3D-cardiomics: A spatial transcriptional atlas of the mammalian heart.

Understanding the spatial gene expression and regulation in the heart is key to uncovering its developmental and physiological processes, during homeostasis and disease. Numerous techniques exist to gain gene expression and regulation information in organs such as the heart, but few utilize intuitive true-to-life three-dimensional representations to analyze and visualise results. Here we combined transcriptomics with 3D-modelling to interrogate spatial gene expression in the mammalian heart. For this, we microdissected and sequenced transcriptome-wide 18 anatomical sections of the adult mouse heart. Our study has unveiled known and novel genes that display complex spatial expression in the heart sub-compartments. We have also created 3D-cardiomics, an interface for spatial transcriptome analysis and visualization that allows the easy exploration of these data in a 3D model of the heart. 3D-cardiomics is accessible from http://3d-cardiomics.erc.monash.edu/.

Cross-Priming Dendritic Cells Exacerbate Immunopathology After Ischemic Tissue Damage in the Heart.

BACKGROUND: Ischemic heart disease is a leading cause of heart failure and despite advanced therapeutic options, morbidity and mortality rates remain high. Although acute inflammation in response to myocardial cell death has been extensively studied, subsequent adaptive immune activity and anti-heart autoimmunity may also contribute to the development of heart failure. After ischemic injury to the myocardium, dendritic cells (DC) respond to cardiomyocyte necrosis, present cardiac antigen to T cells, and potentially initiate a persistent autoimmune response against the heart. Cross-priming DC have the ability to activate both CD4+ helper and CD8+ cytotoxic T cells in response to necrotic cells and may thus be crucial players in exacerbating autoimmunity targeting the heart. This study investigates a role for cross-priming DC in post-myocardial infarction immunopathology through presentation of self-antigen from necrotic cardiac cells to cytotoxic CD8+ T cells. METHODS: We induced type 2 myocardial infarction-like ischemic injury in the heart by treatment with a single high dose of the ß-adrenergic agonist isoproterenol. We characterized the DC population in the heart and mediastinal lymph nodes and analyzed long-term cardiac immunopathology and functional decline in wild type and Clec9a-depleted mice lacking DC cross-priming function. RESULTS: A diverse DC population, including cross-priming DC, is present in the heart and activated after ischemic injury. Clec9a-/- mice deficient in DC cross-priming are protected from persistent immune-mediated myocardial damage and decline of cardiac function, likely because of dampened activation of cytotoxic CD8+ T cells. CONCLUSION: Activation of cytotoxic CD8+ T cells by cross-priming DC contributes to exacerbation of postischemic inflammatory damage of the myocardium and corresponding decline in cardiac function. Importantly, this provides novel therapeutic targets to prevent postischemic immunopathology and heart failure.

Recent Advances in Single-Cell Profiling and Multispecific Therapeutics: Paving the Way for a New Era of Precision Medicine Targeting Cardiac Fibroblasts.

PURPOSE OF REVIEW: Cardiac fibroblast activation contributes to fibrosis, maladaptive remodeling and heart failure progression. This review summarizes the latest findings on cardiac fibroblast activation dynamics derived from single-cell transcriptomic analyses and discusses how this information may aid the development of new multispecific medicines. RECENT FINDINGS: Advances in single-cell gene expression technologies have led to the discovery of distinct fibroblast subsets, some of which are more prevalent in diseased tissue and exhibit temporal changes in response to injury. In parallel to the rapid development of single-cell platforms, the advent of multispecific therapeutics is beginning to transform the biopharmaceutical landscape, paving the way for the selective targeting of diseased fibroblast subpopulations. Insights gained from single-cell technologies reveal critical cardiac fibroblast subsets that play a pathogenic role in the progression of heart failure. Combined with the development of multispecific therapeutic agents that have enabled access to previously "undruggable" targets, we are entering a new era of precision medicine.

View from the heart: cardiac fibroblasts in development, scarring and regeneration.

In the adult, tissue repair after injury is generally compromised by fibrosis, which maintains tissue integrity with scar formation but does not restore normal architecture and function. The process of regeneration is necessary to replace the scar and rebuild normal functioning tissue. Here, we address this problem in the context of heart disease, and discuss the origins and characteristics of cardiac fibroblasts, as well as the crucial role that they play in cardiac development and disease. We discuss the dual nature of cardiac fibroblasts, which can lead to scarring, pathological remodelling and functional deficit, but can also promote heart function in some contexts. Finally, we review current and proposed approaches whereby regeneration could be fostered by interventions that limit scar formation.

Congenital valvular defects associated with deleterious mutations in the PLD1 gene.

BACKGROUND: The underlying molecular aetiology of congenital heart defects is largely unknown. The aim of this study was to explore the genetic basis of non-syndromic severe congenital valve malformations in two unrelated families. METHODS: Whole-exome analysis was used to identify the mutations in five patients who suffered from severe valvular malformations involving the pulmonic, tricuspid and mitral valves. The significance of the findings was assessed by studying sporulation of yeast carrying a homologous Phospholipase D (PLD1) mutation, in situ hybridisation in chick embryo and echocardiography and histological examination of hearts of PLD1 knockout mice. RESULTS: Three mutations, p.His442Pro, p.Thr495fs32* and c.2882+2T>C, were identified in the PLD1 gene. The mutations affected highly conserved sites in the PLD1 protein and the p.His442Pro mutation produced a strong loss of function phenotype in yeast homologous mutant strain. Here we show that in chick embryos PLD1 expression is confined to the forming heart (E2-E8) and homogeneously expressed all over the heart during days E2-E3. Thereafter its expression decreases, remaining only adjacent to the atrioventricular valves and the right ventricular outflow tract. This pattern of expression follows the known dynamic patterning of apoptosis in the developing heart, consistent with the known role of PLD1 in the promotion of apoptosis. In hearts of PLD1 knockout mice, we detected marked tricuspid regurgitation, right atrial enlargement, and increased flow velocity, narrowing and thickened leaflets of the pulmonic valve. CONCLUSIONS: The findings support a role for PLD1 in normal heart valvulogenesis.

Mutations in the Katnb1 gene cause left-right asymmetry and heart defects.

BACKGROUND: The microtubule-severing protein complex katanin is composed two subunits, the ATPase subunit, KATNA1, and the noncatalytic regulatory subunit, KATNB1. Recently, the Katnb1 gene has been linked to infertility, regulation of centriole and cilia formation in fish and mammals, as well as neocortical brain development. KATNB1 protein is expressed in germ cells in humans and mouse, mitotic/meiotic spindles and cilia, although the full expression pattern of the Katnb1 gene has not been described. RESULTS: Using a knockin-knockout mouse model of Katnb1 dysfunction we demonstrate that Katnb1 is ubiquitously expressed during embryonic development, although a stronger expression is seen in the crown cells of the gastrulation organizer, the murine node. Furthermore, null and hypomorphic Katnb1 gene mutations show a novel correlation between Katnb1 dysregulation and the development of impaired left-right signaling, including cardiac malformations. CONCLUSIONS: Katanin function is a critical regulator of heart development in mice. These findings are potentially relevant to human cardiac development. Developmental Dynamics 246:1027-1035, 2017. © 2017 Wiley Periodicals, Inc.

Properties and Immune Function of Cardiac Fibroblasts.

This chapter will discuss the role of cardiac fibroblasts as a target of various immunological inputs as well as an immunomodulatory hub of the heart through interaction with immune cell types and chemokine or cytokine signaling. While the purpose of this chapter is to explore the immunomodulatory properties of cardiac fibroblasts, it is important to note that cardiac fibroblasts are not a homogeneous cell type, but have a unique embryological origin and molecular identity. Specific properties of cardiac fibroblasts may influence the way they interact with the heart microenvironment to promote healthy homeostatic function or respond to pathological insults. Therefore, we will briefly discuss these aspects of cardiac fibroblast biology and then focus on their immunomodulatory role in the heart.

The cardiac fibroblast: Origin, identity and role in homeostasis and disease.

The mammalian heart is responsible for supplying blood to two separate circulation circuits in a parallel manner. This design provides efficient oxygenation and nutrients to the whole body through the left-sided pump, while the right-sided pump delivers blood to the pulmonary circulation for re-oxygenation. In order to achieve this demanding job, the mammalian heart evolved into a highly specialised organ comprised of working contractile cells or cardiomyocytes, a directional and insulated conduction system, capable of independently generating and conducting electric impulses that synchronises chamber contraction, valves that allow the generation of high pressure and directional blood flow into the circulation, coronary circulation, that supplies oxygenated blood for the heart muscle high metabolically active pumping role and inlet/outlet routes, as the venae cavae and pulmonary veins, aorta and pulmonary trunk. This organization highlights the complexity and compartmentalization of the heart. This review will focus on the cardiac fibroblast, a cell type until recently ignored, but that profoundly influences heart function in its various compartments. We will discuss current advances on definitions, molecular markers and function of cardiac fibroblasts in heart homeostasis and disease.

A novel conditional mouse model for Nkx2-5 reveals transcriptional regulation of cardiac ion channels.

Nkx2-5 is one of the master regulators of cardiac development, homeostasis and disease. This transcription factor has been previously associated with a suite of cardiac congenital malformations and impairment of electrical activity. When disease causative mutations in transcription factors are considered, NKX2-5 gene dysfunction is the most common abnormality found in patients. Here we describe a novel mouse model and subsequent implications of Nkx2-5 loss for aspects of myocardial electrical activity. In this work we have engineered a new Nkx2-5 conditional knockout mouse in which flox sites flank the entire Nkx2-5 locus, and validated this line for the study of heart development, differentiation and disease using a full deletion strategy. While our homozygous knockout mice show typical embryonic malformations previously described for the lack of the Nkx2-5 gene, hearts of heterozygous adult mice show moderate morphological and functional abnormalities that are sufficient to sustain blood supply demands under homeostatic conditions. This study further reveals intriguing aspects of Nkx2-5 function in the control of cardiac electrical activity. Using a combination of mouse genetics, biochemistry, molecular and cell biology, we demonstrate that Nkx2-5 regulates the gene encoding Kcnh2 channel and others, shedding light on potential mechanisms generating electrical abnormalities observed in patients bearing NKX2-5 dysfunction and opening opportunities to the study of novel therapeutic targets for anti-arrhythmogenic therapies.

Cardiogenic genes expressed in cardiac fibroblasts contribute to heart development and repair.

RATIONALE: Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment, but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers. OBJECTIVE: To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration. METHODS AND RESULTS: High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical mesenchymal stem cell and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. While genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, T-box 20, caused marked myocardial dysmorphology and perturbations in scar formation on myocardial infarction. CONCLUSIONS: The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs, and direct contribution to cardiac development and repair provoke alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies.

VISIONET: intuitive visualisation of overlapping transcription factor networks, with applications in cardiogenic gene discovery.

BACKGROUND: Existing de novo software platforms have largely overlooked a valuable resource, the expertise of the intended biologist users. Typical data representations such as long gene lists, or highly dense and overlapping transcription factor networks often hinder biologists from relating these results to their expertise. RESULTS: VISIONET, a streamlined visualisation tool built from experimental needs, enables biologists to transform large and dense overlapping transcription factor networks into sparse human-readable graphs via numerically filtering. The VISIONET interface allows users without a computing background to interactively explore and filter their data, and empowers them to apply their specialist knowledge on far more complex and substantial data sets than is currently possible. Applying VISIONET to the Tbx20-Gata4 transcription factor network led to the discovery and validation of Aldh1a2, an essential developmental gene associated with various important cardiac disorders, as a healthy adult cardiac fibroblast gene co-regulated by cardiogenic transcription factors Gata4 and Tbx20. CONCLUSIONS: We demonstrate with experimental validations the utility of VISIONET for expertise-driven gene discovery that opens new experimental directions that would not otherwise have been identified.

Loss of Cited2 causes congenital heart disease by perturbing left-right patterning of the body axis.

Cited2 is a transcriptional coactivator that is required for normal development of the embryo and placenta. Cited2-null mice die during gestation with fully penetrant heart defects and partially penetrant laterality defects. The laterality defects occur due to the loss of Nodal expression in the left lateral plate mesoderm (LPM). The cause of the heart defects that arise independently of laterality defects is unknown; they might occur due to an intrinsic requirement for Cited2 in the developing heart, or to disturbances in left-right patterning of the early embryo. Herein it is established that deletion of Cited2 from the heart progenitors does not alter development, and that heart defects in Cited2-null embryos arise due to an extra-cardiac requirement for Cited2 in establishing the left-right body axis. In addition, we provide evidence supporting a role for Cited2 in tissues of the embryo vital for left-right patterning (the node and LPM). Molecular and genetic analysis reveals that Cited2 is required for the initiation, but not propagation of, the left-sided determinant Nodal in the LPM. Moreover, a new role for Cited2 is identified as a potentiator of bone morphogenetic protein (BMP) signalling, counteracting the initiation of Nodal expression in the LPM. These data define Cited2 as a key regulator of left-right patterning in the mammalian embryo, and reveal that the role of Cited2 in cardiac development lies in its extra-cardiac functions. The clinical relevance of these findings lies in the fact that heterozygous mutation of human CITED2 is associated with congenital heart disease and laterality defects.

Targeting Immune-Fibroblast Crosstalk in Myocardial Infarction and Cardiac Fibrosis.

Inflammation and tissue fibrosis co-exist and are causally linked to organ dysfunction. However, the molecular mechanisms driving immune-fibroblast crosstalk in human cardiac disease remains unexplored and there are currently no therapeutics to target fibrosis. Here, we performed multi-omic single-cell gene expression, epitope mapping, and chromatin accessibility profiling in 38 donors, acutely infarcted, and chronically failing human hearts. We identified a disease-associated fibroblast trajectory marked by cell surface expression of fibroblast activator protein (FAP), which diverged into distinct myofibroblasts and pro-fibrotic fibroblast populations, the latter resembling matrifibrocytes. Pro-fibrotic fibroblasts were transcriptionally similar to cancer associated fibroblasts and expressed high levels of collagens and periostin (POSTN), thymocyte differentiation antigen 1 (THY-1), and endothelin receptor A (EDNRA) predicted to be driven by a RUNX1 gene regulatory network. We assessed the applicability of experimental systems to model tissue fibrosis and demonstrated that 3 different in vivo mouse models of cardiac injury were superior compared to cultured human heart and dermal fibroblasts in recapitulating the human disease phenotype. Ligand-receptor analysis and spatial transcriptomics predicted that interactions between C-C chemokine receptor type 2 (CCR2) macrophages and fibroblasts mediated by interleukin 1 beta (IL-1ß) signaling drove the emergence of pro-fibrotic fibroblasts within spatially defined niches. This concept was validated through in silico transcription factor perturbation and in vivo inhibition of IL-1ß signaling in fibroblasts where we observed reduced pro-fibrotic fibroblasts, preferential differentiation of fibroblasts towards myofibroblasts, and reduced cardiac fibrosis. Herein, we show a subset of macrophages signal to fibroblasts via IL-1ß and rewire their gene regulatory network and differentiation trajectory towards a pro-fibrotic fibroblast phenotype. These findings highlight the broader therapeutic potential of targeting inflammation to treat tissue fibrosis and restore organ function.

Characterization of Pitx2c expression in the mouse heart using a reporter transgene.

To aid in detection and tracking of cells targeted by the left-right (LR) pathway in the heart throughout morphogenesis, expression from a Pitx2c-lacZ transgene (P2Ztg) was analysed in detail. ß-galactosidase expression from P2Ztg was robust, allowing reliable visualisation of low-level Pitx2c expression, and was virtually entirely dependent upon NODAL signalling in the heart. P2Ztg showed expression in trabecular and septal, as well as non-trabecular, myocardium, and a strong expression bias in myocardium associated with individual endocardial cushions of the atrioventricular canal and outflow tract, which are essential for cardiac septation. Expression on the ventral surface of the outflow tract evolved to a specific stripe that could be used to track the future aorta during outflow tract spiralling and remodelling. Our data show that the P2Ztg transgene is a useful resource for detection of molecular disturbances in the LR cascade, as well as morphogenetic defects associated with other cardiac congenital disorders.

Adult mouse fibroblasts retain organ-specific transcriptomic identity.

Organ fibroblasts are essential components of homeostatic and diseased tissues. They participate in sculpting the extracellular matrix, sensing the microenvironment, and communicating with other resident cells. Recent studies have revealed transcriptomic heterogeneity among fibroblasts within and between organs. To dissect the basis of interorgan heterogeneity, we compare the gene expression of murine fibroblasts from different tissues (tail, skin, lung, liver, heart, kidney, and gonads) and show that they display distinct positional and organ-specific transcriptome signatures that reflect their embryonic origins. We demonstrate that expression of genes typically attributed to the surrounding parenchyma by fibroblasts is established in embryonic development and largely maintained in culture, bioengineered tissues and ectopic transplants. Targeted knockdown of key organ-specific transcription factors affects fibroblast functions, in particular genes involved in the modulation of fibrosis and inflammation. In conclusion, our data reveal that adult fibroblasts maintain an embryonic gene expression signature inherited from their organ of origin, thereby increasing our understanding of adult fibroblast heterogeneity. The knowledge of this tissue-specific gene signature may assist in targeting fibrotic diseases in a more precise, organ-specific manner.

Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction.

Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.

Dynamic Interstitial Cell Response during Myocardial Infarction Predicts Resilience to Rupture in Genetically Diverse Mice.

Cardiac ischemia leads to the loss of myocardial tissue and the activation of a repair process that culminates in the formation of a scar whose structural characteristics dictate propensity to favorable healing or detrimental cardiac wall rupture. To elucidate the cellular processes underlying scar formation, here we perform unbiased single-cell mRNA sequencing of interstitial cells isolated from infarcted mouse hearts carrying a genetic tracer that labels epicardial-derived cells. Sixteen interstitial cell clusters are revealed, five of which were of epicardial origin. Focusing on stromal cells, we define 11 sub-clusters, including diverse cell states of epicardial- and endocardial-derived fibroblasts. Comparing transcript profiles from post-infarction hearts in C57BL/6J and 129S1/SvImJ inbred mice, which displays a marked divergence in the frequency of cardiac rupture, uncovers an early increase in activated myofibroblasts, enhanced collagen deposition, and persistent acute phase response in 129S1/SvImJ mouse hearts, defining a crucial time window of pathological remodeling that predicts disease outcome.

Variable outcomes of human heart attack recapitulated in genetically diverse mice.

Clinical variation in patient responses to myocardial infarction (MI) has been difficult to model in laboratory animals. To assess the genetic basis of variation in outcomes after heart attack, we characterized responses to acute MI in the Collaborative Cross (CC), a multi-parental panel of genetically diverse mouse strains. Striking differences in post-MI functional, morphological, and myocardial scar features were detected across 32 CC founder and recombinant inbred strains. Transcriptomic analyses revealed a plausible link between increased intrinsic cardiac oxidative phosphorylation levels and MI-induced heart failure. The emergence of significant quantitative trait loci for several post-MI traits indicates that utilizing CC strains is a valid approach for gene network discovery in cardiovascular disease, enabling more accurate clinical risk assessment and prediction.

Doxorubicin-Induced Cardiotoxicity in Collaborative Cross (CC) Mice Recapitulates Individual Cardiotoxicity in Humans.

Anthracyclines cause progressive cardiotoxicity whose ultimate severity is individual to the patient. Genetic determinants contributing to this variation are difficult to study using current mouse models. Our objective was to determine whether a spectrum of anthracycline induced cardiac disease can be elicited across 10 Collaborative Cross mouse strains given the same dose of doxorubicin. Mice from ten distinct strains were given 5 mg/kg of doxorubicin intravenously once weekly for 5 weeks (total 25 mg/kg). Mice were killed at acute or chronic timepoints. Body weight was assessed weekly, followed by terminal complete blood count, pathology and a panel of biomarkers. Linear models were fit to assess effects of treatment, sex, and sex-by-treatment interactions for each timepoint. Impaired growth and cardiac pathology occurred across all strains. Severity of these varied by strain and sex, with greater severity in males. Cardiac troponin I and myosin light chain 3 demonstrated strain- and sex-specific elevations in the acute phase with subsequent decline despite ongoing progression of cardiac disease. Acute phase cardiac troponin I levels predicted the ultimate severity of cardiac pathology poorly, whereas myosin light chain 3 levels predicted the extent of chronic cardiac injury in males. Strain- and sex-dependent renal toxicity was evident. Regenerative anemia manifested during the acute period. We confirm that variable susceptibility to doxorubicin-induced cardiotoxicity observed in humans can be modeled in a panel of CC strains. In addition, we identified a potential predictive biomarker in males. CC strains provide reproducible models to explore mechanisms contributing to individual susceptibility in humans.