Decision letter: Human cardiac fibroblasts adaptive responses to controlled combined mechanical strain and oxygen changes in vitro

Upon cardiac pathological conditions such as ischemia, microenvironmental changes instruct a series of cellular responses that trigger cardiac fibroblasts-mediated tissue adaptation and inflammation. A comprehensive model of how early environmental changes may induce cardiac fibroblasts (CF) pathological responses is far from being elucidated, partly due to the lack of approaches involving complex and simultaneous environmental stimulation. Here, we provide a first analysis of human primary CF behavior by means of a multi-stimulus microdevice for combined application of cyclic mechanical strain and controlled oxygen tension. Our findings elucidate differential human CFs responses to different combinations of the above stimuli. Individual stimuli cause proliferative effects (PHH3+ mitotic cells, YAP translocation, PDGF secretion) or increase collagen presence. Interestingly, only the combination of hypoxia and a simulated loss of contractility (2% strain) is able to additionally induce increased CF release of inflammatory and pro-fibrotic cytokines and matrix metalloproteinases.

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Cardiac fibroblast mechanoresponse guides anisotropic organization of hiPSC-derived cardiomyocytes in vitro

10.1101/2021.02.16.431369 ◽

2021 ◽

Author(s):

Dylan Mostert ◽

Leda Klouda ◽

Mark C. van Turnhout ◽

Nicholas A. Kurniawan ◽

Carlijn V.C Bouten

Keyword(s):

Cardiac Fibroblasts ◽

Mechanical Strain ◽

Cardiac Injury ◽

Cyclic Strain ◽

Cell Types ◽

Tissue Organization ◽

A Cell ◽

Human Cardiac Fibroblasts ◽

And Function

ABSTRACTThe human myocardium is a mechanically active tissue typified by the anisotropic organization of cells and extracellular matrix (ECM). Upon injury, the composition of the myocardium changes, resulting in disruption of tissue organization and loss of coordinated contraction. Understanding how anisotropic organization in the adult myocardium is shaped and disrupted by environmental cues is thus critical, not only for unravelling the processes taking place during disease progression, but also for developing regenerative strategies to recover tissue function. Here, we decoupled in vitro the two major physical cues that are inherent in the myocardium: structural ECM and mechanical strain. We show that patterned ECM proteins control the orientation of the two main cell types in the myocardium: human cardiac fibroblasts (cFBs) and cardiomyocytes (hiPSC-CMs), despite their different mechanosensing machinery. Uniaxial cyclic strain, mimicking the local anisotropic deformation of the myocardium, did not affect hiPSC-CMs orientation. It did however induce a reorientation of cFBs, perpendicular to the strain direction, albeit this strain-avoidance response was overruled in the presence of anisotropic structural cues. These findings reveal that the mechanoresponsiveness of cFBs may be a critical handle in controlling myocardial tissue structure and function. To test this, we co-cultured hiPSC-CMs and cFBs in varying cell ratios to reconstruct normal and pathological myocardium. Contrary to the hiPSC-CM monoculture, the co-cultures adopted an anisotropic organization under uniaxial cyclic strain, regardless of the cell ratio. Together, these results identify the cFBs as a therapeutic target to mechanically restore structural organization of the tissue in cardiac regenerative therapies.SIGNIFICANCE STATEMENTUpon cardiac injury, adverse remodeling commonly leads to loss of the anisotropy that is typically found in human adult myocardium. Understanding the role of biophysical cues in shaping and disrupting the anisotropic tissue organization is essential to aid in the progress of cardiac regenerative strategies. Here, we report that the mechanoresponsiveness of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (cFBs) differs significantly, resulting in a strain-induced reorganization response for cFBs but not for hiPSC-CMs. In co-culture with varying cell ratios of cFBs and hiPSC-CMs, the co-cultures adopted an anisotropic organization upon cyclic strain administration. Thus, our study proposes the mechanoresponse of cFBs, a cell type often overlooked in cardiac regenerative strategies, as a handle to restore myocardial architecture and function.

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In vitro differentiation of human cardiac fibroblasts into myofibroblasts: characterization using electrical impedance

Biomedical Physics & Engineering Express ◽

10.1088/2057-1976/ac12e1 ◽

2021 ◽

Author(s):

Amelie Degache ◽

Florence Poulletier de Gannes ◽

André Garenne ◽

Rémy Renom ◽

Yann Percherancier ◽

...

Keyword(s):

Electrical Impedance ◽

Cardiac Fibroblasts ◽

In Vitro Differentiation ◽

Human Cardiac Fibroblasts

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Human Cardiac Organoids for Modeling Genetic Cardiomyopathy

Cells ◽

10.3390/cells9071733 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1733 ◽

Cited By ~ 1

Author(s):

Michele Filippo Buono ◽

Lisa von Boehmer ◽

Jaan Strang ◽

Simon P. Hoerstrup ◽

Maximilian Y. Emmert ◽

...

Keyword(s):

Cardiac Fibroblasts ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Promising Tool ◽

Vitro Model ◽

Human Cardiac Fibroblasts ◽

Induced Pluripotent ◽

First Time ◽

Design And Testing

Genetic cardiomyopathies are characterized by changes in the function and structure of the myocardium. The development of a novel in vitro model could help to better emulate healthy and diseased human heart conditions and may improve the understanding of disease mechanisms. In this study, for the first time, we demonstrated the generation of cardiac organoids using a triculture approach of human induced pluripotent stem-cell-derived cardiomyocytes (hiPS-CMs)—from healthy subjects and cardiomyopathy patients—human cardiac microvascular endothelial cells (HCMECs) and human cardiac fibroblasts (HCFs). We assessed the organoids’ suitability as a 3D cellular model for the representation of phenotypical features of healthy and cardiomyopathic hearts. We observed clear differences in structure and beating behavior between the organoid groups, depending on the type of hiPS-CMs (healthy versus cardiomyopathic) used. Organoids may thus prove a promising tool for the design and testing of patient-specific treatments as well as provide a platform for safer and more efficacious drug development.

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(Letter to the Editor) Response to: protective role of peroxiredoxin-4 in heart failure

Clinical Science ◽

10.1042/cs20191184 ◽

2020 ◽

Vol 134 (1) ◽

pp. 73-74

Author(s):

Natalia López-Andrés

Keyword(s):

Heart Failure ◽

Dilated Cardiomyopathy ◽

Cardiac Fibroblasts ◽

Direct Consequence ◽

Protective Role ◽

Mitochondrial Proteins ◽

Galectin 3 ◽

Human Cardiac Fibroblasts

Abstract We thank Ahmed et al. for their letter regarding our study ‘Galectin-3 down-regulates antioxidant peroxiredoxin-4 in human cardiac fibroblasts’ [1]. As emphasized by Ahmed et al., Prx-4 levels decrease [2] whereas MFN-2, OPA-1 and PGC-1α levels increase [3] in dilated cardiomyopathy (DCM). Moreover, Gal-3 expression is also increased in DCM [4]. In our study, we showed in vitro that Gal-3 decreased Prx-4 without modifying MFN-2 or PGC-1α levels in human cardiac fibroblasts. Although cardiac Prx-4 decrease could be a direct consequence of Gal-3 effects on cardiac fibroblasts, we cannot exclude the possibility that other factors increase MFN-2, OPA-1 and PGC-1α levels in both cardiac fibroblasts or cardiomyocytes in the context of DCM. Further studies are needed to clarify the association between Prx-4 decrease and the increase in other mitochondrial proteins in DCM.

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Abstract 15671: Adipocyte-derived Exosomal Lncrna Nbr2 Promotes Diabetic Myocardial Fibrosis Through Regulating the Ikba/nf-kb Pathway

Circulation ◽

10.1161/circ.142.suppl_3.15671 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Yue Guo ◽

Xingfeng Xu ◽

Lingling Wu ◽

Xiaodong Zhuang ◽

Xinxue Liao

Keyword(s):

Myocardial Fibrosis ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Epigenetic Mechanism ◽

Ang Ii ◽

Intramyocardial Injection ◽

Underlying Mechanisms ◽

Human Cardiac Fibroblasts ◽

And Function

Introduction: The activation of NF-κB is the dominant process that correlates with the pathogenesis of diabetic cardiomyopathy (DCM). Recently, accumulating evidence shows that long noncoding RNAs (lncRNAs) play crucial roles in sustaining the NF-κB pathway. However, the underlying mechanisms remain unclear. In this study, we identified the upregulated expressed lncRNA NBR2 in adipocyte-derived exosomes (AdEXO) and investigated its regulatory role in diabetic myocardial fibrosis. Hypothesis: We hypothesized that AdEXO-NBR2 promotes diabetic myocardial fibrosis through regulating the IκBα/NF-κB pathway. Methods: We examined the effect of exosomes from diabetic (db/db) mice-derived adipocytes on ANG-II-induced cardiac fibrosis and function in non-diabetic (C57BL/6J mice). In the invitro study, HG (33mmol/L)-stimulated AdEXO were cultured with adult human cardiac fibroblasts (aHCFs). Differentially expressed lncRNAs in AdEXO were screened using lncRNA sequencing. Results: Intramyocardial injection of diabetic AdEXO in the non-diabetic heart significantly exacerbated myocardial fibrosis, as evidenced by poorer cardiac function and enhancer collagen deposition. Whereas administration of a exosomes biogenesis inhibitor mitigated cardiac fibrosis in diabetic mice. We found lncRNA-NBR2 is a common molecule significantly increased in diabetic AdEXO and HG-stimulated non-diabetic AdEXO. After four weeks of ANG II infusion, EXO-db/dbWT-injected mice displayed fibrosis in the heart. However, interestingly, mice receiving NBR2-deficient db/db-EXO showed a decrease in cardiac fibrosis. Similarly, AdEXO-NBR2 promoted aHCFs proliferation and transformation capabilities in vitro. Mechanistically, NBR2 was loaded to AdEXO by directly interacting with heterogeneous nuclear ribonucleoprotein K (hnRNPK). Subsequently, AdEXO-NBR2 was internalized by aHCFs and epigenetically downregulated IκBα expression by recruitment of hnRNPK/SETDB1 and increasing the H3K9 trimethylation level in the IκBα promoter, ultimately activating the NF-κB pathway. Conclusions: Our findings highlight a novel epigenetic mechanism of AdEXO lncRNA-mediated diabetic cardiac fibrosis and identify NBR2 as a therapeutic target of DCM.

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In vitro models of human cardiac fibrotic tissue on ‘bioartificial’ scaffolds

Biomedical Science and Engineering ◽

10.4081/bse.2019.122 ◽

2020 ◽

Author(s):

Alice Zoso ◽

Irene Carmagnola ◽

Gerardina Ruocco ◽

Mattia Spedicati ◽

Valeria Chiono

Keyword(s):

Cardiac Tissue ◽

Cell Attachment ◽

Cardiac Fibroblasts ◽

Cardiac Regeneration ◽

In Vitro Models ◽

Myocardial Tissue ◽

Fibrotic Tissue ◽

Human Cardiac Fibroblasts ◽

Infarcted Tissue

Cardiac infarction is a global burden worldwide that leads to fibrotic and not contractile myocardial tissue. In this work, in vitro models of infarcted tissue were developed as tools to test novel therapies for cardiac regeneration in the future. Human cardiac fibroblasts were cultured on scaffolds, with different compositions and architectures, as to mimic structural and chemical features of infarcted cardiac tissue. Early findings from in vitro cell tests were reported, showing an enhancement of cell attachment and proliferation in the case of “bioartificial” scaffolds, i.e. scaffolds based on a synthetic and a bioactive polymer.

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Beneficial Effects of Mineralocorticoid Receptor Antagonism on Myocardial Fibrosis in an Experimental Model of the Myxomatous Degeneration of the Mitral Valve

International Journal of Molecular Sciences ◽

10.3390/ijms21155372 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5372

Author(s):

Jaime Ibarrola ◽

Mattie Garaikoetxea ◽

Amaia Garcia-Peña ◽

Lara Matilla ◽

Eva Jover ◽

...

Keyword(s):

Mitral Valve ◽

Myocardial Fibrosis ◽

Mineralocorticoid Receptor ◽

Collagen Type ◽

Cardiac Fibroblasts ◽

Collagen Type I ◽

Type I ◽

Myxomatous Degeneration ◽

Human Cardiac Fibroblasts

Mitral valve prolapse (MVP) patients develop myocardial fibrosis that is not solely explained by volume overload, but the pathophysiology has not been defined. Mineralocorticoid receptor antagonists (MRAs) improve cardiac function by decreasing cardiac fibrosis in other heart diseases. We examined the role of MRA in myocardial fibrosis associated with myxomatous degeneration of the mitral valve. Myocardial fibrosis has been analyzed in a mouse model of mitral valve myxomatous degeneration generated by pharmacological treatment with Nordexfenfluramine (NDF) in the presence of the MRA spironolactone. In vitro, adult human cardiac fibroblasts were treated with NDF and spironolactone. In an experimental mouse, MRA treatment reduced interstitial/perivascular fibrosis and collagen type I deposition. MRA administration blunted NDF-induced cardiac expression of vimentin and the profibrotic molecules galectin-3/cardiotrophin-1. In parallel, MRA blocked the increase in cardiac non-fibrillar proteins such as fibronectin, aggrecan, decorin, lumican and syndecan-4. The following effects are blocked by MRA: in vitro, in adult human cardiac fibroblasts, NDF-treatment-induced myofibroblast activation, collagen type I and proteoglycans secretion. Our findings demonstrate, for the first time, the contribution of the mineralocorticoid receptor (MR) to the development of myocardial fibrosis associated with mitral valve myxomatous degeneration. MRA could be a therapeutic approach to reduce myocardial fibrosis associated with MVP.

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SKI activates the Hippo pathway via LIMD1 to inhibit cardiac fibroblast activation

Basic Research in Cardiology ◽

10.1007/s00395-021-00865-9 ◽

2021 ◽

Vol 116 (1) ◽

Author(s):

Natalie M. Landry ◽

Sunil G. Rattan ◽

Krista L. Filomeno ◽

Thomas W. Meier ◽

Simon C. Meier ◽

...

Keyword(s):

Cardiac Fibroblasts ◽

Cardiac Fibroblast ◽

Negative Regulator ◽

Expression Vectors ◽

Cell Phenotype ◽

Hippo Signaling ◽

Fibroblast Activation ◽

Elastic Substrates ◽

Human Cardiac Fibroblasts

AbstractWe have previously shown that overexpression of SKI, an endogenous TGF-β1 repressor, deactivates the pro-fibrotic myofibroblast phenotype in the heart. We now show that SKI also functions independently of SMAD/TGF-β signaling, by activating the Hippo tumor-suppressor pathway and inhibiting the Transcriptional co-Activator with PDZ-binding motif (TAZ or WWTR1). The mechanism(s) by which SKI targets TAZ to inhibit cardiac fibroblast activation and fibrogenesis remain undefined. A rat model of post-myocardial infarction was used to examine the expression of TAZ during acute fibrogenesis and chronic heart failure. Results were then corroborated with primary rat cardiac fibroblast cell culture performed both on plastic and on inert elastic substrates, along with the use of siRNA and adenoviral expression vectors for active forms of SKI, YAP, and TAZ. Gene expression was examined by qPCR and luciferase assays, while protein expression was examined by immunoblotting and fluorescence microscopy. Cell phenotype was further assessed by functional assays. Finally, to elucidate SKI’s effects on Hippo signaling, the SKI and TAZ interactomes were captured in human cardiac fibroblasts using BioID2 and mass spectrometry. Potential interactors were investigated in vitro to reveal novel mechanisms of action for SKI. In vitro assays on elastic substrates revealed the ability of TAZ to overcome environmental stimuli and induce the activation of hypersynthetic cardiac myofibroblasts. Further cell-based assays demonstrated that SKI causes specific proteasomal degradation of TAZ, but not YAP, and shifts actin cytoskeleton dynamics to inhibit myofibroblast activation. These findings were supported by identifying the bi-phasic expression of TAZ in vivo during post-MI remodeling and fibrosis. BioID2-based interactomics in human cardiac fibroblasts suggest that SKI interacts with actin-modifying proteins and with LIM Domain-containing protein 1 (LIMD1), a negative regulator of Hippo signaling. Furthermore, we found that LATS2 interacts with TAZ, whereas LATS1 does not, and that LATS2 knockdown prevented TAZ downregulation with SKI overexpression. Our findings indicate that SKI’s capacity to regulate cardiac fibroblast activation is mediated, in part, by Hippo signaling. We postulate that the interaction between SKI and TAZ in cardiac fibroblasts is arbitrated by LIMD1, an important intermediary in focal adhesion-associated signaling pathways. This study contributes to the understanding of the unique physiology of cardiac fibroblasts, and of the relationship between SKI expression and cell phenotype.

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