Scleraxis and fibrosis in the pressure-overloaded heart.

AIMS: In response to pro-fibrotic signals, scleraxis regulates cardiac fibroblast activation in vitro via transcriptional control of key fibrosis genes such as collagen and fibronectin; however, its role in vivo is unknown. The present study assessed the impact of scleraxis loss on fibroblast activation, cardiac fibrosis, and dysfunction in pressure overload-induced heart failure. METHODS AND RESULTS: Scleraxis expression was upregulated in the hearts of non-ischemic dilated cardiomyopathy patients, and in mice subjected to pressure overload by transverse aortic constriction (TAC). Tamoxifen-inducible fibroblast-specific scleraxis knockout (Scx-fKO) completely attenuated cardiac fibrosis, and significantly improved cardiac systolic function and ventricular remodelling, following TAC compared to Scx+/+ TAC mice, concomitant with attenuation of fibroblast activation. Scleraxis deletion, after the establishment of cardiac fibrosis, attenuated the further functional decline observed in Scx+/+ mice, with a reduction in cardiac myofibroblasts. Notably, scleraxis knockout reduced pressure overload-induced mortality from 33% to zero, without affecting the degree of cardiac hypertrophy. Scleraxis directly regulated transcription of the myofibroblast marker periostin, and cardiac fibroblasts lacking scleraxis failed to upregulate periostin synthesis and secretion in response to pro-fibrotic transforming growth factor ß. CONCLUSION: Scleraxis governs fibroblast activation in pressure overload-induced heart failure, and scleraxis knockout attenuated fibrosis and improved cardiac function and survival. These findings identify scleraxis as a viable target for the development of novel anti-fibrotic treatments.

Canadian Contributions in Fibroblast Biology.

Fibroblasts are stromal cells found in virtually every tissue and organ of the body. For many years, these cells were often considered to be secondary in functional importance to parenchymal cells. Over the past 2 decades, focused research into the roles of fibroblasts has revealed important roles for these cells in the homeostasis of healthy tissue, and has demonstrated that activation of fibroblasts to myofibroblasts is a key step in disease initiation and progression in many tissues, with fibrosis now recognized as not only an outcome of disease, but also a central contributor to tissue dysfunction, particularly in the heart and lungs. With a growing understanding of both fibroblast and myofibroblast heterogeneity, and the deciphering of the humoral and mechanical cues that impact the phenotype of these cells, fibroblast biology is rapidly becoming a major focus in biomedical research. In this review, we provide an overview of fibroblast and myofibroblast biology, particularly in the heart, and including a discussion of pathophysiological processes such as fibrosis and scarring. We then discuss the central role of Canadian researchers in moving this field forwards, particularly in cardiac fibrosis, and highlight some of the major contributions of these individuals to our understanding of fibroblast and myofibroblast biology in health and disease.

Regulation of Cardiac Fibroblast GLS1 Expression by Scleraxis.

Fibrosis is an energy-intensive process requiring the activation of fibroblasts to myofibroblasts, resulting in the increased synthesis of extracellular matrix proteins. Little is known about the transcriptional control of energy metabolism in cardiac fibroblast activation, but glutaminolysis has been implicated in liver and lung fibrosis. Here we explored how pro-fibrotic TGFß and its effector scleraxis, which drive cardiac fibroblast activation, regulate genes involved in glutaminolysis, particularly the rate-limiting enzyme glutaminase (GLS1). The GLS1 inhibitor CB-839 attenuated TGFß-induced fibroblast activation. Cardiac fibroblast activation to myofibroblasts by scleraxis overexpression increased glutaminolysis gene expression, including GLS1, while cardiac fibroblasts from scleraxis-null mice showed reduced expression. TGFß induced GLS1 expression and increased intracellular glutamine and glutamate levels, indicative of increased glutaminolysis, but in scleraxis knockout cells, these measures were attenuated, and the response to TGFß was lost. The knockdown of scleraxis in activated cardiac fibroblasts reduced GLS1 expression by 75%. Scleraxis transactivated the human GLS1 promoter in luciferase reporter assays, and this effect was dependent on a key scleraxis-binding E-box motif. These results implicate scleraxis-mediated GLS1 expression as a key regulator of glutaminolysis in cardiac fibroblast activation, and blocking scleraxis in this process may provide a means of starving fibroblasts of the energy required for fibrosis.

What's in a name? On fibroblast phenotype and nomenclature 1.

Fibroblasts have long been recognized as important stromal cells, playing key roles in synthesizing and maintaining the extracellular matrix, but historically were treated as a relatively uniform cell type. Studies in recent years have revealed a surprising level of heterogeneity of fibroblasts across tissues, and even within organs such as the skin and heart. This heterogeneity may have functional consequences, including during stress and disease. While the field has moved forward quickly to begin to address the scientific import of this heterogeneity, the descriptive language used for these cells has not kept pace, particularly when considering the phenotype changes that occur as fibroblasts convert to myofibroblasts in response to injury. We discuss here the nature and sources of the heterogeneity of fibroblasts, and review how our understanding of the complexity of the fibroblast to myofibroblast phenotype conversion has changed with increasing scrutiny. We propose that the time is opportune to reevaluate how we name and describe these cells, particularly as they transition to myofibroblasts through discrete stages. A standardized nomenclature is essential to address the confusion that currently exists in the literature as to the usage of terms like myofibroblast and the description of fibroblast phenotype changes in disease.

Scleraxis regulates Twist1 and Snai1 expression in the epithelial-to-mesenchymal transition.

Numerous physiological and pathological events, from organ development to cancer and fibrosis, are characterized by an epithelial-to-mesenchymal transition (EMT), whereby adherent epithelial cells convert to migratory mesenchymal cells. During cardiac development, proepicardial organ epithelial cells undergo EMT to generate fibroblasts. Subsequent stress or damage induces further phenotype conversion of fibroblasts to myofibroblasts, causing fibrosis via synthesis of an excessive extracellular matrix. We have previously shown that the transcription factor scleraxis is both sufficient and necessary for the conversion of cardiac fibroblasts to myofibroblasts and found that scleraxis knockout reduced cardiac fibroblast numbers by 50%, possibly via EMT attenuation. Scleraxis induced expression of the EMT transcriptional regulators Twist1 and Snai1 via an unknown mechanism. Here, we report that scleraxis binds to E-box consensus sequences within the Twist1 and Snai1 promoters to transactivate these genes directly. Scleraxis upregulates expression of both genes in A549 epithelial cells and in cardiac myofibroblasts. Transforming growth factor-ß induces EMT, fibrosis, and scleraxis expression, and we found that transforming growth factor-ß-mediated upregulation of Twist1 and Snai1 completely depends on the presence of scleraxis. Snai1 knockdown upregulated the epithelial marker E-cadherin; however, this effect was lost after scleraxis overexpression, suggesting that scleraxis may repress E-cadherin expression. Together, these results indicate that scleraxis can regulate EMT via direct transactivation of the Twist1 and Snai1 genes. Given the role of scleraxis in also driving the myofibroblast phenotype, scleraxis appears to be a critical controller of fibroblast genesis and fate in the myocardium and thus may play key roles in wound healing and fibrosis. NEW & NOTEWORTHY The molecular mechanism by which the transcription factor scleraxis mediates Twist1 and Snai1 gene expression was determined. These results reveal a novel means of transcriptional regulation of epithelial-to-mesenchymal transition and demonstrate that transforming growth factor-ß-mediated epithelial-to-mesenchymal transition is dependent on scleraxis, providing a potential target for controlling this process.

Regulation of cardiac fibroblast MMP2 gene expression by scleraxis.

Remodeling of the cardiac extracellular matrix is responsible for a number of the detrimental effects on heart function that arise secondary to hypertension, diabetes and myocardial infarction. This remodeling consists both of an increase in new matrix protein synthesis, and an increase in the expression of matrix metalloproteinases (MMPs) that degrade existing matrix structures. Previous studies utilizing knockout mice have demonstrated clearly that MMP2 plays a pathogenic role during matrix remodeling, thus it is important to understand the mechanisms that regulate MMP2 gene expression. We have shown that the transcription factor scleraxis is an important inducer of extracellular matrix gene expression in the heart that may also control MMP2 expression. In the present study, we demonstrate that scleraxis directly transactivates the proximal MMP2 gene promoter, resulting in increased histone acetylation, and identify a specific E-box sequence in the promoter to which scleraxis binds. Cardiac myo-fibroblasts isolated from scleraxis knockout mice exhibited dramatically decreased MMP2 expression; however, scleraxis over-expression in knockout cells could rescue this loss. We further show that regulation of MMP2 gene expression by the pro-fibrotic cytokine TGFß occurs via a scleraxis-dependent mechanism: TGFß induces recruitment of scleraxis to the MMP2 promoter, and TGFß was unable to up-regulate MMP2 expression in cells lacking scleraxis due to either gene knockdown or knockout. These results reveal that scleraxis can exert control over both extracellular matrix synthesis and breakdown, and thus may contribute to matrix remodeling in wound healing and disease.