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Purpose of review The first computer-assisted personalized sedation (CAPS) device was developed to address the growing demand for routine endoscopy procedures in the United States in the early 2000s. This review will describe the environment that gave rise to CAPS and summarize the design of that first device. It will then discuss the market forces that led to the fall of CAPS, with sales of the device ending 2 years after commercialization. Recent findings CAPS was initially conceived as a means to enable proceduralists to administer conscious sedation with propofol safely. In the nearly 20 years since its conception, the expectations of patients and proceduralists for endoscopy sedation, have evolved from conscious sedation to deep. Due to the increased risk inherent in deep sedation, future CAPS devices should be tools for anesthesiologists, not proceduralists. Summary Over $2 billion are spent annually for anesthesia services in routine endoscopic procedures for low-risk patients; a spending rate that is not sustainable. CAPS, in an 'anesthesia oversight' model similar to medical supervision, has a future as a cost-efficient means for anesthesia services to provide sedation in endoscopy and other nonoperating room venues. Anesthesiologists should work with medical device companies and payers to develop a CAPS 'anesthesia oversight' model.

Human heart failure, a leading cause of death worldwide, is a prominent example of a chronic disease that may result from poor cell renewal. The Hippo signaling pathway is an inhibitory kinase cascade that represses adult heart muscle cell (cardiomyocyte) proliferation and renewal after myocardial infarction in genetically modified mice. Here, we investigated an adeno-associated virus 9 (AAV9)–based gene therapy to locally knock down the Hippo pathway gene Salvador (Sav) in border zone cardiomyocytes in a pig model of ischemia/reperfusion-induced myocardial infarction. Two weeks after myocardial infarction, when pigs had left ventricular systolic dysfunction, we administered AAV9-Sav–short hairpin RNA (shRNA) or a control AAV9 viral vector carrying green fluorescent protein (GFP) directly into border zone cardiomyocytes via catheter-mediated subendocardial injection. Three months after injection, pig hearts treated with a high dose of AAV9-Sav-shRNA exhibited a 14.3% improvement in ejection fraction (a measure of left ventricular systolic function), evidence of cardiomyocyte division, and reduced scar sizes compared to pigs receiving AAV9-GFP. AAV9-Sav-shRNA–treated pig hearts also displayed increased capillary density and reduced cardiomyocyte ploidy. AAV9-Sav-shRNA gene therapy was well tolerated and did not induce mortality. In addition, liver and lung pathology revealed no tumor formation. Local delivery of AAV9-Sav-shRNA gene therapy to border zone cardiomyocytes in pig hearts after myocardial infarction resulted in tissue renewal and improved function and may have utility in treating heart failure.

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 causes

Spatial organization of cell-types, their colocalization patterns, and intercellular communication mechanisms are critical aspects of brain architecture and function. Understanding these aspects requires integrating data from single-cell RNA-Seq (scRNA-seq) and spatial transcriptomics (ST),the two cutting edge technologies that offer complementary insights into tissue architecture and function. Integrating these data types is non-trivial since they differ widely in the number of profiled genes and often do not share marker genes for given cell-types. We developed STANN, a neural network model that overcomes these methodological challenges for single-cell resolution ST data. We applied STANN to assign cell-types in a recent ST dataset (seqFISH+) of mouse olfactory bulb (MOB). Our analysis of STANN’s assigned cell-types revealed principles of MOB architecture and intercellular communication in unprecedented detail and beyond the conventional morphological layer-based description. We find that the proportions of cell-types are consistent within individual morphological layers of MOB, but vary widely between the layers. Surprisingly, even within a morphological layer, we find high spatial variation in cellular colocalization patterns and intercellular communication mechanisms. These observations imply the formation of spatially localized gene regulatory networks, and suggest that spatially localized receptor-ligand usage refines subtypes of major cell-types and play an important role in determining their colocalization patterns.

RATIONALE: Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1, Tbx3, and Shox2, have been identified, the cis-regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified. OBJECTIVE: To define the epigenetic profile of PCs using comparative ATAC-seq and to identify novel enhancers involved in SAN gene regulation, development and function. METHODS AND RESULTS: We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched ATAC-seq peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at E8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations. CONCLUSIONS: (1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) Cis-regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human ISL1 enhancer may regulate human SAN function.

Intercellular communication and spatial organization of cells are two critical aspects of a tissue’s function. Understanding these aspects requires integrating data from single-cell RNA-Seq (scRNA-seq) and spatial transcriptomics (ST), the two cutting edge technologies that offer complementary insights into tissue composition, architecture, and function. Integrating these data types is non-trivial since they differ widely in the number of profiled genes and often do not share marker genes for given cell-types. We developed STANN, a neural network model that overcomes these methodological challenges. Given ST and scRNA-seq data of a tissue, STANN models cell-types in the scRNA-seq dataset from the genes that are profiled by both ST and scRNA-seq. The trained STANN model then assigns cell-types to the ST dataset. We apply STANN to assign cell-types in a recent ST dataset (SeqFISH+) of mouse olfactory bulb (MOB). Our analysis of STANN’s assigned cell-types revealed principles of tissue architecture and intercellular communication at unprecedented detail. We find that cell-type compositions are disproportionate in the tissue, yet their relative proportions are spatially consistent within individual morphological layers. Surprisingly, within a morphological layer, there is a high spatial variation in cell-type colocalization patterns and intercellular communication mechanisms. Our analysis suggests that spatially localized gene regulatory networks may account for such variability in intercellular communication mechanisms.

The adult mammalian heart's potential for regeneration is very inefficient. Importantly, adult mammalian cardiomyocytes (CMs) are characterized as a cell population with very limited mitotic potential. Conversely, the neonatal mouse heart possesses a brief, yet robust, regenerative capacity within the first week of life. Cell type-specific enrichment procedures are essential for characterizing the full spectrum of epigenomic landscapes and gene regulatory networks deployed by mammalian CMs. In this chapter, we describe a protocol useful for purifying CM nuclei from mammalian cardiac tissue. Furthermore, we detail a low-input procedure suitable for the parallel genome-wide profiling of chromatin accessibility, histone modifications, and transcription factor-binding sites. The CM nuclei purified using this process are suitable for multi-omic profiling approaches.

The adult mammalian heart's potential for regeneration is very inefficient. Importantly, adult mammalian cardiomyocytes (CMs) are characterized as a cell population with very limited mitotic potential. Conversely, the neonatal mouse heart possesses a brief, yet robust, regenerative capacity within the first week of life. Cell type-specific enrichment procedures are essential for characterizing the full spectrum of epigenomic landscapes and gene regulatory networks deployed by mammalian CMs. In this chapter, we describe a protocol useful for purifying CM nuclei from mammalian cardiac tissue. Furthermore, we detail a low-input procedure suitable for the parallel genome-wide profiling of chromatin accessibility, histone modifications, and transcription factor-binding sites. The CM nuclei purified using this process are suitable for multi-omic profiling approaches.

Heart failure (HF), a devastating disease and a leading cause of death worldwide, is an unmet clinical need. The Hippo pathway, an inhibitory kinase cascade, represses adult cardiomyocyte (CM) proliferation post-myocardial infarction (MI) in rodent models. Here, we investigated an Adeno Associated Virus 9 (AAV9) gene therapy that knocks down the Hippo pathway gene, Sav, in a pig model of MI after ischemic reperfusion (I/R), to closely model the human condition. Two weeks post-MI, when pigs had established left ventricular (LV) systolic dysfunction, we delivered an AAV9- Sav -shRNA or control virus by subendocardial injections to border zone (BZ) CMs. Three months post viralinjections, AAV9- Sav -shRNA injected pigs had improved LV systolic function, evidence of CM division, and reduced scar size. In addition, AAV9- Sav- shRNA treated hearts had more capillaries and reduced CM ploidy. AAV9- Sav -shRNA therapy was well tolerated without any mortality and pathologic examination of liver and lungs failed to uncover evidence for tumor formation revealing that AAV9- Sav -shRNA gene therapy is safe within the timeframe of this study. Our findings indicate that gene therapy, knocking down the Hippo pathway post-MI, in a pig model improves heart function and may have utility for humans.

Precise control of organ growth and patterning is executed through a balanced regulation of progenitor self-renewal and differentiation. In the auditory sensory epithelium—the organ of Corti—progenitor cells exit the cell cycle in a coordinated wave between E12.5 and E14.5 before the initiation of sensory receptor cell differentiation, making it a unique system for studying the molecular mechanisms controlling the switch between proliferation and differentiation. Here we identify the Yap/Tead complex as a key regulator of the self-renewal gene network in organ of Corti progenitor cells. We show that Tead transcription factors bind directly to the putative regulatory elements of many stemness- and cell cycle-related genes. We also show that the Tead coactivator protein, Yap, is degraded specifically in the Sox2-positive domain of the cochlear duct, resulting in down-regulation of Tead gene targets. Further, conditional loss of the Yap gene in the inner ear results in the formation of significantly smaller auditory and vestibular sensory epithelia, while conditional overexpression of a constitutively active version of Yap , Yap5SA , is sufficient to prevent cell cycle exit and to prolong sensory tissue growth. We also show that viral gene delivery of Yap5SA in the postnatal inner ear sensory epithelia in vivo drives cell cycle reentry after hair cell loss. Taken together, these data highlight the key role of the Yap/Tead transcription factor complex in maintaining inner ear progenitors during development, and suggest new strategies to induce sensory cell regeneration.

Background: Surviving cells in the postinfarction border zone are subjected to intense fluctuations of their microenvironment. Recently, border zone cardiomyocytes have been specifically implicated in cardiac regeneration. Here, we defined their unique transcriptional and regulatory properties, and comprehensively validated new molecular markers, including Nppb , encoding B-type natriuretic peptide, after infarction. Methods: Transgenic reporter mice were used to identify the Nppb -positive border zone after myocardial infarction. Transcriptome analysis of remote, border, and infarct zones and of purified cardiomyocyte nuclei was performed using RNA-sequencing. Top candidate genes displaying border zone spatial specificity were histologically validated in ischemic human hearts. Mice in which Nppb was deleted by genome editing were subjected to myocardial infarction. Chromatin accessibility landscapes of border zone and control cardiomyocyte nuclei were assessed by using assay for transposase-accessible chromatin using sequencing. Results: We identified the border zone as a spatially confined region transcriptionally distinct from the remote myocardium. The transcriptional response of the border zone was much stronger than that of the remote ventricular wall, involving acute downregulation of mitochondrial oxidative phosphorylation, fatty acid metabolism, calcium handling, and sarcomere function, and the activation of a stress-response program. Analysis of infarcted human hearts revealed that the transcriptionally discrete border zone is conserved in humans, and led to the identification of novel conserved border zone markers including NPPB , ANKRD1 , DES , UCHL1, JUN , and FOXP1 . Homozygous Nppb mutant mice developed acute and lethal heart failure after myocardial infarction, indicating that B-type natriuretic peptide is required to preserve postinfarct heart function. Assay for transposase-accessible chromatin using sequencing revealed thousands of cardiomyocyte lineage-specific MEF2-occupied regulatory elements that lost accessibility in the border zone. Putative injury-responsive enhancers that gained accessibility were highly associated with AP-1 (activator protein 1) binding sites. Nuclear c-Jun, a component of AP-1, was observed specifically in border zone cardiomyocytes. Conclusions: Cardiomyocytes in a discrete zone bordering the infarct switch from a MEF2-driven homeostatic lineage-specific to an AP-1–driven injury-induced gene expression program. This program is conserved between mouse and human, and includes Nppb expression, which is required to prevent acute heart failure after infarction.

Lack of blood flow to the lower extremities in peripheral arterial disease causes oxygen and nutrient deprivation in ischemic skeletal muscles, leading to functional impairment. Treatment options for muscle regeneration in this scenario are lacking. Here, we selectively targeted the Hippo pathway in myofibers, which provide architectural support for muscle stem cell niches, to facilitate functional muscle recovery in ischemic extremities by promoting angiogenesis, neovascularization, and myogenesis. We knocked down the core Hippo pathway component, Salvador (SAV1), by using an adeno-associated virus 9 (AAV9) vector expressing a miR30-based triple short-hairpin RNA (shRNA), controlled by a muscle-specific promoter. In a mouse hindlimb-ischemia model, AAV9 SAV1 shRNA administration in ischemic muscles induced nuclear localization of the Hippo effector YAP, accelerated perfusion restoration, and increased exercise endurance. Intravascular lectin labeling of the vasculature revealed enhanced angiogenesis. Using 5-ethynyl-2′-deoxyuridine to label replicating cellular DNA in vivo, we found SAV1 knockdown concurrently increased paired box transcription factor Pax7+ muscle satellite cell and CD31+ endothelial cell proliferation in ischemic muscles. To further study Hippo suppression in skeletal muscle regeneration, we used a cardiotoxin-induced muscle damage model in adult (12-15 weeks old) and aged mice (26-month old). Two weeks after delivery of AAV9 SAV1 shRNA into injured muscles, the distribution of regenerative myofibers shifted toward a larger cross-sectional area and increased capillary density compared with mice receiving AAV9 control. Together, these findings suggest our approach may have clinical promise in regenerative therapy for leg ischemia and muscle injury.

Transcriptional enhancers commonly work over long genomic distances to precisely regulate spatiotemporal gene expression patterns. Dissecting the promoters physically contacted by these distal regulatory elements is essential for understanding developmental processes as well as the role of disease-associated risk variants. Modern proximity-ligation assays, like HiChIP and ChIA-PET, facilitate the accurate identification of long-range contacts between enhancers and promoters. However, these assays are technically challenging, expensive, and time-consuming, making it difficult to investigate enhancer topologies, especially in uncharacterized cell types. To overcome these shortcomings, we therefore designed LoopPredictor, an ensemble machine learning model, to predict genome topology for cell types which lack long-range contact maps. To enrich for functional enhancer-promoter loops over common structural genomic contacts, we trained LoopPredictor with both H3K27ac and YY1 HiChIP data. What’s more, the integration of several related multi-omics features facilitated identifying and annotating the predicted loops. LoopPredictor is able to efficiently identify cell type-specific enhancer mediated loops, and promoter-promoter interactions, with a modest feature input requirement. Comparable to experimentally generated H3K27ac HiChIP data, we found that LoopPredictor was able to identify functional enhancer loops. Furthermore, to explore the cross-species prediction capability of LoopPredictor, we fed mouse multi-omics features into a model trained on human data and found that the predicted enhancer loops outputs were highly conserved. LoopPredictor enables the dissection of cell type-specific long-range gene regulation, and can accelerate the identification of distal disease-associated risk variants.

Within the realm of zoological study, the question of how an organism reaches a specific size has been largely unexplored. Recently, studies performed to understand the regulation of organ size have revealed that both cellular signals and external cues contribute toward the determination of total cell mass within each organ. The establishment of final organ size requires the precise coordination of cell growth, proliferation, and survival throughout development and postnatal life. In the mammalian heart, the regulation of size is biphasic. During development, cardiomyocyte proliferation predominantly determines cardiac growth, whereas in the adult heart, total cell mass is governed by signals that regulate cardiac hypertrophy. Here, we review the current state of knowledge regarding the extrinsic factors and intrinsic mechanisms that control heart size during development. We also discuss the metabolic switch that occurs in the heart after birth and precedes homeostatic control of postnatal heart size.

SUMMARY Cellular heterogeneity within the mammalian brain poses a challenge toward understanding its complex functions. Within the olfactory bulb, odor information is processed by subtypes of inhibitory interneurons whose heterogeneity and functionality are influenced by ongoing adult neurogenesis. To investigate this cellular heterogeneity and better understand adult-born neuron development, we utilized single-cell RNA sequencing and computational modeling to reveal diverse and transcriptionally distinct neuronal and nonneuronal cell types. We also analyzed molecular changes during adult-born interneuron maturation and uncovered developmental programs within their gene expression profiles. Finally, we identified that distinct neuronal subtypes are differentially affected by sensory experience. Together, these data provide a transcriptome-based foundation for investigating subtype-specific neuronal function in the olfactory bulb (OB), charting the molecular profiles that arise during the maturation and integration of adult-born neurons and how they dynamically change in an activity-dependent manner., In Brief Using single-cell sequencing, Tepe et al. describe cellular heterogeneity in the mouse olfactory bulb, uncover markers for each cell type, and reveal differentially regulated genes in adult-born neurons. These findings provide a framework for studying cell-type-specific functions and circuit integration in the mammalian brain., Graphical Aabstract

Infectious endocarditis (IE) is a rare illness with high morbidity and mortality. Incidence of IE is on the rise in industrialized countries, particularly as those with congenital heart defects are living longer and the use of indwelling central catheters increases. With the 2007 American Heart Association guidelines, there has been a shift in recommending antibiotic prophylaxis only to high-risk patient populations. This clinical review will highlight the changing epidemiology and etiology of IE, followed by an emphasis on the appropriate indications for antibiotic prophylaxis in high-risk populations undergoing specific procedures.

Single-cell spatial transcriptomics (sc-ST) holds the promise to elucidate architectural aspects of complex tissues. Such analyses require modeling cell types in sc-ST datasets through their integration with single-cell RNA-seq datasets. However, this integration, is nontrivial since the two technologies differ widely in the number of profiled genes, and the datasets often do not share many marker genes for given cell types. We developed a neural network model, spatial transcriptomics cell-types assignment using neural networks (STANN), to overcome these challenges. Analysis of STANN's predicted cell types in mouse olfactory bulb (MOB) sc-ST data delineated MOB architecture beyond its morphological layer-based conventional description. We find that cell-type proportions remain consistent within individual morphological layers but vary significantly between layers. Notably, even within a layer, cellular colocalization patterns and intercellular communication mechanisms show high spatial variations. These observations imply a refinement of major cell types into subtypes characterized by spatially localized gene regulatory networks and receptor-ligand usage.

Deletion of the Hippo pathway component Salvador in mouse hearts with established ischaemic heart failure after myocardial infarction induces a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function. Previous work has shown that interfering with Hippo signalling during myocardial injury improves heart function in mice. Clinical outcomes of acute myocardial infarction in humans have improved as a result of better emergency care, but chronic heart failure, whereby the heart tissue undergoes pathological remodelling, remains a leading cause of death. James Martin and colleagues now show that the failing heart has a previously unrecognized capacity for repair. They show that blocking Hippo signalling can rescue established heart failure in mice. Deletion of the Hippo pathway component Salvador (Salv) or virus-mediated delivery of Salv short hairpin RNA when ischaemic heart failure is established can improve heart function in mice. The authors attribute the effect to the induction of a reparative genetic program, including increased expression of stress response genes and proliferative genes and preservation of mitochondrial quality control. Mammalian organs vary widely in regenerative capacity. Poorly regenerative organs, such as the heart are particularly vulnerable to organ failure. Once established, heart failure commonly results in mortality1. The Hippo pathway, a kinase cascade that prevents adult cardiomyocyte proliferation and regeneration2, is upregulated in human heart failure. Here we show that deletion of the Hippo pathway component Salvador (Salv) in mouse hearts with established ischaemic heart failure after myocardial infarction induces a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function compared with controls. Using translating ribosomal affinity purification, we isolate cardiomyocyte-specific translating messenger RNA. Hippo-deficient cardiomyocytes have increased expression of proliferative genes and stress response genes, such as the mitochondrial quality control gene, Park2. Genetic studies indicate that Park2 is essential for heart repair, suggesting a requirement for mitochondrial quality control in regenerating myocardium. Gene therapy with a virus encoding Salv short hairpin RNA improves heart function when delivered at the time of infarct or after ischaemic heart failure following myocardial infarction was established. Our findings indicate that the failing heart has a previously unrecognized reparative capacity involving more than cardiomyocyte renewal.

The regenerative capacity of the adult mammalian heart is limited, because of the reduced ability of cardiomyocytes to progress through mitosis. Endogenous cardiomyocytes have regenerative capacity at birth but this capacity is lost postnatally, with subsequent organ growth occurring through cardiomyocyte hypertrophy. The Hippo pathway, a conserved kinase cascade, inhibits cardiomyocyte proliferation in the developing heart to control heart size and prevents regeneration in the adult heart. The dystrophin-glycoprotein complex (DGC), a multicomponent transmembrane complex linking the actin cytoskeleton to extracellular matrix, is essential for cardiomyocyte homeostasis. DGC deficiency in humans results in muscular dystrophy, including the lethal Duchenne muscular dystrophy. Here we show that the DGC component dystroglycan 1 (Dag1) directly binds to the Hippo pathway effector Yap to inhibit cardiomyocyte proliferation in mice. The Yap-Dag1 interaction was enhanced by Hippo-induced Yap phosphorylation, revealing a connection between Hippo pathway function and the DGC. After injury, Hippo-deficient postnatal mouse hearts maintained organ size control by repairing the defect with correct dimensions, whereas postnatal hearts deficient in both Hippo and the DGC showed cardiomyocyte overproliferation at the injury site. In the hearts of mature Mdx mice (which have a point mutation in Dmd)-a model of Duchenne muscular dystrophy-Hippo deficiency protected against overload-induced heart failure.

SummaryCardiomyocyte (CM) loss after injury results in adverse remodelling and fibrosis, which inevitably lead to heart failure. ERBB2-Neuregulin and Hippo-YAP signaling pathways are key mediators of CM proliferation and regeneration, yet the crosstalk between these pathways is unclear. Here, we demonstrate in adult mice that transient over-expression (OE) of activated ERBB2 in CMs promotes cardiac regeneration in a heart failure model. OE CMs present an EMT-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration, and ECM turnover. Molecularly, we identified YAP as a critical mediator of ERBB2 signaling. In OE CMs, YAP interacts with nuclear envelope and cytoskeletal components, reflecting the altered mechanic state elicited by ERBB2. Hippo-independent activating phosphorylation on YAP at S352 and S274 were enriched in OE CMs, peaking during metaphase, and viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Taken together, we demonstrate a potent ERBB2-mediated YAP mechanosensory signaling, involving EMT-like characteristics, resulting in heart regeneration.HighlightsERBB2-driven regeneration of scarred hearts recapitulates core-EMT processesYAP is activated and required downstream to ERBB2 signaling in CMsYAP activity is mechanically driven by cytoskeleton and nuclear envelope remodelingYAP S274 and S352 phosphorylation is essential for CM mitosis

As with all glial cells, the major role of retinal Müller glia (MG) is to provide essential neuronal support. However, the MG of some non-mammalian species have the additional ability to generate new retinal neurons capable of sight restoration. Unfortunately, mammalian MG do not possess this ability. However, if we could understand the reasons why, we may be able to devise strategies to confer regenerative potential. The recent discovery that the Hippo signaling pathway acts as an intrinsic block to mammalian MG proliferation, along with reports of adeno-associated virus (AAV)-based MG reprogramming and functional photoreceptor differentiation, may indicate a watershed moment in the field of mammalian retinal regeneration. However, as researchers delve deeper into the cellular and molecular mechanisms, and further refine MG reprogramming strategies, we should recall past misinterpretations of data in this field and proceed with caution. Here, we provide a summary of these emerging data and a discussion of technical concerns specific to AAV-mediated reprogramming experiments that must be addressed in order for the field to move forward.

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 Pitx2c 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.

SummaryMost disease-associated variants identified by population based genetic studies are non-coding, which compromises finding causative genes and mechanisms. Presumably they interact through looping with nearby genes to modulate transcription. Hi-C provides the most complete and unbiased method for genome-wide identification of potential regulatory interactions, but finding chromatin loops in Hi-C data remains difficult and tissue specific data are limited. We have generated Hi-C data from primary cardiac tissue and developed a method, peakHiC, for sensitive and quantitative loop calling to uncover the human heart regulatory interactome. We identify complex CTCF-dependent and -independent contact networks, with loops between coding and non-coding gene promoters, shared enhancers and repressive sites. Across the genome, enhancer interaction strength correlates with gene transcriptional output and loop dynamics follows CTCF, cohesin and H3K27Ac occupancy levels. Finally, we demonstrate that intersection of the human heart regulatory interactome with cardiovascular disease variants facilitates prioritizing disease-causative genes.

Cardiac fibroblasts (CFs) respond to injury by transitioning through multiple cell states, including resting CFs, activated CFs, and myofibroblasts. We report here that Hippo signaling cell-autonomously regulates CF fate transitions and proliferation, and non-cell-autonomously regulates both myeloid and CF activation in the heart. Conditional deletion of Hippo pathway kinases, Lats1 and Lats2, in uninjured CFs initiated a self-perpetuating fibrotic response in the adult heart that was exacerbated by myocardial infarction (MI). Single cell transcriptomics showed that uninjured Lats1/2 mutant CFs spontaneously transitioned to a myofibroblast cell state. Through gene regulatory network reconstruction, we found that Hippo-deficient myofibroblasts deployed a network of transcriptional regulators of endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) consistent with elevated secretory activity. We observed an expansion of myeloid cell heterogeneity in uninjured Lats1/2 CKO hearts with similarity to cells recovered from control hearts post-MI. Integrated genome-wide analysis of Yap chromatin occupancy revealed that Yap directly activates myofibroblast cell identity genes, the proto-oncogene Myc, and an array of genes encoding pro-inflammatory factors through enhancer–promoter looping. Our data indicate that Lats1/2 maintain the resting CF cell state through restricting the Yap-induced injury response.

The Pitx2 gene encodes a homeobox transcription factor that is required for mammalian development. Disruption of PITX2 expression in humans causes congenital heart diseases and is associated with atrial fibrillation; however, the cellular and molecular processes dictated by Pitx2 during cardiac ontogeny remain unclear. To characterize the role of Pitx2 during murine heart development we sequenced over 75,000 single cardiac cell transcriptomes between two key developmental timepoints in control and Pitx2 null embryos. We found that cardiac cell composition was dramatically altered in mutants at both E10.5 and E13.5. Interestingly, the differentiation dynamics of both anterior and posterior second heart field-derived progenitor cells were disrupted in Pitx2 mutants. We also uncovered evidence for defects in left-right asymmetry within atrial cardiomyocyte populations. Furthermore, we were able to detail defects in cardiac outflow tract and valve development associated with Pitx2. Our findings offer insight into Pitx2 function and provide a compilation of gene expression signatures for further detailing the complexities of heart development that will serve as the foundation for future studies of cardiac morphogenesis, congenital heart disease and arrhythmogenesis.

After myocardial injury, cardiomyocyte loss cannot be corrected by using currently available clinical treatments. In recent years, considerable effort has been made to develop cell-based cardiac repair therapies aimed at correcting for this loss. An exciting crop of recent studies reveals that inducing endogenous repair and proliferation of cardiomyocytes may be a viable option for regenerating injured myocardium. Here, we review current heart failure treatments, the state of cardiomyocyte renewal in mammals, and the molecular signals that stimulate cardiomyocyte proliferation. These signals include growth factors, intrinsic signaling pathways, microRNAs, and cell cycle regulators. Animal model cardiac regeneration studies reveal that modulation of exogenous and cell-intrinsic signaling pathways can induce reentry of adult cardiomyocytes into the cell cycle. Using direct myocardial injection, epicardial patch delivery, or systemic administration of growth molecules, these studies show that inducing endogenous cardiomyocytes to self-renew is an exciting and promising therapeutic strategy to treat cardiac injury in humans.

The sustained delivery of extracellular vesicles, secreted by induced-pluripotent-stem-cell-derived cardiomyocytes, through a hydrogel patch promotes cardiac recovery after myocardial infarction in rats.

The mammalian cochlea loses its ability to regenerate new hair cells prior to the onset of hearing. In contrast, the adult vestibular system can produce new hair cells in response to damage, or by reprogramming of supporting cells with the hair cell transcription factor Atoh1. We used RNA-seq and ATAC-seq to probe the transcriptional and epigenetic responses of utricle supporting cells to damage and Atoh1 transduction. We show that the regenerative response of the utricle correlates with a more accessible chromatin structure in utricle supporting cells compared to their cochlear counterparts. We also provide evidence that Atoh1 transduction of supporting cells is able to promote increased transcriptional accessibility of some hair cell genes. Our study offers a possible explanation for regenerative differences between sensory organs of the inner ear, but shows that additional factors to Atoh1 may be required for optimal reprogramming of hair cell fate.

Previous work from ours and other laboratories have shown that steroid receptor coactivators (SRCs) are involved in heart development and in mitigating cardiac dysfunction in cardiac injury models. Members of the p160 SRC family, SRC-1 (NCOA1), SRC-2 (NCOA2/TIF2/GRIP1) and SRC-3 (NCOA3/AIB1/ACTR/pCIP), interact with nuclear receptors and other transcription factors to drive target gene expression by assembling transcriptional coactivator complexes to increase transcription. This indicates a potential for SRC targeting drugs pertinent to cell migration, proliferation and survival-promoting paracrine interactions in cardiac tissue injury responses. We have identified a small molecule activator of SRCs (MCB-613) that selectively and reversibly binds to SRCs as shown by surface plasmon resonance and is a potent SRC stimulator that acts to greatly enhance SRC transcriptional activity with no apparent toxicity in mice. We postulated that MCB-613 could enable wound repair and preservation of cardiac function after an acute MI by reducing the extent of injury-related fibrosis and the subsequent chronic loss of cardiac function associated with non-contracting scar tissue. We thus tested the effect of MCB-613 on the cardiac injury response by administering MCB-613 two hours after ischemic injury in a mouse model of MI. Along with measurements of functional cardiac output and damage, we sought to identify the cell-type specific responses responsible for MCB-613’s cardio-protective effects by utilizing single cell transcriptomics of cardiac interstitial cells to characterize the effects of SRC stimulation on cardiac function post-MI. 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: (i) inhibition of macrophage inflammatory signaling and IL-1 signaling which attenuates the acute inflammatory response, (ii) attenuation of fibroblast differentiation, and (iii) promotion of Tsc22d3 expressing macrophages - all of which may limit inflammatory damage. Our results indicate MCB-613 controls the cellular interstitial cardiac repair response to ischemia. Distinct molecular and cellular mechanisms related to stimulation of SRC-3 have been identified that pave the way for the further exploration of SRCs as drug targets that can be engaged to improve the management of myocardial injury response outcomes. SRC stimulation with MCB-613 (and derivatives) is a potential novel therapeutic approach for inhibiting cardiac dysfunction after MI.

In the adult liver, the Hippo pathway mammalian STE20-like protein kinases 1 and 2 and large tumor suppressor homologs 1 and 2 (LATS1/2) control activation of the transcriptional coactivators Yes-associated protein (YAP) and WW domain containing transcription regulator 1 (TAZ) in hepatocytes and biliary epithelial cells, thereby regulating liver cell proliferation, differentiation, and malignant transformation. Less is known about the contribution of Hippo signaling to liver development. We used conditional mutagenesis to show that the Hippo signaling pathway kinases LATS1 and LATS2 are redundantly required during mouse liver development to repress YAP and TAZ in both the biliary epithelial and hepatocyte lineages. In the absence of LATS1/2, biliary epithelial cells exhibit excess proliferation while hepatoblasts fail to mature into hepatocytes, defects that result in perinatal lethality. Using an in vitro hepatocyte differentiation assay, we demonstrate that YAP activity decreases and Hippo pathway kinase activities increase upon differentiation. In addition, we show that YAP activation in vitro, resulting from either depletion of its negative regulators LATS1/2 or expression of a mutant form of YAP that is less efficiently phosphorylated by LATS1/2, results in transcriptional suppression of genes that normally accompany hepatocyte maturation. Moreover, we provide evidence that YAP activity is repressed by Hippo pathway activation upon hepatocytic maturation in vitro. Finally, we examine the localization of YAP during fetal liver development and show that higher levels of YAP are found in biliary epithelial cells, while in hepatocytes YAP levels decrease with hepatocyte maturation. Conclusion Hippo signaling, mediated by the LATS1 and LATS2 kinases, is required to restrict YAP and TAZ activation during both biliary and hepatocyte differentiation. These findings suggest that dynamic regulation of the Hippo signaling pathway plays an important role in differentiation and functional maturation of the liver. (Hepatology 2016;64:1757-1772).

Clefting of the lip, with or without palatal involvement (CLP), is associated with a higher incidence of developmental tooth abnormalities, including hypodontia and supernumerary teeth, aberrant crown and root morphologies, and enamel defects, although the underlying mechanistic link is poorly understood. As most CLP genes are expressed throughout the oral epithelium, the authors hypothesized that the expression of CLP genes may persist in the dental epithelium and thus, in addition to their earlier role in labiopalatine development, may play an important functional role in subsequent tooth patterning and amelogenesis. To address this, the authors generated a unique conditional knockout model involving the major CLP gene, Irf6, that overcomes the previously reported perinatal lethality to enable assessment of any posteruption dental phenotypes. A dental epithelium–specific Irf6 conditional knockout ( Irf6-cKO) mouse was generated via a Pitx2-Cre driver line. Dental development was analyzed by microcomputed tomography, scanning electron microscopy, histology, immunohistochemistry, and quantitative polymerase chain reaction. Irf6-cKO mice displayed variable hypodontia, occasional supernumerary incisors and molars, as well as crown and root patterning anomalies, including peg-shaped first molars and taurodontic and C-shaped mandibular second molars. Enamel density was reduced in preeruption Irf6-cKO mice, and some shearing of enamel rods was noted in posteruption incisors. There was also rapid attrition of Irf6-cKO molars following eruption. Histologically, Irf6-cKO ameloblasts exhibited disturbances in adhesion and polarity, and delayed enamel formation was confirmed immunohistochemically. Altered structure of Hertwig’s epithelial root sheath was also observed. These data support a role for IRF6 in tooth number, crown and root morphology and amelogenesis that is likely due to a functional role of Irf6 in organization and polarity of epithelial cell types. This data reinforce the notion that various isolated tooth defects could be considered part of the CLP spectrum in relatives of an affected individual.

A leading cause of death and decrease in the quality of life in the USA is myocardial infarction (MI). Molecular and genetic analyses have revealed a plethora of information about critical processes involved with MI. However, there is a lack of accompanying information about cardiac tissue biomechanical properties, which may provide additional critical information to understand the tissue remodeling process after MI. In this work, we utilize two complementary noncontact optical coherence elastography (OCE) techniques to assess the changes in mouse cardiac tissue biomechanical properties 6 weeks after the MI was induced. A focused micro air-pulse induced localized displacements, which were detected by a phase-sensitive OCE (PhS-OCE) system. The localized tissue displacement was modeled by a spring-mass damper model to quantify the tissue stiffness. Additionally, the propagation of an air-pulse induced elastic wave was measured at various meridional angles as a complementary measurement of tissue stiffness and anisotropy. The damping results show that the MI caused a decrease in the stiffness of the cardiac tissue. Similarly, the analysis of elastic wave propagation showed that the cardiac tissue became softer and more isotropic after MI. These results show that OCE can detect the changes in cardiac tissue biomechanical properties after MI. OCE may also be useful for developing targeted therapies by identifying regions of cardiac tissue affected by MI.

Myocardial infarction (MI) is a leading cause of death and decrease of quality of life in the USA. An immense amount of research and development has been focused on the molecular mechanisms associated with MI, which have led to numerous therapies for treating and repairing cardiac tissue after MI. However, there is a relative lack of information about the changes in cardiac tissue biomechanical properties due to MI. Therefore, there is a direct need for techniques that can measure cardiac tissue biomechanical properties, which would help further develop our understanding of tissue biomechanical dynamics associated with MI as well as aid in the development of therapies that consider biomechanical properties. In this work we utilize noncontact dynamic optical coherence elastography (OCE) to evaluate the changes in cardiac biomechanical properties 6 weeks after MI in a mouse model. We performed complementary analysis based on elastic wave propagation and damping analyses. Our results show that the left ventricle cardiac tissue became more isotropic and softer after 6 weeks in the MI-affected mice as compared to the sham mice based on the elastic wave propagation measurements. The damping measurements also showed that the MI-affected mice had softer left ventricles as compared to the sham mice. Moreover, the damping analysis was able to localize the boundary of the MI-damaged region. These results show that OCE can be a powerful tool for understanding the dynamics in biomechanical changes in murine cardiac tissue and could potentially reveal diseased areas for targeted therapies.

The Pitx2 gene encodes a homeobox transcription factor that is required for mammalian development. Disruption of PITX2 expression in humans causes congenital heart diseases and is associated with atrial fibrillation; however, the cellular and molecular processes dictated by Pitx2 during cardiac ontogeny remain unclear. To characterize the role of Pitx2 during murine heart development we sequenced over 75,000 single cardiac cell transcriptomes between two key developmental timepoints in control and Pitx2 null embryos. We found that cardiac cell composition was dramatically altered in mutants at both E10.5 and E13.5. Interestingly, the differentiation dynamics of both anterior and posterior second heart field-derived progenitor cells were disrupted in Pitx2 mutants. We also uncovered evidence for defects in left-right asymmetry within atrial cardiomyocyte populations. Furthermore, we were able to detail defects in cardiac outflow tract and valve development associated with Pitx2. Our findings offer insight into Pitx2 function and provide a compilation of gene expression signatures for further detailing the complexities of heart development that will serve as the foundation for future studies of cardiac morphogenesis, congenital heart disease and arrhythmogenesis.

SUMMARY In response to retinal damage, the Müller glial cells (MGs) of the zebrafish retina have the ability to undergo a cellular reprogramming event in which they enter the cell cycle and divide asymmetrically, thereby producing multipotent retinal progenitors capable of regenerating lost retinal neurons. However, mammalian MGs do not exhibit such a proliferative and regenerative ability. Here, we identify Hippo pathway-mediated repression of the transcription cofactor YAP as a core regulatory mechanism that normally blocks mammalian MG proliferation and cellular reprogramming. MG-specific deletion of Hippo pathway components Lats1 and Lats2, as well as transgenic expression of a Hippo non-responsive form of YAP (YAP5SA), resulted in dramatic Cyclin D1 upregulation, loss of adult MG identity, and attainment of a highly proliferative, progenitor-like cellular state. Our results reveal that mammalian MGs may have latent regenerative capacity that can be stimulated by repressing Hippo signaling., In Brief Rueda et al. identify the Hippo pathway as an endogenous molecular mechanism normally preventing mammalian Müller glial reprogramming to a proliferative, progenitor-like state., Graphical Abstract

New strategies in the field of cardiac regeneration are directed at identifying proliferation-inducing substances to induce regrowth of myocardium. Current screening assays utilize neonatal cardiomyocytes and markers for cytokinesis, such as Aurora B-kinase. However, detection of cardiomyocyte division is complicated because of cell cycle variants, in particular, binucleation.To analyze the process of cardiomyocyte binucleation to identify definitive discriminators for cell cycle variants and authentic cardiomyocyte division.Herein, we demonstrate by direct visualization of the contractile ring and midbody in Myh6 (myosin, heavy chain 6)-eGFP (enhanced green fluorescent protein)-anillin transgenic mice that cardiomyocyte binucleation starts by formation of a contractile ring. This is followed by irregular positioning of the midbody and movement of the 2 nuclei into close proximity to each other. In addition, the widespread used marker Aurora B-kinase was found to also label binucleating cardiomyocytes, complicating the interpretation of existing screening assays. Instead, atypical midbody positioning and the distance of daughter nuclei on karyokinesis are bona fide markers for cardiomyocyte binucleation enabling to unequivocally discern such events from cardiomyocyte division in vitro and in vivo.The 2 criteria provide a new method for identifying cardiomyocyte division and should be considered in future studies investigating cardiomyocyte turnover and regeneration after injury, in particular in the postnatal heart to prevent the assignment of false positive proliferation events.