BRG1-SWI/SNF-dependent regulation of the Wt1 transcriptional landscape mediates epicardial activity during heart development and disease

Epicardium-derived cells (EPDCs) contribute cardiovascular cell types during development and in adulthood respond to Thymosin \u3b24 (T\u3b24) and myocardial infarction (MI) by reactivating a fetal gene programme to promote neovascularization and cardiomyogenesis. The mechanism for epicardial gene (re-)activation remains elusive. Here we reveal that BRG1, the essential ATPase subunit of the SWI/SNF chromatin-remodelling complex, is required for expression of Wilms' tumour 1 (Wt1), fetal EPDC activation and subsequent differentiation into coronary smooth muscle, and restores Wt1 activity upon MI. BRG1 physically interacts with T\u3b24 and is recruited by CCAAT/enhancer-binding protein \u3b2 (C/EBP\u3b2) to discrete regulatory elements in the Wt1 locus. BRG1-T\u3b24 co-operative binding promotes optimal transcription of Wt1 as the master regulator of embryonic EPDCs. Moreover, chromatin immunoprecipitation-sequencing reveals BRG1 binding at further key loci suggesting SWI/SNF activity across the fetal epicardial gene programme. These findings reveal essential functions for chromatin-remodelling in the activation of EPDCs during cardiovascular development and repair

Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair

The Role of Scleraxis in Heart Valve Development and Disease

Mitral valve prolapse (MVP) affects more than 2% of the population in the United States, and is the most common cause of chronic mitral valve regurgitation (MVR) in developed countries. It is estimated that MVP affects more than 150 million people worldwide. When left untreated, MVP can lead to severe MVR resulting in a myriad of cardiac complications. The mortality rate of MVP patients with severe MVR is over 6% per year. Despite clinical significance, there remains a lack of medical therapies for MVP, with surgical intervention being the most effective treatment to date. Therefore, understanding normal heart valve development, and how it is altered during disease, may provide novel insights into new therapeutic targets. MVP associated with myxomatous mitral valve degeneration (MMVD) is the most common cause of MVR requiring surgery. Healthy mature heart valves are highly organized and composed of stratified layers of elastins, collagens and proteoglycans that collectively provide all of the necessary biomechanical properties for structure-function relationships throughout life. In contrast, diseased valves that suffer from MMVD are pathologically thickened and characterized by an abnormal abundance of extracellular matrix (ECM) proteoglycans, which prevents the valves from closing properly and leads to regurgitation and functional prolapse. Although the ECM composition of mature tri-laminar valves has been well described, little is known about the molecular mechanisms that establish and maintain these highly organized structures. However, the etiology of MVP has historically been associated with genetic connective tissue disorders including Marfan syndrome (MFS). A deeper understanding of ECM regulation in normal valve development will help elucidate conserve pathways in disease as a potential means of therapy. In the current studies, we show that the basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) regulates ECM deposition, with a loss of Scx being largely attributed to a significant decrease in the expression and contribution of chondroitin sulfate proteoglycans (CSPGs) to the mature valve leaflets. In addition, we determine that Scx is sufficient to promote CSPG expression in both embryonic and mature valve cells. We further delineate that canonical Tgf-beta-Smad signaling positively regulates Scx and CSPG expression, while activated MAPK attenuates this pathway in a Tgf-beta-independent manner. We show that MAPK activation is sufficient to stabilize the bHLH activator/repressor Twist1, however conclude that Twist1 does not bind to or transcriptionally regulate Scx. We also show that Scx is increased in a MFS mouse model of MMVD, and overexpression can promote myxomatous phenotypes in otherwise normal human mitral valve interstitial cells. Using this model, we explore the idea that reduced Scx function may potentially rescue myxomatous valve phenotypes in vivo. Furthermore, we have identified previously unappreciated protein-coding and non-protein-coding mRNAs that are differentially expressed in the absence of Scx during valve remodeling stages. We report an enrichment of mRNAs associated with processes related to gene regulation and cellular development. Furthermore, bioinformatics analysis predicted known (Tgf-beta-2) and novel (Onecut1) upstream regulators of Scx during valve remodeling. In addition, we show that the loss of Scx leads to differential changes in mRNA transcripts and alternative splicing of several genes. Together, these findings provide insights into molecular signaling pathways that regulate Scx, and identify novel genes and hierarchical networks that are regulated by Scx during valve development, which may be altered in MMVD

RNA-Seq Analysis to Identify Novel Roles of Scleraxis during Embryonic Mouse Heart Valve Remodeling

<div><p>Heart valve disease affects up to 30% of the population and has been shown to have origins during embryonic development. Valvulogenesis begins with formation of endocardial cushions in the atrioventricular canal and outflow tract regions. Subsequently, endocardial cushions remodel, elongate and progressively form mature valve structures composed of a highly organized connective tissue that provides the necessary biomechanical function throughout life. While endocardial cushion formation has been well studied, the processes required for valve remodeling are less well understood. The transcription factor Scleraxis (Scx) is detected in mouse valves from E15.5 during initial stages of remodeling, and expression remains high until birth when formation of the highly organized mature structure is complete. Heart valves from <i>Scx^-/-</i> mice are abnormally thick and develop fibrotic phenotypes similar to human disease by juvenile stages. These phenotypes begin around E15.5 and are associated with defects in connective tissue organization and valve interstitial cell differentiation. In order to understand the etiology of this phenotype, we analyzed the transcriptome of remodeling valves isolated from E15.5 <i>Scx^-/-</i> embryos using RNA-seq. From this, we have identified a profile of protein and non-protein mRNAs that are dependent on Scx function and using bioinformatics we can predict the molecular functions and biological processes affected by these genes. These include processes and functions associated with gene regulation (methyltransferase activity, DNA binding, Notch signaling), vitamin A metabolism (retinoic acid biosynthesis) and cellular development (cell morphology, cell assembly and organization). In addition, several mRNAs are affected by alternative splicing events in the absence of <i>Scx</i>, suggesting additional roles in post-transcriptional modification. In summary, our findings have identified transcriptome profiles from abnormal heart valves isolated from E15.5 <i>Scx^-/-</i> embryos that could be used in the future to understand mechanisms of heart valve disease in the human population.</p></div

Top 25 most differentially expressed protein-coding mRNAs (>1.5-fold change, p<0.05) in <i>Scx^-/-</i> atrioventricular canal samples compared to <i>Scx^+/+</i> controls.

<p>Top 25 most differentially expressed protein-coding mRNAs (>1.5-fold change, p<0.05) in <i>Scx^-/-</i> atrioventricular canal samples compared to <i>Scx^+/+</i> controls.</p

Exon-level splicing indices of mRNAs affected by alternative splicing events in <i>Scx^-/-</i> samples at E15.5.

<p>Splicing indices of the top 8 mRNAs affected by changes in exon abundance (at least 10 exons) in the absence of Scx. * indicate significant differences in exon abundance (p<0.05).</p

Bioinformatics pathway analysis to predict molecular functions and biological processes significantly affected by the loss of Scx in heart valves at E15.5.

<p>(GO, Gene Ontology).</p

Validation of RNA-seq analysis.

<p>(A) Fold changes in gene expression of <i>Actn4, Aldha1</i> and <i>Dot1L</i> based on RNA-seq analysis. Grey line indicates normalized gene expression levels in <i>Scx^+/+</i> controls (set at 1). (B) qPCR to show fold changes in <i>Scx, Actn4, Aldha1</i> and <i>Dot1L</i> expression in rat VICs infected with an adenovirus overexpressing Scx (AdV-Scx) compared to AdV-GFP infected controls. *, p<0.05, n = 3. Grey line indicates normalized gene expression levels in AdV-GFP treated rat VICs (set at 1). (C-H) Fluorescent immunohistochemistry to detect Type IV Collagen (Col4) (C, D at 10x magnification), Fibromodulin (Fmod) (E, F at 20x magnification) and Heparin sulfate proteoglycan 2 (Hspg2) (G, H at 20x magnification) in mitral valves (highlighted by white line) from E15.5 <i>Scx^+/+</i> controls (C, E, G) and <i>Scx^-/-</i> (D, F, H) embryos. Note reduced expression in tissue sections from <i>Scx^-/-</i> embryos. mv, mitral valve; IVS, interventricular septum.</p