SPARC regulates collagen interaction with cardiac fibroblast cell surfaces

Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [3H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α1(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.

Download Full-text

Loss of Function in Dopamine D3 Receptor Attenuates Left Ventricular Cardiac Fibroblast Migration and Proliferation in vitro

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.732282 ◽

2021 ◽

Vol 8 ◽

Author(s):

Andrew Kisling ◽

Shannon Byrne ◽

Rohan U. Parekh ◽

Deepthy Melit-Thomas ◽

Lisandra E. de Castro Brás ◽

...

Keyword(s):

Cardiac Tissue ◽

Cardiac Fibroblasts ◽

Fibroblast Cell ◽

Left Ventricular ◽

Cardiac Fibroblast ◽

Loss Of Function ◽

Fibroblast Migration ◽

And Migration ◽

Proliferation And Migration

Evidence suggests the existence of an intracardiac dopaminergic system that plays a pivotal role in regulating cardiac function and fibrosis through G-protein coupled receptors, particularly mediated by dopamine receptor 3 (D3R). However, the expression of dopamine receptors in cardiac tissue and their role in cardiac fibroblast function is unclear. In this brief report, first we determined expression of D1R and D3R both in left ventricle (LV) tissue and fibroblasts. Then, we explored the role of D3R in the proliferation and migration of fibroblast cell cultures using both genetic and pharmaceutical approaches; specifically, we compared cardiac fibroblasts isolated from LV of wild type (WT) and D3R knockout (D3KO) mice in response to D3R-specific pharmacological agents. Finally, we determined if loss of D3R function could significantly alter LV fibroblast expression of collagen types I (Col1a1) and III (Col3a1). Cardiac fibroblast proliferation was attenuated in D3KO cells, mimicking the behavior of WT cardiac fibroblasts treated with D3R antagonist. In response to scratch injury, WT cardiac fibroblasts treated with the D3R agonist, pramipexole, displayed enhanced migration compared to control WT and D3KO cells. Loss of function in D3R resulted in attenuation of both proliferation and migration in response to scratch injury, and significantly increased the expression of Col3a1 in LV fibroblasts. These findings suggest that D3R may mediate cardiac fibroblast function during the wound healing response. To our knowledge this is the first report of D3R's expression and functional significance directly in mouse cardiac fibroblasts.

Download Full-text

Cardiac myofibroblasts differentiated in 3D culture exhibit distinct changes in collagen I production, processing, and matrix deposition

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00153.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. H2924-H2932 ◽

Cited By ~ 40

Author(s):

Felicitta Poobalarahi ◽

Catalin F. Baicu ◽

Amy D. Bradshaw

Keyword(s):

Cardiac Fibroblasts ◽

Three Dimensional ◽

3D Culture ◽

Pressure Overload ◽

Fibroblast Cell ◽

Collagen I ◽

Matrix Deposition ◽

Nylon Mesh ◽

Ecm Proteins ◽

Cardiac Myofibroblasts

Myofibroblasts are a differentiated fibroblast cell type characterized by increased contractile capacity and elevated production of extracellular matrix (ECM) proteins. In the heart, myofibroblast expression is implicated in fibrosis associated with pressure-overload hypertrophy, among other pathologies. Although enhanced expression of ECM proteins by myofibroblasts is established, few studies have addressed the nature of the ECM deposited by myofibroblasts. To characterize ECM production and assembly by cardiac myofibroblasts, we developed a three-dimensional (3D) culture system using primary cardiac fibroblasts seeded into a nylon mesh that allows us to reversibly interconvert between myofibroblast and fibroblast phenotypes. We report that an increase in collagen I production by myofibroblasts was accompanied by a significant increase in collagen deposition into insoluble ECM. Furthermore, myofibroblasts exhibited increased levels of procollagen α1(I) with C-propeptide attached (and N-propeptide removed) relative to procollagen α1(I) compared with fibroblast cultures. An increase in production of the myofibroblast-associated splice variant of fibronectin (EDA-Fn) was seen in myofibroblast 3D cultures. Because the regulation of procollagen I processing is known to have profound effects on ECM assembly, differences in procollagen I secretion and maturation coupled with expression of EDA-Fn are shown to contribute to the production of a distinct ECM by the cardiac myofibroblast.

Download Full-text

Abstract 277: Microrna-33 Promotes Cardiac Fibrosis by Maintaining Cellular Lipid Homeostasis

Circulation Research ◽

10.1161/res.119.suppl_1.277 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Masataka Nishiga ◽

Takahiro Horie ◽

Yasuhide Kuwabara ◽

Osamu Baba ◽

Tetsushi Nakao ◽

...

Keyword(s):

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Pressure Overload ◽

Cholesterol Content ◽

Atp Binding Cassette Transporter ◽

Primary Fibroblasts ◽

Proliferation In Vitro ◽

Altered Lipid

Background: A highly conserved microRNA, miR-33 is considered as a potential therapeutic target for atherosclerosis, because recent reports, including ours, indicated miR-33 has atherogenic effects by reducing HDL-C. However, the functions of miR-33 in heart failure remain to be elucidated. Methods and results: To clarify the functions of miR-33 involved in cardiac hypertrophy and fibrosis in vivo, we investigated the responses to pressure overload by transverse aortic constriction (TAC) in miR-33 deficient (KO) mice. When subjected to TAC, miR-33 expression level was significantly up-regulated in wild-type (WT) left ventricles, whereas miR-33 KO hearts displayed no less hypertrophic responses than WT hearts. However, interestingly, histological and gene expression analyses showed ameliorated cardiac fibrosis in miR-33 KO hearts compared to WT hearts. Furthermore, we generated cardiac fibroblast specific miR-33 deficient mice, which also showed ameliorated cardiac fibrosis when they were subjected to TAC. We also found that cardiac fibroblasts were mainly responsible for miR-33 expression in the heart, because its expression was about 4-folds higher in isolated primary cardiac fibroblasts than cardiomyocytes. Deficiency of miR-33 impaired cell proliferation in primary fibroblasts, which was considered due to altered lipid raft cholesterol content by up-regulated ATP-binding cassette transporter A1/G1. Conclusion: Deficiency of miR-33 impaired fibroblast proliferation in vitro, and ameliorated cardiac fibrosis induced by pressure overload in vivo.

Download Full-text

Abstract 292: TRPA1 Promoting Myofibroblast Differentiation Mediate Pressure Overload-Induced Cardiac Fibrosis

Circulation ◽

10.1161/circ.138.suppl_2.292 ◽

2018 ◽

Vol 138 (Suppl_2) ◽

Author(s):

Shuang Li ◽

Dong Han ◽

Dachun Yang

Keyword(s):

Knockout Mice ◽

Molecular Mechanisms ◽

Cardiac Fibrosis ◽

Transient Receptor Potential ◽

Transforming Growth Factor ◽

Ventricular Remodeling ◽

Cardiac Fibroblasts ◽

Gene Transfection ◽

Pressure Overload

Background: Hypertensive ventricular remodeling is a common cause of heart failure. Activation and accumulation of cardiac fibroblasts is the key contributors to this progression. Our previous studies indicate that transient receptor potential ankyrin 1 (TRPA1), a Ca 2+ channel necessary and sufficient, play a prominent role in ventricular remodeling. However, the molecular mechanisms regulating remain poorly understood. Methods: We used TRPA1 agonists cinnamaldehyde (CA) pretreatment and TRPA1 knockout mice to understand the role of TRPA1 in ventricular remodeling of hypertensive heart. We also examine the mechanisms through gene transfection and in vitro experiments. Results: TRPA1 overexpression fully activated myofibroblast transformation, while fibroblasts lacking TRPA1 were refractory to transforming growth factor β (TGF-β) -induced transdifferentiation. TRPA1 knockout mice showed hypertensive ventricular remodeling reversal following pressure overload. We found that the TGF-β induced TRPA1 expression through calcineurin-NFAT-Dyrk1A signaling pathway via the TRPA1 promoter. Once induced, TRPA1 activates the Ca 2+ -responsive protein phosphatase calcineurin, which itself induced myofibroblast transdifferentiation. Moreover, inhibition of calcineurin prevented TRPA1-dependent transdifferentiation. Conclusion: Our study provides the first evidence that TRPA1 regulation in cardiac fibroblasts transformation in response to hypertensive stimulation. The results suggesting a comprehensive pathway for myofibroblast formation in conjunction with TGF-β, Calcineurin, NFAT and Dyrk1A. Furthermore, these data indicate that negative modulation of cardiac fibroblast TRPA1 may represent a therapeutic strategy against hypertensive cardiac remodeling.

Download Full-text

Abstract 89: 17beta-Estradiol-activated Estrogen Receptors Attenuate Cardiac Fibrosis Through Inhibition of Collagen I and III Expression in Female Hearts

Circulation Research ◽

10.1161/res.119.suppl_1.89 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Elke Dworatzek ◽

Shokoufeh Mahmoodzadeh ◽

Christina Westphal ◽

Daniela Fliegner ◽

Vera Regitz-Zagrosek

Keyword(s):

Estrogen Receptors ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Pressure Overload ◽

Left Ventricular ◽

Collagen I ◽

Sham Operation ◽

Female Rat ◽

Female Mice ◽

Gene And Protein Expression

Objectives: Female pressure-overloaded hearts show less fibrosis compared with males. 17β-Estradiol (E2) attenuates cardiac fibrosis in female mice. Whether this is mediated by direct E2-effects on collagen synthesis is still unknown. Therefore, we investigated the role of E2 and estrogen receptors (ER) on collagen I and III expression and analyzed involved mechanisms. Methods: Female C57BL/6J mice (7 weeks) underwent sham operation, ovariectomy (OVX), OVX with E2-supplementation (390mg E2-containing pellets) or placebo. After 2 weeks, animals underwent transverse aortic constriction (TAC) or sham surgery. Mice were sacrificed after 9 weeks. Collagen amount, collagen I and III protein in left ventricular tissue were detected by Sirius Red and antibody staining, respectively. Gene and protein expression were determined by quantitative Real-Time PCR and Western blot. Adult female rat cardiac fibroblasts were treated with E2 (10 -8 M), vehicle, ERα- and β-agonists (10 -7 M) for 24h or pre-treated with PD98059 for 1h. ER binding to the collagen I and III promoter was analyzed by chromatin immunoprecipitation assays. Findings: In female OVX mice, undergoing TAC surgery, E2-supplementation significantly reduced collagen deposition, collagen I and III mRNA and protein levels in comparison with mice without E2. In female rat cardiac fibroblasts, E2 significantly down-regulated collagen I and III mRNA and protein level. Specific ER-agonist-treatment showed that E2-mediated regulation of collagen I and III expression was mediated via activation of ERα, but not ERβ. Further, upon E2-treatment, ERα was phosphorylated at Ser118, which occurred by E2-induced activation of ERK1/2 signaling. Furthermore, we could show that ERα and ERβ bind to two putative half-palindromic estrogen response elements within the collagen I and III promoter in female cardiac fibroblasts. Conclusion: E2 inhibits cardiac collagen I and III mRNA and protein in female mice under pressure overload. Data from rat female cardiac fibroblasts suggest that this is mediated via E2-activated ERK1/2 signaling and ERα, which binds with ERβ to the collagen I and III promoter. Understanding of how E2/ER attenuate collagen I and III expression in pathological hypertrophy may improve therapy.

Download Full-text

Novel role for cardiac myocyte-derived β-2 microglobulin in mediating cardiac fibrosis

Clinical Science ◽

10.1042/cs20180681 ◽

2018 ◽

Vol 132 (19) ◽

pp. 2117-2120

Author(s):

Michael J. Boyer ◽

Satoru Eguchi

Keyword(s):

Cardiac Fibrosis ◽

Cardiac Myocyte ◽

Cardiac Fibroblasts ◽

Significant Risk ◽

Growth Factor Receptor ◽

Pressure Overload ◽

Significant Risk Factor ◽

Cardiac Complications

Hypertension is a significant risk factor for the development of cardiovascular ailments, including ischemic heart disease and diastolic dysfunction. In a recent issue of Clinical Science, Li et al. [Clin. Sci. (2018) 132, 1855–1874] report that β-2 microglobulin (β2M) is a novel secreted soluble factor released by cardiac myocytes during pressure overload that promotes profibrotic gene expression in cardiac fibroblasts both in vitro and in vivo. Their study further identifies elevated β2M levels as a possible biomarker for hypertensive patients with cardiac complications. The authors propose a mechanism that mechanically stretched cardiomyocytes release soluble β2M which, through paracrine communication with cardiac fibroblasts, transactivates epidermal growth factor receptor (EGFR) to initiate acute signal transduction and up-regulate profibrotic genes, thereby promoting fibrosis. Here, we will discuss the background, significance of the study, alternative mechanisms, and future directions.

Download Full-text

Sex-specific regulation of collagen I and III expression by 17β-Estradiol in cardiac fibroblasts: role of estrogen receptors

Cardiovascular Research ◽

10.1093/cvr/cvy185 ◽

2018 ◽

Vol 115 (2) ◽

pp. 315-327 ◽

Cited By ~ 25

Author(s):

Elke Dworatzek ◽

Shokoufeh Mahmoodzadeh ◽

Cindy Schriever ◽

Kana Kusumoto ◽

Lisa Kramer ◽

...

Keyword(s):

Estrogen Receptor Alpha ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Pressure Overload ◽

Collagen I ◽

Female Rat ◽

17Β Estradiol ◽

Specific Regulation

Abstract Aims Sex differences in cardiac fibrosis point to the regulatory role of 17β-Estradiol (E2) in cardiac fibroblasts (CF). We, therefore, asked whether male and female CF in rodent and human models are differentially susceptible to E2, and whether this is related to sex-specific activation of estrogen receptor alpha (ERα) and beta (ERβ). Methods and results In female rat CF (rCF), 24 h E2-treatment (10−8 M) led to a significant down-regulation of collagen I and III expression, whereas both collagens were up-regulated in male rCF. E2-induced sex-specific collagen regulation was also detected in human CF, indicating that this regulation is conserved across species. Using specific ERα- and ERβ-agonists (10−7 M) for 24 h, we identified ERα as repressive and ERβ as inducing factor in female and male rCF, respectively. In addition, E2-induced ERα phosphorylation at Ser118 only in female rCF, whereas Ser105 phosphorylation of ERβ was exclusively found in male rCF. Further, in female rCF we found both ER bound to the collagen I and III promoters using chromatin immunoprecipitation assays. In contrast, in male rCF only ERβ bound to both promoters. In engineered connective tissues (ECT) from rCF, collagen I and III mRNA were down-regulated in female ECT and up-regulated in male ECT by E2. This was accompanied by an impaired condensation of female ECT, whereas male ECT showed an increased condensation and stiffness upon E2-treatment, analysed by rheological measurements. Finally, we confirmed the E2-effect on both collagens in an in vivo mouse model with ovariectomy for E2 depletion, E2 substitution, and pressure overload by transverse aortic constriction. Conclusion The mechanism underlying the sex-specific regulation of collagen I and III in the heart appears to involve E2-mediated differential ERα and ERβ signaling in CFs.

Download Full-text

Computational Approaches to Understanding the Role of Fibroblast-Myocyte Interactions in Cardiac Arrhythmogenesis

BioMed Research International ◽

10.1155/2015/465714 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 11

Author(s):

Tashalee R. Brown ◽

Trine Krogh-Madsen ◽

David J. Christini

Keyword(s):

Gap Junctions ◽

Potential Role ◽

Cardiac Tissue ◽

Cardiac Fibroblasts ◽

Cardiac Arrhythmogenesis ◽

Voltage Dependent ◽

Underlying Mechanisms ◽

Slow Propagation

The adult heart is composed of a dense network of cardiomyocytes surrounded by nonmyocytes, the most abundant of which are cardiac fibroblasts. Several cardiac diseases, such as myocardial infarction or dilated cardiomyopathy, are associated with an increased density of fibroblasts, that is, fibrosis. Fibroblasts play a significant role in the development of electrical and mechanical dysfunction of the heart; however the underlying mechanisms are only partially understood. One widely studied mechanism suggests that fibroblasts produce excess extracellular matrix, resulting in collagenous septa. These collagenous septa slow propagation, cause zig-zag conduction paths, and decouple cardiomyocytes resulting in a substrate for arrhythmia. Another emerging mechanism suggests that fibroblasts promote arrhythmogenesis through direct electrical interactions with cardiomyocytes via gap junctions. Due to the challenges of investigating fibroblast-myocyte coupling in native cardiac tissue, computational modeling andin vitroexperiments have facilitated the investigation into the mechanisms underlying fibroblast-mediated changes in cardiomyocyte action potential morphology, conduction velocity, spontaneous excitability, and vulnerability to reentry. In this paper, we summarize the major findings of the existing computational studies investigating the implications of fibroblast-myocyte interactions in the normal and diseased heart. We then present investigations from our group into the potential role of voltage-dependent gap junctions in fibroblast-myocyte interactions.

Download Full-text

Effects of the absence of procollagen C-endopeptidase enhancer-2 on myocardial collagen accumulation in chronic pressure overload

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00227.2012 ◽

2012 ◽

Vol 303 (2) ◽

pp. H234-H240 ◽

Cited By ~ 27

Author(s):

Catalin F. Baicu ◽

Yuhua Zhang ◽

An O. Van Laer ◽

Ludivine Renaud ◽

Michael R. Zile ◽

...

Keyword(s):

Cardiac Fibroblasts ◽

Pressure Overload ◽

Left Ventricular ◽

Bone Morphogenic Protein ◽

Collagen Content ◽

Fibrillar Collagen ◽

Collagen Deposition ◽

Myocardial Stiffness ◽

Collagen Accumulation

Cardiac interstitial fibrillar collagen accumulation, such as that associated with chronic pressure overload (PO), has been shown to impair left ventricular diastolic function. Therefore, insight into cellular mechanisms that mediate excessive collagen deposition in the myocardium is pivotal to this important area of research. Collagen is secreted as a soluble procollagen molecule with NH2- and COOH (C)-terminal propeptides. Cleavage of these propeptides is required for collagen incorporation to insoluble collagen fibrils. The C-procollagen proteinase, bone morphogenic protein 1, cleaves the C-propeptide of procollagen. Procollagen C-endopeptidase enhancer (PCOLCE) 2, an enhancer of bone morphogenic protein-1 activity in vitro, is expressed at high levels in the myocardium. However, whether the absence of PCOLCE2 affects collagen content at baseline or after PO induced by transverse aortic constriction (TAC) has never been examined. Accordingly, in vivo procollagen processing and deposition were examined in wild-type (WT) and PCOLCE2-null mice. No significant differences in collagen content or myocardial stiffness were detected in non-TAC (control) PCOLCE2-null versus WT mice. After TAC-induced PO, PCOLCE2-null hearts demonstrated a lesser collagen content (PCOLCE2-null TAC collagen volume fraction, 0.41% ± 0.07 vs. WT TAC, 1.2% ± 0.3) and lower muscle stiffness compared with WT PO hearts [PCOLCE2-null myocardial stiffness (β), 0.041 ± 0.002 vs. WT myocardial stiffness, 0.065 ± 0.001]. In addition, in vitro, PCOLCE2-null cardiac fibroblasts exhibited reductions in efficiency of C-propeptide cleavage, as demonstrated by increases in procollagen α1(I) and decreased levels of processed collagen α1(I) versus WT cardiac fibroblasts. Hence, PCOLCE2 is required for efficient procollagen processing and deposition of fibrillar collagen in the PO myocardium. These results support a critical role for procollagen processing in the regulation of collagen deposition in the heart.

Download Full-text

Chronic Benzene Exposure Aggravates Pressure Overload-Induced Cardiac Dysfunction

10.1101/2021.08.31.458367 ◽

2021 ◽

Author(s):

Igor N Zelko ◽

Sujith Dassanayaka ◽

Marina V Malovichko ◽

Caitlin M Howard ◽

Lauren F Garrett ◽

...

Keyword(s):

Heart Failure ◽

Endothelial Cells ◽

Cardiac Function ◽

Cardiac Tissue ◽

Pressure Overload ◽

Endothelial Activation ◽

Air Exposure ◽

Benzene Exposure ◽

Increased Risk

Benzene is a ubiquitous environmental pollutant abundant in household products, petrochemicals and cigarette smoke. Benzene is a well-known carcinogen in humans and experimental animals; however, little is known about the cardiovascular toxicity of benzene. Recent population-based studies indicate that benzene exposure is associated with an increased risk for heart failure. Nonetheless, it is unclear whether benzene exposure is sufficient to induce and/or exacerbate heart failure. We examined the effects of benzene (50 ppm, 6 h/day, 5 days/week, 6 weeks) or HEPA-filtered air exposure on transverse aortic constriction (TAC)-induced pressure overload in male C57BL/6J mice. Our data show that benzene exposure had no effect on cardiac function in the Sham group; however, it significantly compromised cardiac function as depicted by a significant decrease in fractional shortening and ejection fraction, as compared with TAC/Air-exposed mice. RNA-seq analysis of the cardiac tissue from the TAC/benzene-exposed mice showed a significant increase in several genes associated with adhesion molecules, cell-cell adhesion, inflammation, and stress response. In particular, neutrophils were implicated in our unbiased analyses. Indeed, immunofluorescence studies showed that TAC/benzene exposure promotes infiltration of CD11b+/S100A8+/myeloperoxidase+-positive neutrophils in the hearts by 3-fold. In vitro, the benzene metabolites, hydroquinone and catechol, induced the expression of P-selectin in cardiac microvascular endothelial cells by 5-fold and increased the adhesion of neutrophils to these endothelial cells by 1.5-2.0-fold. Benzene metabolite-induced adhesion of neutrophils to the endothelial cells was attenuated by anti-P-selectin antibody. Together, these data suggest that benzene exacerbates heart failure by promoting endothelial activation and neutrophil recruitment.

Download Full-text