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.

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.

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/.

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.

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.

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.

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.

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.

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.

Metformin intervention prevents cardiac dysfunction in a murine model of adult congenital heart disease.

OBJECTIVE: Congenital heart disease (CHD) is the most frequent birth defect worldwide. The number of adult patients with CHD, now referred to as ACHD, is increasing with improved surgical and treatment interventions. However the mechanisms whereby ACHD predisposes patients to heart dysfunction are still unclear. ACHD is strongly associated with metabolic syndrome, but how ACHD interacts with poor modern lifestyle choices and other comorbidities, such as hypertension, obesity, and diabetes, is mostly unknown. METHODS: We used a newly characterized mouse genetic model of ACHD to investigate the consequences and the mechanisms associated with combined obesity and ACHD predisposition. Metformin intervention was used to further evaluate potential therapeutic amelioration of cardiac dysfunction in this model. RESULTS: ACHD mice placed under metabolic stress (high fat diet) displayed decreased left ventricular ejection fraction. Comprehensive physiological, biochemical, and molecular analysis showed that ACHD hearts exhibited early changes in energy metabolism with increased glucose dependence as main cardiac energy source. These changes preceded cardiac dysfunction mediated by exposure to high fat diet and were associated with increased disease severity. Restoration of metabolic balance by metformin administration prevented the development of heart dysfunction in ACHD predisposed mice. CONCLUSIONS: This study reveals that early metabolic impairment reinforces heart dysfunction in ACHD predisposed individuals and diet or pharmacological interventions can be used to modulate heart function and attenuate heart failure. Our study suggests that interactions between genetic and metabolic disturbances ultimately lead to the clinical presentation of heart failure in patients with ACHD. Early manipulation of energy metabolism may be an important avenue for intervention in ACHD patients to prevent or delay onset of heart failure and secondary comorbidities. These interactions raise the prospect for a translational reassessment of ACHD presentation in the clinic.

The interstitium in cardiac repair: role of the immune-stromal cell interplay.

Cardiac regeneration, that is, restoration of the original structure and function in a damaged heart, differs from tissue repair, in which collagen deposition and scar formation often lead to functional impairment. In both scenarios, the early-onset inflammatory response is essential to clear damaged cardiac cells and initiate organ repair, but the quality and extent of the immune response vary. Immune cells embedded in the damaged heart tissue sense and modulate inflammation through a dynamic interplay with stromal cells in the cardiac interstitium, which either leads to recapitulation of cardiac morphology by rebuilding functional scaffolds to support muscle regrowth in regenerative organisms or fails to resolve the inflammatory response and produces fibrotic scar tissue in adult mammals. Current investigation into the mechanistic basis of homeostasis and restoration of cardiac function has increasingly shifted focus away from stem cell-mediated cardiac repair towards a dynamic interplay of cells composing the less-studied interstitial compartment of the heart, offering unexpected insights into the immunoregulatory functions of cardiac interstitial components and the complex network of cell interactions that must be considered for clinical intervention in heart diseases.

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.

Combinatorial Ranking of Gene Sets to Predict Disease Relapse: The Retinoic Acid Pathway in Early Prostate Cancer.

BACKGROUND: Quantitative high-throughput data deposited in consortia such as International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) present opportunities and challenges for computational analyses. METHODS: We present a computational strategy to systematically rank and investigate a large number (210-220) of clinically testable gene sets, using combinatorial gene subset generation and disease-free survival (DFS) analyses. This approach integrates protein-protein interaction networks, gene expression, DNA methylation, and copy number data, in association with DFS profiles from patient clinical records. RESULTS: As a case study, we applied this pipeline to systematically analyze the role of ALDH1A2 in prostate cancer (PCa). We have previously found this gene to have multiple roles in disease and homeostasis, and here we investigate the role of the associated ALDH1A2 gene/protein networks in PCa, using our methodology in combination with PCa patient clinical profiles from ICGC and TCGA databases. Relationships between gene signatures and relapse were analyzed using Kaplan-Meier (KM) log-rank analysis and multivariable Cox regression. Relative expression versus pooled mean from diploid population was used for z-statistics calculation. Gene/protein interaction network analyses generated 11 core genes associated with ALDH1A2; combinatorial ranking of the power set of these core genes identified two gene sets (out of 211 - 1 = 2,047 combinations) with significant correlation with disease relapse (KM log rank p < 0.05). For the more significant of these two sets, referred to as the optimal gene set (OGS), patients have median survival 62.7 months with OGS alterations compared to >150 months without OGS alterations (p = 0.0248, hazard ratio = 2.213, 95% confidence interval = 1.1-4.098). Two genes comprising OGS (CYP26A1 and RDH10) are strongly associated with ALDH1A2 in the retinoic acid (RA) pathways, suggesting a major role of RA signaling in early PCa progression. Our pipeline complements human expertise in the search for prognostic biomarkers in large-scale datasets.

Point mutations in murine Nkx2-5 phenocopy human congenital heart disease and induce pathogenic Wnt signaling.

Mutations in the Nkx2-5 gene are a main cause of congenital heart disease. Several studies have addressed the phenotypic consequences of disrupting the Nkx2-5 gene locus, although animal models to date failed to recapitulate the full spectrum of the human disease. Here, we describe a new Nkx2-5 point mutation murine model, akin to its human counterpart disease-generating mutation. Our model fully reproduces the morphological and physiological clinical presentations of the disease and reveals an understudied aspect of Nkx2-5-driven pathology, a primary right ventricular dysfunction. We further describe the molecular consequences of disrupting the transcriptional network regulated by Nkx2-5 in the heart and show that Nkx2-5-dependent perturbation of the Wnt signaling pathway promotes heart dysfunction through alteration of cardiomyocyte metabolism. Our data provide mechanistic insights on how Nkx2-5 regulates heart function and metabolism, a link in the study of congenital heart disease, and confirms that our models are the first murine genetic models to our knowledge to present all spectra of clinically relevant adult congenital heart disease phenotypes generated by NKX2-5 mutations in patients.

An atypical role for the myeloid receptor Mincle in central nervous system injury.

The C-type lectin Mincle is implicated in innate immune responses to sterile inflammation, but its contribution to associated pathologies is not well understood. Herein, we show that Mincle exacerbates neuronal loss following ischemic but not traumatic spinal cord injury. Loss of Mincle was beneficial in a model of transient middle cerebral artery occlusion but did not alter outcomes following heart or gut ischemia. High functional scores in Mincle KO animals using the focal cerebral ischemia model were accompanied by reduced lesion size, fewer infiltrating leukocytes and less neutrophil-derived cytokine production than isogenic controls. Bone marrow chimera experiments revealed that the presence of Mincle in the central nervous system, rather than recruited immune cells, was the critical regulator of a poor outcome following transient middle cerebral artery occlusion. There was no evidence for a direct role for Mincle in microglia or neural activation, but expression in a subset of macrophages resident in the perivascular niche provided new clues on Mincle's role in ischemic stroke.

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.

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.