the authors generated mouse models of a human syndrome due to mutations in RONIN (THAP11) and HCFC1, and show that this syndrome is both an inborn error of vitamin B12 metabolism and displays some features of ribosomopathy.

The cardiac conduction system is a network of heterogeneous cell population that initiates and propagates electric excitations in the myocardium. Purkinje fibers, a network of specialized myocardial cells, comprise the distal end of the conduction system in the ventricles. The developmental origins of Purkinje fibers and their roles during cardiac physiology and arrhythmia have been reported. However, it is not clear if they play a role during ischemic injury and heart regeneration. Here we introduce a novel tamoxifen-inducible Cre allele that specifically labels a broad range of components in the cardiac conduction system while excludes other cardiac cell types and vital organs. Using this new allele, we investigated the cellular and molecular response of Purkinje fibers to myocardial injury. In a neonatal mouse myocardial infarction model, we observed significant increase in Purkinje cell number in regenerating myocardium. RNA-Seq analysis using laser-captured Purkinje fibers showed a unique transcriptomic response to myocardial infarction. Our finds suggest a novel role of cardiac Purkinje fibers in heart injury.

Rationale: During neonatal heart regeneration, the fibrotic response, which is required to prevent cardiac rupture, resolves via poorly understood mechanisms. Deletion of the Hippo pathway gene Sav in adult cardiomyocytes increases Yap activity and promotes cardiac regeneration, partly by inducing fibrosis resolution. Deletion of Yap in neonatal cardiomyocytes leads to increased fibrosis and loss of neonatal heart regeneration, suggesting that Yap inhibits fibrosis by regulating intercellular signaling from cardiomyocytes to cardiac fibroblasts (CFs). Objective: We investigated the role of Wntless ( Wls ), which is a direct target gene of Yap, in communication between cardiomyocytes and CFs during neonatal heart regeneration. Methods and Results: We generated 2 mouse models to delete Wls specifically in cardiomyocytes ( Myh6-Cas9 combined with AAV9-Wls-g RNAs, and Myh6 cre-ERT2/+ ; Wls flox/flox ). Reanalysis of single-cell RNA sequencing data revealed that Wnt ligands are expressed in cardiomyocytes, whereas Wnt receptors are expressed in CFs, suggesting that Wnt signaling is directional from cardiomyocytes to CFs during neonatal heart regeneration. Wls deletion in neonatal cardiomyocytes disrupted Wnt signaling, revealed by reduced noncanonical Wnt signaling in non-cardiomyocytes. Four weeks after neonatal heart myocardial infarction, heart function was measured by echocardiography. Wls deletion in neonatal cardiomyocytes after myocardial infarction impairs neonatal heart regeneration, marked by decreased contractile function and increased fibrosis. Wls mutant hearts display CF activation, characterized by increased extracellular matrix secretion, inflammation, and CF proliferation. Conclusions: These data indicate that during neonatal heart regeneration, intercellular signaling from cardiomyocytes to CFs occurs via noncanonical Wnt signaling to rebuild cardiac architecture after myocardial infarction.

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 β-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 β-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–β-catenin pathway.

RationaleThe most significantly associated atrial fibrillation (AF) risk loci in humans map to a noncoding gene desert upstream of the evolutionarily conserved left-right (LR) transcription factor Pitx2, a master regulator of LR asymmetric organ development. Pitx2 dosage is fundamentally linked to the development of sinus node dysfunction (SND) and AF, the most common cardiac arrhythmia affecting adults, but the mechanistic basis for this remains obscure. We identified a conserved long noncoding RNA (lncRNA), Playrr, which is exclusively transcribed on the embryo’s right side, opposite to Pitx2 on the left, that participates in mutually antagonistic transcriptional regulation with Pitx2.ObjectiveThe objective of this study was to investigate a role of Playrr in regulating Pitx2 transcription and protecting against the development of cardiac rhythm disturbances.Methods and ResultsPlayrr expression in the developing heart was analyzed with RNA in situ hybridization. Playrr was expressed asymmetrically (on the right) to Pitx2 (on the left) in developing mouse embryos, including in mouse embryonic sinoatrial node cells. We utilized CRISPR/Cas9 genome editing in mice to target Playrr, generating mice lacking Playrr RNA transcript (PlayrrEx1sj allele). Using qRT-PCR we detected upregulation of the cardiac isoform, Pitx2c, during visceral organ morphogenesis in PlayrrEx1sj mutant embryos. Surface ECG (AliveCor®) and 24-hour telemetry ECG detected bradycardia and irregular interbeat (R-R) intervals suggestive of SND in PlayrrEx1sj mutant adults. Programmed stimulation of PlayrrEx1sj mutant adults resulted in pacing-induced AF. Within the right atrium of PlayrrEx1sj mutant hearts, Masson’s trichrome stain revealed increased collagen deposition indicative of fibrosis, and immunofluorescence demonstrated mis-localization of Connexin 43 in atrial cardiomyocytes. These findings suggested an altered atrial substrate in PlayrrEx1sj adult mice. Finally, transcriptomic analysis by chromatin run-on and sequencing (ChRO-seq) in atria of PlayrrEx1sj mutant mice compared to wild type controls revealed differential expression of genes involved in cell-cell adhesion and motility, fibrosis, and dysregulation of the key cardiac genes Tbx5 and Hcn1.ConclusionsAdult mice lacking functional Playrr lncRNA transcript have baseline bradyarrhythmia and increased susceptibility to AF. These cardiac phenotypes are similar to those observed in Pitx2 heterozygous mice. Interactions between Pitx2 and Playrr may provide a genetic mechanism for modulating Pitx2 dosage and susceptibility to SND and AF.

Cardiovascular diseases and cancer are the leading causes of death worldwide. Recurrent mutations of metabolic enzymes in cancer cells provide benefits for tumor growth, while creating a systemic metabolic phenotype and increasing the risk for adverse cardiac events. We recently showed that tumors producing the oncometabolite D-2-hydroxyglutarate increase ATP citrate lyase activity and histone acetylation in the heart. How cancer cells drive metabolic remodeling in the heart, and whether ATP citrate lyase is critical for cardiac adaptation during cancer, remains poorly understood. We co-cultured wildtype adult mouse ventricular cardiomyocytes with colorectal cancer cells overexpressing ATP citrate lyase. Co-culture of cardiomyocytes with colorectal cancer cells was associated with reduced expression of ATP citrate lyase, pyruvate dehydrogenase, and mitochondrial complex I, suggesting that the cancer cell environment impaired mitochondrial function in vitro. In vitro modulation of ATP citrate lyase activity abrogated the metabolic alterations in cultured cardiomyocytes indicating that ATP citrate lyase drives remodeling during cancer. Accordingly, we created mice lacking ATP citrate lyase in the heart using CRISPR-Cas 9 genomic knockout. Loss of ATP citrate lyase leads to left ventricular systolic dysfunction and causes global gene and protein expression changes in the heart. Targeted mass spectrometry-based metabolomics and lipidomics showed decreased acetyl-CoA and succinyl-CoA levels in isolated cardiomyocytes, while the abundance of cholesterol esters increased. In vivo [ 18 F]-FDG-positron emission tomography revealed that animals lacking ATP citrate lyase increased plasma glucose uptake compared to animals with cardiac ATP citrate lyase expression. These findings are consistent with maladaptive metabolic remodeling and impaired oxidative metabolism during heart failure. Manipulating the activity of ATP citrate lyase using BMS303141 (0.5 μM) in ex vivo perfused wildtype rat hearts caused a rapid decline in cardiac power and increased utilization of glucose confirming our in vivo mouse studies. Conversely, inhibition of ATP citrate lyase during oncometabolic stress via D-2-hydroxyglutarate improved cardiac contractile function via redirection of Krebs cycle flux and provision of ATP levels. Together these data demonstrate that ATP citrate lyase regulates oxidative metabolism and structural remodeling in the heart and creates a targetable metabolic liability during cancer.

Background: The evolutionary conserved Hippo signaling pathway (HSP) is an inhibitor of tissue regeneration, including the Lats1/2 kinases that phosphorylate and inactivate YAP and TAZ. Emerging data indicate that HSP regulates innate immunity, inflammation, and autoimmune diseases. However, the role of HSP in post-MI inflammation is poorly understood. Methods: Single-cell RNA-sequencing (scRNA-Seq) and spatial transcriptomics were utilized in cardiac fibroblast (CF)-specific-Lats1/2 knockout (Lats CKO) mouse heart. Flow cytometry, immunofluorescence (IF), and echocardiogram (Echo) were used to describe the phenotype in ventricle of Lats CKO mouse heart with/out Ccr2 knockout. Results: We integrated our scRNA-Seq data in Lats CKO mouse heart with three published data sets of MI mouse heart. A CF subcluster with elevated YAP activity and expressing chemokines Ccl2 and Ccl7 (Ccl2/Ccl7+ CFs) and macrophages (Macs) expressing Ccr2, the receptor for Ccl2/Ccl7 (Ccr2+ Macs), were identified, which suggests a signaling transduction from Ccl2/Ccl7+ CFs to Ccr2+ Macs in post-MI hearts. To figure out the function and mechanism of this specific cell communication, we employed a Lats CKO mice model which mimics the post-MI heart and exhibits high YAP activity and conformed the cell communication between Ccl2/Ccl7+ CFs and Ccr2+ Macs by spatial transcriptomics. Furthermore, Ccr2 loss of function in Lats CKO mice suppressed Mac expansion and CF proliferation. Consistently, Ccr2 knockout reversed the phenotype presenting smaller left ventricle chamber in Lats CKO mouse heart because of CF proliferation. Conclusion: YAP target genes Ccl2 and Ccl7 in CFs are responsible for Ccr2+ Macs recruitment and the recruited Ccr2+ Macs in turn promote CF proliferation post-MI.

Summary Histone variants contribute to the complexity of the chromatin landscape and play an integral role in defining DNA domains and regulating gene expression. The histone H3 variant H3.3 is incorporated into genic elements independent of DNA replication by its chaperone HIRA. Here we demonstrate that Hira is required for the self-renewal of adult hematopoietic stem cells (HSCs) and to restrain erythroid differentiation. Deletion of Hira led to rapid depletion of HSCs while differentiated hematopoietic cells remained largely unaffected. Depletion of HSCs after Hira deletion was accompanied by increased expression of bivalent and erythroid genes, which was exacerbated upon cell division and paralleled increased erythroid differentiation. Assessing H3.3 occupancy identified a subset of polycomb-repressed chromatin in HSCs that depends on HIRA to maintain the inaccessible, H3.3-occupied state for gene repression. HIRA-dependent H3.3 incorporation thus defines distinct repressive chromatin that represses erythroid differentiation of HSCs., Graphical abstract, Highlights • Deletion of Hira depletes adult HSCs but not fetal liver HSCs • HIRA represses hematopoietic development and erythropoiesis genes in HSCs • Cell division exacerbates gene derepression in Hira-deficient HSCs • H3.3 deposition and the closed state of polycomb-repressed chromatin depend on HIRA, Murdaugh et al. demonstrate that deletion of the histone H3.3 chaperone Hira depletes adult but not fetal liver HSCs by inducing differentiation. Hira regulates H3.3 deposition at a subset of polycomb-repressed chromatin that encodes genes involved in hematopoietic development, which becomes accessible and derepressed in the absence of Hira.

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.

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.

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.

Significance We are at an exciting era of identification of the cell and molecular processes necessary for tissue remodeling and repair. Unlike current systemic therapeutics, our studies reveal pharmacologic stimulation of SRCs modulates macrophage and fibrotic reparative cell responses to promote more effective repair and lasting beneficial remodeling after myocardial infarction., Progressive remodeling of the heart, resulting in cardiomyocyte (CM) loss and increased inflammation, fibrosis, and a progressive decrease in cardiac function, are hallmarks of myocardial infarction (MI)-induced heart failure. We show that MCB-613, a potent small molecule stimulator of steroid receptor coactivators (SRCs) attenuates pathological remodeling post-MI. MCB-613 decreases infarct size, apoptosis, hypertrophy, and fibrosis while maintaining significant cardiac function. MCB-613, when given within hours post MI, induces lasting protection from adverse remodeling concomitant with: 1) inhibition of macrophage inflammatory signaling and interleukin 1 (IL-1) signaling, which attenuates the acute inflammatory response, 2) attenuation of fibroblast differentiation, and 3) promotion of Tsc22d3-expressing macrophages—all of which may limit inflammatory damage. SRC stimulation with MCB-613 (and derivatives) is a potential therapeutic approach for inhibiting cardiac dysfunction after MI.

Cardiomyocyte loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. The ERBB2-Neuregulin and Hippo-YAP signalling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient overexpression of activated ERBB2 in cardiomyocytes (OE CMs) promotes cardiac regeneration in a heart failure model. OE CMs present an epithelial-mesenchymal transition (EMT)-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration and extracellular matrix turnover. We identified YAP as a critical mediator of ERBB2 signalling. In OE CMs, YAP interacts with nuclear-envelope and cytoskeletal components, reflecting an altered mechanical state elicited by ERBB2. We identified two YAP-activating phosphorylations on S352 and S274 in OE CMs, which peak during metaphase, that are ERK dependent and Hippo independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Together, we reveal a potent ERBB2-mediated YAP mechanotransduction signalling, involving EMT-like characteristics, resulting in robust heart regeneration.

Genome-wide association studies have uncovered over a 100 genetic loci associated with atrial fibrillation (AF), the most common arrhythmia. Many of the top AF-associated loci harbor key cardiac transcription factors, including PITX2, TBX5, PRRX1, and ZFHX3. Moreover, the vast majority of the AF-associated variants lie within noncoding regions of the genome where causal variants affect gene expression by altering the activity of transcription factors and the epigenetic state of chromatin. In this review, we discuss a transcriptional regulatory network model for AF defined by effector genes in Genome-wide association studies loci. We describe the current state of the field regarding the identification and function of AF-relevant gene regulatory networks, including variant regulatory elements, dose-sensitive transcription factor functionality, target genes, and epigenetic states. We illustrate how altered transcriptional networks may impact cardiomyocyte function and ionic currents that impact AF risk. Last, we identify the need for improved tools to identify and functionally test transcriptional components to define the links between genetic variation, epigenetic gene regulation, and atrial function.

Human genetic studies identified a strong association between loss of function mutations in RBFOX2 and hypoplastic left heart syndrome (HLHS). There are currently no Rbfox2 mouse models that recapitulate HLHS. Therefore, it is still unknown how RBFOX2 as an RNA binding protein contributes to heart development. To address this, we conditionally deleted Rbfox2 in embryonic mouse hearts and found profound defects in cardiac chamber and yolk sac vasculature formation. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS. To determine the molecular drivers of these cardiac defects, we performed RNA-sequencing in Rbfox2 mutant hearts and identified dysregulated alternative splicing (AS) networks that affect cell adhesion to extracellular matrix (ECM) mediated by Rho GTPases. We identified two Rho GTPase cycling genes as targets of RBFOX2. Modulating AS of these two genes using antisense oligos led to cell cycle and cell-ECM adhesion defects. Consistently, Rbfox2 mutant hearts displayed cell cycle defects and inability to undergo endocardial-mesenchymal transition, processes dependent on cell-ECM adhesion and that are seen in HLHS. Overall, our work not only revealed that loss of Rbfox2 leads to heart development defects resembling HLHS, but also identified RBFOX2-regulated AS networks that influence cell-ECM communication vital for heart development.

Background The conversion of fibroblasts into induced cardiomyocytes may regenerate myocardial tissue from cardiac scar through in situ cell transdifferentiation. The efficiency transdifferentiation is low, especially for human cells. We explored the leveraging of Hippo pathway intermediates to enhance induced cardiomyocyte generation. Methods and Results We screened Hippo effectors Yap (yes‐associated protein), Taz (transcriptional activator binding domain), and Tead1 (TEA domain transcription factor 1; Td) for their reprogramming efficacy with cardio‐differentiating factors Gata4, Mef2C, and Tbx5 (GMT). Td induced nearly 3‐fold increased expression of cardiomyocyte marker cTnT (cardiac troponin T) by mouse embryonic and adult rat fibroblasts versus GMT administration alone ( P P P Conclusions These data suggest that the Hippo pathway intermediate Tead1 is an important regulator of cardiac reprogramming that increases the efficiency of maturate induced cardiomyocytes generation and may be a vital component of human cardiodifferentiation strategies.

Significant phenotypic differences exist between the vascular endothelium of different organs, including cell-cell junctions, paracellular fluid transport, shape, and mural cell coverage. These organ-specific morphological features ultimately manifest as different functional capacities, as demonstrated by the dramatic differences in capillary permeability between the leaky vessels of the liver compared to the almost impermeable vasculature found in the brain. While these morphological and functional differences have been long appreciated, the molecular basis of endothelial organ specialization remains unclear. To determine the epigenetic and transcriptional mechanisms driving this functional heterogeneity, we profiled accessible chromatin, as well as gene expression, in six different organs, across three distinct time points, during murine development and in adulthood. After identifying both common, and organ-specific DNA motif usage and transcriptional signatures, we then focused our studies on the endothelium of the central nervous system. Using single cell RNA-seq, we identified key gene regulatory networks governing brain blood vessel maturation, including TCF/LEF and FOX transcription factors. Critically, these unique regulatory regions and gene expression signatures are evolutionarily conserved in humans. Collectively, this work provides a valuable resource for identifying the transcriptional regulators controlling organ-specific endothelial specialization and provides novel insight into the gene regulatory networks governing the maturation and maintenance of the cerebrovasculature.

The Hippo pathway is an evolutionarily and functionally conserved signalling pathway that controls organ size by regulating cell proliferation, apoptosis, and differentiation. Emerging evidence has shown that the Hippo pathway plays critical roles in cardiac development, homeostasis, disease, and regeneration. Targeting the Hippo pathway has tremendous potential as a therapeutic strategy for treating intractable cardiovascular diseases, such as heart failure. In this review, we summarize the function of the Hippo pathway in the heart. Particularly, we highlight the post-translational modification of Hippo pathway components, including the core kinases LATS1/2 and their downstream effectors YAP/TAZ, in different contexts, which has provided new insights and avenues in cardiac research.

DNA methylation and demethylation play an important role in shaping the epigenetic landscape and chromatin accessibility to control gene expression during development in mammals. Ten-eleven Translocation (Tet1, Tet2 and Tet3) is a family of dioxygenases that catalyze DNA methylation oxidation with ultimate DNA demethylation. Our previous study showed that cardiac-specific deletion of Tet2 and Tet3 could disrupt YY1-mediated long range chromatin interactions during heart development and lead to ventricular non-compaction cardiomyopathy. However, it is still unclear whether and how Tet protein mediated epigenetic modifications contribute to cardiac lineage specification during embryonic development. In this study, we generated cardiac specific Tet1-3 triple deficient (Tet-TKO) mouse lines using various cardiac specific Cres to evaluate the function of Tet protein in regulating cardiac lineage specification. We observed developmental defects at outflow tract (OFT) in Tet-TKO embryos, suggesting that Tet deficiency affects the second heart field (SHF) development. Single cell RNA-seq analysis further revealed the accumulation of multipotent SHF progenitors and subsequent halt of myocyte differentiation upon Tet depletion. At the molecular level, we found that Tet ablation perturbs the transcriptional network of Islet1, a transcription factor that is crucial for cardiac development in embryos. Overall, our study demonstrates a critical role of Tet-mediated epigenetic regulation for embryonic cardiac development.

While the intestine is the critical interface between cholesterol absorption and excretion, surprisingly little is known about the role of de novo cholesterol synthesis in this organ and how it affects whole body cholesterol homeostasis. The mevalonate pathway is most well-known for the production of cholesterol, but it is also required for the production of essential non-sterol isoprenoids. 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway, is regulated by a three-protein complex made up of INSIG, SCAP, and SREBP2. Intestine specific knockouts of Scap and Srebp2 both result in severe enteropathy and reduced mouse survival. Here, we assessed the hypothesis that Hmgcr is required for enterocyte viability. Mice harboring floxed alleles for Hmgcr were bred with the Villin-Cre transgene to specifically knock out this enzyme in the villus and crypt epithelial cells of the small intestine (i-KO). The i-KO mice are viable through adulthood and fertile. Hmgcr is efficiently deleted based on analysis via drop digital PCR and qPCR. RNA sequencing shows reduction in all SREBP2 target genes throughout the mevalonate pathway in intestinal epithelial cells. Lipidomics confirms substantial reductions in abundance of all sterol and non-sterol isoprenoids, except 7-dehydrocholesterol and cholesterol. Cholesterol is likely maintained through reabsorption of biliary cholesterol or increased uptake from the circulation. Circulating cholesterol levels and cholesterol absorption are not altered in i-KO mice, but triglyceride absorption is increased through compensatory changes in bile acid composition and intestinal growth. At the cellular level, the intestine rapidly compensates for loss of Hmgcr via dramatic expansion of the stem cell compartment within the crypts. In conclusion, the mechanisms by which the intestine compensates for genetic loss of Hmgcr include altered triglyceride absorption, bile acid composition, increased absorptive surface area, and expansion of the resident stem cell compartment. Together these studies provide insight into the effects of HMGCR knockout in intestinal development and lipid metabolism.

After birth, cycling mammalian CMs (cardiomyocytes) progressively lose the ability to undergo cytokinesis and hence they become binucleated, which leads to cell cycle exit and loss of regenerative capacity. During late embryonic and early postnatal heart growth, CM development is accompanied by an expansion of the cardiac fibroblast (cFb) population and compositional changes in the ECM (extracellular matrix). Whether and how these changes influence cardiomyocyte cytokinesis is currently unknown.To elucidate the role of postnatal cFbs and the ECM in cardiomyocyte cytokinesis and identify ECM proteins that promote cardiomyocyte cytokinesis.Using primary rat cardiomyocyte cultures, we found that a proportion of postnatal, but not embryonic, cycling cardiomyocytes fail to progress through cytokinesis and subsequently binucleate, consistent with published reports of in vitro and in vivo observations. Direct coculture with postnatal cFbs increased cardiomyocyte binucleation, which could be inhibited by RGD peptide treatment. In contrast, cFb-conditioned medium or transwell coculture did not significantly increase cardiomyocyte binucleation, suggesting that cFbs inhibit cardiomyocyte cytokinesis through ECM modulation rather than by secreting diffusible factors. Furthermore, we found that both embryonic and postnatal CMs binucleate at a significantly higher rate when cultured on postnatal cFb-derived ECM compared with embryonic cFb-derived ECM. These cytokinetic defects correlate with cardiomyocyte inefficiency in mitotic rounding, a process which is key to successful cytokinesis. To identify ECM proteins that modulate cardiomyocyte cytokinesis, we compared the composition of embryonic and postnatal cFb-derived ECM by mass spectrometry followed by functional assessment. We found that 2 embryonically enriched ECM proteins, SLIT2 and NPNT (nephronectin), promote cytokinesis of postnatal CMs in vitro and in vivo.We identified the postnatal cardiac ECM as a nonpermissive environment for cardiomyocyte cytokinesis and uncovered novel functions for the embryonic ECM proteins SLIT2 and NPNT (nephronectin) in promoting postnatal cardiomyocyte cytokinesis. Graphic Abstract: A graphic abstract is available for this article.

Ischemic heart disease is one of the leading causes of mortality. Myocardial infarction causes loss of cardiomyocytes in the injury area accompanied by formation of a fibrotic scar. This initiates a cascade of events including further loss of myocyte, increased fibrosis, and pathological cardiac hypertrophy, eventually leading to the heart failure. Cardiomyocytes in mammals have limited regenerative potential due to post mitotic nature of cardiomyocytes. Recently, multiple studies have provided substantial insights in to the molecular pathways governing this block in adult cardiomyocyte proliferation, and successfully employed that understanding to achieve cardiac regeneration. These strategies include directly reprograming the cardiomyocytes or manipulating the cardiac interstitium to repair the injured heart. In this review, we discuss the recent advances made in the field in the past two years.

Genome-wide association studies found that increased risk for atrial fibrillation (AF), the most common human heart arrhythmia, is associated with noncoding sequence variants located in proximity to PITX2 . Cardiomyocyte-specific epigenomic and comparative genomics uncovered 2 AF-associated enhancers neighboring PITX2 with varying conservation in mice. Chromosome conformation capture experiments in mice revealed that the Pitx2c promoter directly contacted the AF-associated enhancer regions. CRISPR/Cas9-mediated deletion of a 20-kb topologically engaged enhancer led to reduced Pitx2c transcription and AF predisposition. Allele-specific chromatin immunoprecipitation sequencing on hybrid heterozygous enhancer knockout mice revealed that long-range interaction of an AF-associated region with the Pitx2c promoter was required for maintenance of the Pitx2 c promoter chromatin state. Long-range looping was mediated by CCCTC-binding factor (CTCF), since genetic disruption of the intronic CTCF-binding site caused reduced Pitx2c expression, AF predisposition, and diminished active chromatin marks on Pitx2 . AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory functions directed at PITX2 .

Disease-associated genetic variants that lie in non-coding regions found by genome-wide association studies are thought to alter the functionality of transcription regulatory elements and target gene expression. To uncover causal genetic variants, variant regulatory elements and their target genes, here we cross-reference human transcriptomic, epigenomic and chromatin conformation datasets. Of 104 genetic variant regions associated with atrial fibrillation candidate target genes are prioritized. We optimize EMERGE enhancer prediction and use accessible chromatin profiles of human atrial cardiomyocytes to more accurately predict cardiac regulatory elements and identify hundreds of sub-threshold variants that co-localize with regulatory elements. Removal of mouse homologues of atrial fibrillation-associated regions in vivo uncovers a distal regulatory region involved in Gja1 (Cx43) expression. Our analyses provide a shortlist of genes likely affected by atrial fibrillation-associated variants and provide variant regulatory elements in each region that link genetic variation and target gene regulation, helping to focus future investigations.

Oncogenic mutations confer on cells the ability to propagate indefinitely, but whether oncogenes alter the cell fate of these cells is unknown. Here, we show that the transcriptional regulator PRDM16s causes oncogenic fate conversion by transforming cells fated to form platelets and erythrocytes into myeloid leukemia stem cells (LSCs). Prdm16s expression in megakaryocyte-erythroid progenitors (MEPs), which normally lack the potential to generate granulomonocytic cells, caused AML by converting MEPs into LSCs. Prdm16s blocked megakaryocytic/erythroid potential by interacting with super enhancers and activating myeloid master regulators, including PU.1. A CRISPR dropout screen confirmed that PU.1 is required for Prdm16s-induced leukemia. Ablating PU.1 attenuated leukemogenesis and reinstated the megakaryocytic/erythroid potential of leukemic MEPs in mouse models and human AML with PRDM16 rearrangement. Thus, oncogenic PRDM16s expression gives MEPs an LSC fate by activating myeloid gene regulatory networks.