Human cardiac fibrosis-on-a-chip model recapitulates disease hallmarks and can serve as a platform for drug screening

AbstractWhile interstitial fibrosis plays a significant role in heart failure, our understanding of disease progression in humans is limited. To address this limitation, we have engineered a cardiac-fibrosis-on-a-chip model consisting of a microfabricated device with live force measurement capabilities using co-cultured human cardiac fibroblasts and pluripotent stem cell-derived cardiomyocytes. Transforming growth factor-β was used as a trigger for fibrosis. Here, we have reproduced the classic hallmarks of fibrosis-induced heart failure including high collagen deposition, increased tissue stiffness, BNP secretion, and passive tension. Force of contraction was significantly decreased in fibrotic tissues that displayed a transcriptomic signature consistent with human cardiac fibrosis/heart failure. Treatment with an anti-fibrotic drug decreased tissue stiffness and BNP secretion, with corresponding changes in the transcriptomic signature. This model represents an accessible approach to study human heart failure in vitro, and allows for testing anti-fibrotic drugs while facilitating the real-time assessment of cardiomyocyte function.

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High doses of TGF-β potently suppress type I collagen via the transcription factor CUX1

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0669 ◽

2011 ◽

Vol 22 (11) ◽

pp. 1836-1844 ◽

Cited By ~ 26

Author(s):

Maria Fragiadaki ◽

Tetsurou Ikeda ◽

Abigail Witherden ◽

Roger M Mason ◽

David Abraham ◽

...

Keyword(s):

Type I Collagen ◽

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Transforming Growth Factor Β ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Type I ◽

Aberrant Expression

Transforming growth factor-β (TGF-β) is an inducer of type I collagen, and uncontrolled collagen production leads to tissue scarring and organ failure. Here we hypothesize that uncovering a molecular mechanism that enables us to switch off type I collagen may prove beneficial in treating fibrosis. For the first time, to our knowledge, we provide evidence that CUX1 acts as a negative regulator of TGF-β and potent inhibitor of type I collagen transcription. We show that CUX1, a CCAAT displacement protein, is associated with reduced expression of type I collagen both in vivo and in vitro. We show that enhancing the expression of CUX1 results in effective suppression of type I collagen. We demonstrate that the mechanism by which CUX1 suppresses type I collagen is through interfering with gene transcription. In addition, using an in vivo murine model of aristolochic acid (AA)-induced interstitial fibrosis and human AA nephropathy, we observe that CUX1 expression was significantly reduced in fibrotic tissue when compared to control samples. Moreover, silencing of CUX1 in fibroblasts from kidneys of patients with renal fibrosis resulted in increased type I collagen expression. Furthermore, the abnormal CUX1 expression was restored by addition of TGF-β via the p38 mitogen-activated protein kinase pathway. Collectively, our study demonstrates that modifications of CUX1 expression lead to aberrant expression of type I collagen, which may provide a molecular basis for fibrogenesis.

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Abstract 95: The Role of TGFβ Signaling in a Fibrotic cMyBP-C HCM/HF Model

Circulation Research ◽

10.1161/res.119.suppl_1.95 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Qinghang Meng ◽

Bidur Bhandary ◽

Md. Shenuarin Bhuiyan ◽

Hanna Osinska ◽

Jeffrey Robbins

Keyword(s):

Transgenic Mice ◽

Life Span ◽

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Transforming Growth Factor Β ◽

Tgfβ Signaling ◽

Gene Encoding ◽

Western Blots

Purpose: Hypertrophic cardiomyopathy (HCM) is considered one of the most common genetic heart disorders with a prevalence of about 1 in 500 people, with 35% of those affected being attributed to mutations within the gene encoding cardiac myosin-binding protein C (cMyBP-C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40kDa fragment in mouse cardiomyocytes ( Mybp3 40kDa ) leads to HCM, fibrosis and heart failure, mimicking human disease progression. The transforming growth factor-β (TGFβ) signaling pathway has been implicated in a variety of fibrotic processes. The goal of this study is to define the role of TGFβ signaling in distinct cell populations, the cardiomyocyte and fibroblast, in the cMyBP-C HCM/HF model. Methods and results: Masson’s Trichrome staining, PCR arrays, immunohistochemistry and western blots were performed to characterize the fibrotic progression in Mybp3 40kDa transgenic mice. Cardiac fibrosis was initially detected 4 weeks after transgene expression. Extensive interstitial fibrosis and severe atrial fibrosis were detected at 16 weeks. Both canonical and non-canonical TGFβ pathways were active during fibrotic progression. To specifically block TGFβ signaling in Mybp3 40kDa transgenic mice, compound mutant mice were generated, in which the tgfbr1 or tgfbr2 alleles were ablated, either in cardiomyocytes or in activated fibroblasts (myofibroblasts) by αMHC-Cre or Periostin-MerCreMer-Cre respectively. Blockage of TGFβ signaling in either cardiomyocytes or myofibroblasts alleviated cardiac fibrosis. Furthermore, treatment with the non-canonical TGFβ signaling inhibitor MMI-0100 also alleviated cardiac fibrosis and increased the life span of the Mybp3 40kDa transgenic mice. Conclusions: TGFβ signaling is activated in the Mybp3 40kD HCM/HF model. Genetic or pharmaceutical inhibition of TGFβ signaling inhibited fibrosis and increased the life span in this model.

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Abstract 74: The Role of Fibroblast-specific Canonical Tgfβ Signaling in Cardiac Fibrosis

Circulation Research ◽

10.1161/res.117.suppl_1.74 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Hadi Khalil ◽

Onur Kanisicak ◽

Robert N. Correll ◽

Michelle Sargent ◽

Jeffery D. Molkentin

Keyword(s):

Heart Failure ◽

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Contractile Activity ◽

Cardiac Fibroblasts ◽

Pressure Overload ◽

Type I ◽

Tgfβ Signaling ◽

Chronic Heart Disease

Heart failure is a progressive disease characterized by cardiomyocyte loss, interstitial fibrosis, and chamber remodeling. During physiological conditions cardiac fibroblasts contribute to the homeostatic maintenance of myocardial structure as well as the maintenance of biochemical, mechanical and electrical properties of the heart. Injury and/or cytokine stimulation activate fibroblasts which transdifferentiate into myofibroblasts. These newly formed cells secrete extracellular matrix (ECM) for wound healing and tissue remodeling through their contractile activity. Fibrosis mediated by these cells can initially be a beneficial response that acutely scarifies areas after an infarct to prevent wall rupture. However, during chronic disease states such as heart failure, persistent recruitment and activation of fibroblasts leads to excessive deposition of ECM that results in stiffening and pathological remodeling of the ventricles. During chronic heart disease, cardiomyocytes, immune cells and fibroblasts secrete the cytokine transforming growth factor-TGFβ, which activates fibroblasts and promotes their conversion to myofibroblasts. Manipulation of TGFβ by losartan, which antagonizes angiotensin II (AngII) and aspects of TGFβ signaling, has shown some anti-fibrotic effects in cardiovascular remodeling. Also deletion of Tgfbr1 (type I TGFβ receptor) in cardiomyocytes or a TGFβ blocking antibody reduced the fibrotic response after pressure overload. However heart failure was not improved because deleterious TGFβ signaling in fibroblasts persisted. We therefore utilized a novel fibroblast-specific inducible Cre-expressing mouse line (Periostin-MerCreMer) to examine the canonical (Smad2/3) TGFβ signaling within fibroblasts to determine how these cells and their activation mediate disease in heart failure. Our data indicate that fibroblast-specific deletion of Smad3 but not Smad2 was sufficient to significantly inhibit myocardial fibrosis. Smad2/3 double nulls were also generated and analyzed, as were TGFBR1 and TGFBR2 loxp targeted mice, also crossed with the Postn-MerCreMer knockin allele to achieve specificity in activated fibroblasts.

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Cardiomyocyte-specific loss of RNA polymerase II subunit 5-mediating protein causes myocardial dysfunction and heart failure

Cardiovascular Research ◽

10.1093/cvr/cvy307 ◽

2018 ◽

Vol 115 (11) ◽

pp. 1617-1628

Author(s):

Jian Zhang ◽

Jingyi Sheng ◽

Liwei Dong ◽

Yinli Xu ◽

Liming Yu ◽

...

Keyword(s):

Heart Failure ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Genome Stability ◽

Transforming Growth Factor ◽

Myocardial Dysfunction ◽

Transforming Growth Factor Β ◽

And Function ◽

Deficient Mice

AbstractAimsMyocardial dysfunction is an important cause of heart failure (HF). RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP) is a transcriptional mediating protein which co-ordinates cellular processes including gene expression, metabolism, proliferation, and genome stability. However, its role in cardiac disease remains unknown. We aimed to determine the role and regulatory mechanisms of RMP in cardiomyocyte function and the development of HF.Methods and resultsMyocardial RMP expression was examined in human heart tissues from healthy controls and patients with advanced HF. Compared to normal cardiac tissues, RMP levels were significantly decreased in the myocardium of patients with advanced HF. To investigate the role of RMP in cardiac function, Cre-loxP recombinase technology was used to generate tamoxifen-inducible cardiomyocyte-specific Rmp knockout mice. Unexpectedly, cardiomyocyte-specific deletion of Rmp in mice resulted in contractile dysfunction, cardiac dilatation, and fibrosis. Furthermore, the lifespan of cardiac-specific Rmp-deficient mice was significantly shortened when compared with littermates. Mechanistically, we found that chronic HF in Rmp-deficient mice was associated with impaired mitochondrial structure and function, which may be mediated via a transforming growth factor-β/Smad3-proliferator-activated receptor coactivator1α (PGC1α)-dependent mechanism. PGC1α overexpression partially rescued chronic HF in cardiomyocyte-specific Rmp-deficient mice, and Smad3 blockade protected against the loss of PGC1α and adenosine triphosphate content that was induced by silencing RMP in vitro.ConclusionsRMP plays a protective role in chronic HF. RMP may protect cardiomyocytes from injury by maintaining PGC1α-dependent mitochondrial biogenesis and function. The results from this study suggest that RMP may be a potential therapeutic agent for treating HF.

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Noncanonical TGF-β pathways, mTORC1 and Abl, in renal interstitial fibrogenesis

AJP Renal Physiology ◽

10.1152/ajprenal.00320.2009 ◽

2010 ◽

Vol 298 (1) ◽

pp. F142-F149 ◽

Cited By ~ 81

Author(s):

Shinong Wang ◽

Mark C. Wilkes ◽

Edward B. Leof ◽

Raimund Hirschberg

Keyword(s):

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Mammalian Target Of Rapamycin ◽

Mtor Inhibitors ◽

Transforming Growth Factor Β ◽

Renal Diseases ◽

Obstructive Nephropathy ◽

Renal Fibrogenesis

Renal interstitial fibrosis is a major determinant of renal failure in the majority of chronic renal diseases. Transforming growth factor-β (TGF-β) is the single most important cytokine promoting renal fibrogenesis. Recent in vitro studies identified novel non-smad TGF-β targets including p21-activated kinase-2 (PAK2), the abelson nonreceptor tyrosine kinase (c-Abl), and the mammalian target of rapamycin (mTOR) that are activated by TGF-β in mesenchymal cells, specifically in fibroblasts but less in epithelial cells. In the present studies, we show that non-smad effectors of TGF-β including PAK2, c-Abl, Akt, tuberin (TSC2), and mTOR are activated in experimental unilateral obstructive nephropathy in rats. Treatment with c-Abl or mTOR inhibitors, imatinib mesylate and rapamycin, respectively, each blocks noncanonical (non-smad) TGF-β pathways in the kidney in vivo and diminishes the number of interstitial fibroblasts and myofibroblasts as well as the interstitial accumulation of extracellular matrix proteins. These findings indicate that noncanonical TGF-β pathways are activated during the early and rapid renal fibrogenesis of obstructive nephropathy. Moreover, the current findings suggest that combined inhibition of key regulators of these non-smad TGF-β pathways even in dose-sparing protocols are effective treatments in renal fibrogenesis.

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Enhanced Bioactive Myocardial Transforming Growth Factor-β in Advanced Human Heart Failure

Circulation Journal ◽

10.1253/circj.cj-14-0511 ◽

2014 ◽

Vol 78 (11) ◽

pp. 2711-2718 ◽

Cited By ~ 17

Author(s):

Shaukat Khan ◽

Jennifer Joyce ◽

Kenneth B. Margulies ◽

Takeshi Tsuda

Keyword(s):

Heart Failure ◽

Growth Factor ◽

Human Heart ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Human Heart Failure

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Abstract P475: Microrna-129-5p Rescues Myocardial Fibrosis And Calcification By Targeting Asporin And Sox9 In Cardiac Fibroblasts

Circulation Research ◽

10.1161/res.129.suppl_1.p475 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Lejla Medzikovic ◽

Laila Aryan ◽

Gregoire Ruffenach ◽

Min Li ◽

Nicoletta Savalli ◽

...

Keyword(s):

Myocardial Fibrosis ◽

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Systolic Dysfunction ◽

Transforming Growth Factor Β ◽

Calcium Deposition ◽

Systemic Delivery ◽

Myocardial Calcification

Myocardial fibrosis promotes heart failure (HF) progression by impairing myocardial compliance, but also may predispose to myocardial calcification, further impairing cardiac function. Transition of resident cardiac fibroblast (CF) to pro-fibrotic myofibroblasts (MF) and osteogenic cell fates (OF) are key events which are partially controlled by microRNAs (miRs). To discover novel miRs involved in myocardial fibrosis and calcification, we compared online-available microarray datasets of left ventricles (LV) from failing human and mouse hearts. Assessing differentially-expressed miRs known to regulate fibrosis and calcification genes revealed that miR-129-5p is significantly downregulated in HF LV. Bioinformatic target analysis revealed small leucin-rich proteoglycan Asporin (Aspn) and SRY-Box Transcription Factor 9 (Sox9) as two novel miR-129-5p targets upregulated in both mouse and human diseased LV. Thus far, nothing is known about miR-129-5p in cardiac fibrosis and calcification. Additionally, the role of Asporin in myocardial fibrosis and the roles of either Asporin or Sox9 in myocardial calcification remain undiscovered. We show that miR-129-5p is expressed in CF in mouse and human hearts and is downregulated in CF of both HF patients and Angiotensin II (AngII)-injured mice, while Asporin and Sox9 are upregulated in CF of HF LV. In vitro , AngII or transforming growth factor-β downregulated miR-129-5p expression in primary adult mouse CF. Overexpression of miR-129-5p in CF inhibited expression of MF and OF transition markers, reduced migration, collagen production and calcium deposition. We validated Asporin and Sox9 as direct targets of miR-129-5p. Accordingly, silencing of Asporin and Sox9 in CF attenuated molecular and functional characteristics of MF and OF transition. Strikingly, systemic delivery of miR-129-5p mimics in mice directly targets CF and is sufficient to rescue preexisting AngII-induced myocardial fibrosis, calcification, diastolic- and systolic dysfunction. In conclusion, miR-129-5p rescues myocardial fibrosis and calcification by attenuating MF and OF transition via inhibition of Asporin and Sox9 in CF and is a promising therapeutic target.

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Activation of SIRT3 by resveratrol ameliorates cardiac fibrosis and improves cardiac function via the TGF-β/Smad3 pathway

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00454.2014 ◽

2015 ◽

Vol 308 (5) ◽

pp. H424-H434 ◽

Cited By ~ 78

Author(s):

Tongshuai Chen ◽

Jingyuan Li ◽

Junni Liu ◽

Na Li ◽

Shujian Wang ◽

...

Keyword(s):

Growth Factor ◽

Cardiac Function ◽

Calorie Restriction ◽

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Transforming Growth Factor Β ◽

Wild Type

Sirtuins [sirtuin (SIRT)1–SIRT7] mediate the longevity-promoting effects of calorie restriction in yeast, worms, flies, and mice. Additionally, SIRT3 is the only SIRT analog whose increased expression has been shown to be associated with longevity in humans. The polyphenol resveratrol (RSV) is the first compound discovered able to mimic calorie restriction by stimulating SIRTs. In the present study, we report that RSV activated SIRT3 in cardiac fibroblasts both in vivo and in vitro. Moreover, in wild-type mice, RSV prevented cardiac hypertrophy in response to hypertrophic stimuli. However, this protective effect was not observed in SIRT3 knockout mice. Additionally, the activation of SIRT3 by RSV ameliorated collagen deposition and improved cardiac function. In isolated cardiac fibroblasts, pretreatment with RSV suppressed fibroblast-to-myoblast transformation by inhibiting the transforming growth factor-β/Smad3 pathway. Therefore, these data indicate that the activation of SIRT3 by RSV could ameliorate cardiac fibrosis and improve cardiac function via the transforming growth factor-β/Smad3 pathway.

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Retinoid X receptor agonists attenuates cardiomyopathy in streptozotocin-induced type 1 diabetes through LKB1-dependent anti-fibrosis effects

Clinical Science ◽

10.1042/cs20190985 ◽

2020 ◽

Vol 134 (6) ◽

pp. 609-628 ◽

Cited By ~ 1

Author(s):

Dajun Chai ◽

Xiaoyan Lin ◽

Qiaowen Zheng ◽

Changsheng Xu ◽

Hong Xie ◽

...

Keyword(s):

Myocardial Fibrosis ◽

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Transforming Growth Factor Β ◽

Underlying Mechanism ◽

Left Ventricular ◽

Retinoid X Receptor ◽

Sprague Dawley

Abstract Diabetic cardiac fibrosis increases ventricular stiffness and facilitates the occurrence of diastolic dysfunction. Retinoid X receptor (RXR) plays an important role in cardiac development and has been implicated in cardiovascular diseases. In the present study, we investigated the effects of RXR agonist treatment on streptozotocin (STZ)-induced diabetic cardiomyopathy (DCM) and the underlying mechanism. Sprague–Dawley (SD) rats induced by STZ injection were treated with either RXR agonist bexarotene (Bex) or vehicle alone. Echocardiography was performed to determine cardiac structure and function. Cardiac fibroblasts (CFs) were treated with high glucose (HG) with or without the indicated concentration of Bex or the RXR ligand 9-cis-retinoic acid (9-cis-RA). The protein abundance levels were measured along with collagen, body weight (BW), blood biochemical indexes and transforming growth factor-β (TGF-β) levels. The effects of RXRα down-regulation by RXRα small interfering RNA (siRNA) were examined. The results showed that bexarotene treatment resulted in amelioration of left ventricular dysfunction by inhibiting cardiomyocyte apoptosis and myocardial fibrosis. Immunoblot with heart tissue homogenates from diabetic rats revealed that bexarotene activated liver kinase B1 (LKB1) signaling and inhibited p70 ribosomal protein S6 kinase (p70S6K). The increased collagen levels in the heart tissues of DCM rats were reduced by bexarotene treatment. Treatment of CFs with HG resulted in significantly reduced LKB1 activity and increased p70S6K activity. RXRα mediated the antagonism of 9-cis-RA on HG-induced LKB1/p70S6K activation changes in vitro. Our findings suggest that RXR agonist ameliorates STZ-induced DCM by inhibiting myocardial fibrosis via modulation of the LKB1/p70S6K signaling pathway. RXR agonists may serve as novel therapeutic agents for the treatment of DCM.

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Investigation of cardiac fibroblasts using myocardial slices

Cardiovascular Research ◽

10.1093/cvr/cvx152 ◽

2017 ◽

Vol 114 (1) ◽

pp. 77-89 ◽

Cited By ~ 21

Author(s):

Filippo Perbellini ◽

Samuel A Watson ◽

Martina Scigliano ◽

Samha Alayoubi ◽

Sebastian Tkach ◽

...

Keyword(s):

Angiotensin Ii ◽

Growth Factor ◽

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Transforming Growth Factor Β ◽

Mechanical Stimuli ◽

New Model ◽

Phenotypic Changes

Abstract Aims Cardiac fibroblasts (CFs) are considered the principal regulators of cardiac fibrosis. Factors that influence CF activity are difficult to determine. When isolated and cultured in vitro, CFs undergo rapid phenotypic changes including increased expression of α-SMA. Here we describe a new model to study CFs and their response to pharmacological and mechanical stimuli using in vitro cultured mouse, dog and human myocardial slices. Methods and results Unloading of myocardial slices induced CF proliferation without α-SMA expression up to 7 days in culture. CFs migrating onto the culture plastic support or cultured on glass expressed αSMA within 3 days. The cells on the slice remained αSMA(−) despite transforming growth factor-β (20 ng/ml) or angiotensin II (200 µM) stimulation. When diastolic load was applied to myocardial slices using A-shaped stretchers, CF proliferation was significantly prevented at Days 3 and 7 (P < 0.001). Conclusions Myocardial slices allow the study of CFs in a multicellular environment and may be used to effectively study mechanisms of cardiac fibrosis and potential targets.

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