Cardiac Fibroblast Proliferation Research Articles

Limb-Bud and Heart (LBH) Upregulation in Cardiomyocytes under Hypoxia Promotes the Activation of Cardiac Fibroblasts via Exosome Secretion.

The activation of cardiac fibroblasts (CFs) after myocardial infarction (MI) is essential for post-MI infarct healing, during which the regulation of transforming growth factor beta1 (TGF-β1) signaling is predominant. We have demonstrated that TGF-β1-mediated upregulation of LBH contributes to post-MI CF activation via modulating αB-crystallin (CRYAB), after being upregulated by TGF-β1. In this study, the effect of LBH-CRYAB signaling on the cardiac microenvironment via exosome communication and the corresponding mechanisms were investigated. The upregulation of LBH and CRYAB was verified in both cardiomyocytes (CMs) and CFs in hypoxic, post-MI peri-infarct tissues. CM-derived exosomes were isolated and identified, and LBH distribution was elevated in exosomes derived from LBH-upregulated CMs under hypoxia. Treatment with LBH+ exosomes promoted cellular proliferation, differentiation, and epithelial-mesenchymal transition-like processes in CFs. Additionally, in primary LBHKO CFs, western blotting showed that LBH knockout partially inhibited TGF-β1-induced CF activation, while LBH-CRYAB signaling affected TGF-β1 expression and secretion through a positive feedback loop. The administration of a Smad3 phosphorylation inhibitor to LBHKO CFs under TGF-β1 stimulation indicated that Smad3 phosphorylation partially accounted for TGF-β1-induced LBH upregulation. In conclusion, LBH upregulation in CMs in post-MI peri-infarct areas correlated with a hypoxic cardiac microenvironment and led to elevated exosomal LBH levels, promoting the activation of recipient CFs, which brings new insights into the studies and therapeutic strategies of post-MI cardiac repair.

SZC-6, a small-molecule activator of SIRT3, attenuates cardiac hypertrophy in mice.

Sirtuin3 (SIRT3), a class III histone deacetylase, is implicated in various cardiovascular diseases as a novel therapeutic target. SIRT3 has been proven to be cardioprotective in a model of Ang II-induced cardiac hypertrophy. However, a few small-molecule compounds targeting deacetylases could activate SIRT3. In this study, we generated a novel SIRT3 activator, 3-(2-bromo-4-hydroxyphenyl)-7-hydroxy-2H-chromen-2-one (SZC-6), through structural optimization of the first SIRT3 agonist C12. We demonstrated that SZC-6 directly bound to SIRT3 with Kd value of 15 μM, and increased SIRT3 deacetylation activity with EC50 value of 23.2 ± 3.3 µM. In neonatal rat cardiomyocytes (NRCMs), pretreatment with SZC-6 (10, 20, 40 µM) dose-dependently attenuated isoproterenol (ISO)-induced hypertrophic responses. Administration of SZC-6 (20, 40 and 60 mg·kg-1·d-1, s.c.) for 2 weeks starting from one week prior ISO treatment dose-dependently reversed ISO-induced impairment of diastolic and systolic cardiac function in wild-type mice, but not in SIRT3 knockdown mice. We showed that SZC-6 (10, 20, 40 µM) dose-dependently inhibited cardiac fibroblast proliferation and differentiation into myofibroblasts, which was abolished in SIRT3-knockdown mice. We further revealed that activation of SIRT3 by SZC-6 increased ATP production and rate of mitochondrial oxygen consumption, and reduced ROS, improving mitochondrial function in ISO-treated NRCMs. We also found that SZC-6 dose-dependently enhanced LKB1 phosphorylation, thereby promoting AMPK activation to inhibit Drp1-dependent mitochondrial fragmentation. Taken together, these results demonstrate that SZC-6 is a novel SIRT3 agonist with potential value in the treatment of cardiac hypertrophy partly through activation of the LKB1-AMPK pathway.

Inhibitory effect of (pro)renin receptor decoy inhibitor PRO20 on endoplasmic reticulum stress during cardiac remodeling.

Background: Ectopic activation of renin-angiotensin-system contributes to cardiovascular and renal diseases. (Pro)renin receptor (PRR) binds to renin and prorenin, participating in the progression of nephrology. However, whether PRR could be considered as a therapeutic target for cardiac remodeling and heart failure remains unknown. Materials and methods: Transverse aortic constriction (TAC) surgery was performed to establish a mouse model of chronic pressure overload-induced cardiac remodeling. Neonatal rat cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were isolated and stimulated by Angiotensin II (Ang II). PRR decoy inhibitor PRO20 was synthesized and used to evaluate its effect on cardiac remodeling. Results: Soluble PRR and PRR were significantly upregulated in TAC-induced cardiac remodeling and Ang II-treated CMs and CFs. Results of In vivo experiments showed that suppression of PRR by PRO20 significantly retarded cardiac remodeling and heart failure indicated by morphological and echocardiographic analyses. In vitro experiments, PRO20 inhibited CM hypertrophy, and also alleviated CF activation, proliferation and extracellular matrix synthesis. Mechanically, PRO20 enhanced intracellular cAMP levels, but not affected cGMP levels in CMs and CFs. Moreover, treatment of PRO20 in CFs markedly attenuated the production of reactive oxygen species and phosphorylation of IRE1 and PERK, two well-identified markers of endoplasmic reticulum (ER) stress. Accordingly, administration of PRO20 reversed ER stressor thapsigargin-induced CM hypertrophy and CF activation/migration. Conclusion: Taken together, these findings suggest that inhibition of PRR by PRO20 attenuates cardiac remodeling through increasing cAMP levels and reducing ER stress in both CMs and CFs.

Intercellular model predicts mechanisms of inflammation-fibrosis coupling after myocardial infarction.

After myocardial infarction (MI), cardiac cells work together to regulate wound healing of the infarct. The pathological response to MI yields cardiac remodelling comprising inflammatory and fibrosis phases, and the interplay of cellular dynamics that underlies these phases has not been elucidated. This study developed a computational model to identify cytokine and cellular dynamics post-MI to predict mechanisms driving post-MI inflammation, resolution of inflammation, andscar formation. Additionally, this study evaluated the interdependence between inflammation and fibrosis. Our model bypassed limitations of in vivo approaches in achieving cellular specificity and performing specific perturbations such as global knockouts of chemical factors. The model predicted that inflammation is a graded response to initial infarct size that is amplified by a positive feedback loop between neutrophils and interleukin 1β (IL-1β). Resolution of inflammation was driven by degradation of IL-1β, matrix metalloproteinase 9, and transforming growth factor β (TGF-β), as well as apoptosis of neutrophils. Inflammation regulated TGFβ secretion directly through immune cell recruitment and indirectly through upregulation of macrophage phagocytosis. Lastly, we found that mature collagen deposition was an ultrasensitive switch in response to inflammation, which was amplified primarily by cardiac fibroblast proliferation. These findings describe the relationship between inflammation and fibrosis and highlight how the two responses work together post-MI. This model revealed that post-MI inflammation and fibrosis are dynamically coupled, which provides rationale for designing novel anti-inflammatory, pro-resolving or anti-fibrotic therapies that may improve the response to MI. KEY POINTS: Inflammation and matrix remodelling are two processes involved in wound healing after a heart attack. Cardiac cells work together to facilitate these processes; this is done by secreting cytokines that then regulate the cells themselves or other cells surrounding them. This study developed a computational model of the dynamics of cardiac cells and cytokines to predict mechanisms through which inflammation and matrix remodelling is regulated. We show the roles of various cytokines and signalling motifs in driving inflammation, resolution of inflammation and fibrosis. The novel concept of inflammation-fibrosis coupling, based on the model prediction that inflammation and fibrosis are dynamically coupled, provides rationale for future studies and for designing therapeutics to improve the response after a heart attack.

Abstract P3075: Hippo Pathway In Cardiac Fibroblast Regulates Macrophage Recruitment Following Injury

Background: The evolutionary conserved Hippo signaling pathway (HSP) is an inhibitor of tissue regeneration, including the Lats1/2 kinases that phosphorylate and inactivate YAP and TAZ. Emerging data indicate that HSP regulates innate immunity, inflammation, and autoimmune diseases. However, the role of HSP in post-MI inflammation is poorly understood. Methods: Single-cell RNA-sequencing (scRNA-Seq) and spatial transcriptomics were utilized in cardiac fibroblast (CF)-specific-Lats1/2 knockout (Lats CKO) mouse heart. Flow cytometry, immunofluorescence (IF), and echocardiogram (Echo) were used to describe the phenotype in ventricle of Lats CKO mouse heart with/out Ccr2 knockout. Results: We integrated our scRNA-Seq data in Lats CKO mouse heart with three published data sets of MI mouse heart. A CF subcluster with elevated YAP activity and expressing chemokines Ccl2 and Ccl7 (Ccl2/Ccl7+ CFs) and macrophages (Macs) expressing Ccr2, the receptor for Ccl2/Ccl7 (Ccr2+ Macs), were identified, which suggests a signaling transduction from Ccl2/Ccl7+ CFs to Ccr2+ Macs in post-MI hearts. To figure out the function and mechanism of this specific cell communication, we employed a Lats CKO mice model which mimics the post-MI heart and exhibits high YAP activity and conformed the cell communication between Ccl2/Ccl7+ CFs and Ccr2+ Macs by spatial transcriptomics. Furthermore, Ccr2 loss of function in Lats CKO mice suppressed Mac expansion and CF proliferation. Consistently, Ccr2 knockout reversed the phenotype presenting smaller left ventricle chamber in Lats CKO mouse heart because of CF proliferation. Conclusion: YAP target genes Ccl2 and Ccl7 in CFs are responsible for Ccr2+ Macs recruitment and the recruited Ccr2+ Macs in turn promote CF proliferation post-MI.

Abstract P1007: Cardiac Fibroblast BAG3 Controls TGFBR2 Signaling And Contributes To Engineered Cardiac Tissue Function.

Introduction: Mutations in BAG3 result in dilated cardiomyopathy with cardiac fibrosis. Currently, the role of BAG3 in non-myocyte cell types is not known. We tested the hypothesis that BAG3 controls cardiac fibroblast (CF) function and that the loss of fibroblast-specific BAG3 is deleterious, using cells and engineered cardiac tissues (ECT) derived from pluripotent stem cells (iPSC). Methods: Isogenic iPSCs containing homozygous knockout (KO) of BAG3 were differentiated into CFs. Mass spectrometry measured the effect of BAG3 loss on CF proteome. EdU staining and flow cytometry were used to assess CF proliferation. Transforming growth factor beta (TGF) signaling pathway activity was measured using a luciferase reporter and Western blotting. Affinity purification mass spectrometry (AP-MS) identified high-confidence interactors of BAG3 in CFs. To assess the role of fibroblast-specific BAG3 in cardiac function, conditional KO ECTs were generated using wild-type cardiomyocytes mixed with either wild-type CFs or KO CFs. ECTs were functionally assessed using video microscopy and immunofluorescence. The effects of CF BAG3 loss on tissue gene expression are under study by single nuclear RNA sequencing. Results: Analysis of proteomics data revealed enrichment of TGF and proliferation pathways in KO cells. KO-CFs had increased proliferation measured by EdU staining and increased SMAD luciferase reporter activity. Western blotting showed that KO CFs have higher expression of TGF beta receptor 2 (TGFBR2) and activation of canonical TGF signaling. AP-MS identified TGFBR2 as an interacting partner of BAG3, suggesting BAG3 mediated TGFBR2 turnover. In ECT, the loss of CF BAG3 caused a reduction in cardiac tissue contractile force (50% reduction, n = 10-12 tissues per group). Immunofluorescence revealed significantly increased tissue fibrosis in CF-KO cardiac tissues quantified by vimentin staining. Conclusions: BAG3 loss results in increased TGFBR2 signaling and proliferation in cardiac fibroblasts. Conditional KO ECTs demonstrate that CF BAG3 affects tissue function. These data suggest that fibrosis related to BAG3 pathology may be a result of a primary deficiency in CFs rather than a secondary response to cardiomyocyte death.

Abstract P2115: Differential Cardiac Remodeling Profile Of Immunosuppression Drugs

Introduction: Heart transplantation provides lifesaving therapy for patients with end-stage heart failure. The longevity of the therapy is limited by Cardiac Graft Dysfunction (CGD), which is an acquired cardiomyopathy affecting transplanted hearts associated with diastolic and/or systolic dysfunction. Some clinical risk factors for CGD have been identified, but none of them are easily modifiable. An unexplored potential contributor to CGD is the choice of immunosuppression agent used despite multiple clinical reports suggesting reduced adverse cardiac remodeling with mammalian target of rapamycin (mTOR) inhibitors compared to calcineurin inhibitors (CNI). This study examines mechanisms of differential cardiac remodeling effects of CNI versus mTOR inhibitors in a human cellular cardiac model. Methods/Results: We utilized 3D cardiac spheres composed of induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells (cardiac organoids). Cardiac organoids were treated with 5 days of vehicle, tacrolimus (CNI), or sirolimus (mTOR inhibitor). We did not observe a significant difference in surrogates of systolic or diastolic function in treated cardiac organoids. We pursued single cell-RNA sequencing of drug-treated cardiac organoids and identified gene expression changes consistent with increased extracellular matrix deposition and fibroblast activity in response to CNI treatment. In addition, CNI-treated cardiac organoids cellular composition was notable for increased proportion of fibroblasts and less cardiomyocytes compared to mTOR inhibitor-treated cardiac organoids. To validate gene expression changes observed, we treated cardiac fibroblasts with drugs and observed an increase in collagen production in response to CNI treatment and a reduction in fibroblast number and collagen production in response to mTOR inhibitor treatment. Furthermore, we observed increased ATP production in CNI-treated cardiac fibroblasts, but a reduction in mTOR-treated counterparts. Conclusion: We identify reduced extracellular matrix deposition and cardiac fibroblast proliferation in response to mTOR inhibitor as a potential mechanism for the more favorable remodeling profile observed clinically.

Abstract P2048: Matricellular Protein Cilp1 Promotes Myocardial Fibrosis In Response To Myocardial Infarction

Rationale: Cartilage intermediate layer protein 1 (Cilp1) is a secreted extracellular matrix (ECM) protein normally associated with bone and cartilage development. Its function and mechanism of action in adult heart disease remain elusive. Objective: To establish the function and mechanism of action of Cilp1 in post-myocardial infarction (MI) cardiac remodeling. Methods and Results: We investigated the expression of Cilp1 in mouse models of pathological cardiac remodeling and human heart failure patients. Cilp1 was expressed predominantly in cardiac fibroblasts and upregulated in response to cardiac injury and in the heart and blood of heart failure patients. We generated Cilp1 knock out (KO) and transgenic (Tg) mice with N-terminal half of the protein (NCilp1) overexpressed in myofibroblasts. Cilp1 KO mice had better cardiac function, reduced number of immune cells and myofibroblasts, and enhanced microvascular survival after MI compared to wild-type (WT) littermates. Conversely, NCilp1-Tg mice had augmented loss of cardiac function, increased number of myofibroblasts and infarct size after the MI injury. RNA-seq and gene ontology analysis indicated that cell proliferation and mTORC1 signaling were downregulated in KO hearts compared to WT hearts. In vivo BrdU labeling and immunofluorescence staining showed that myofibroblast proliferation in the Cilp1 KO heart was downregulated. Biaxial mechanical testing and ECM gene expression analysis indicated that while MI caused significant stiffness in WT hearts it had little effect on KO hearts. Upregulation of collagen expression after MI injury was attenuated in KO hearts. Recombinant CILP1 protein or NCilp1-conditioned medium promoted proliferation of neonatal rat ventricular cardiac fibroblasts via the mTORC1 signaling pathway. Conclusions: Our studies established a pathological role of Cilp1 in promoting post-MI remodeling, identified a novel function of Cilp1 in promoting myofibroblast proliferation, and suggested that Cilp1 may serve as a potential biomarker for pathological cardiac remodeling and target for fibrotic heart disease.

Long Noncoding RNA HLA Complex Group 18 Improves the Cell Proliferation of Myocardial Fibroblasts by Regulating the Hsa-microRNA-133a/Epidermal Growth Factor Receptor Axis.

Hsa-microRNA (has-miR)-133a inactivates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and suppresses the cell proliferation of myocardial fibroblasts by downregulation of the epidermal growth factor receptor (EGFR) expression. Bioinformatics analysis exhibits extended noncoding RNA HLA complex group 18 (lncRNA-HCG18) binds to hsa-miR-133a. The purpose of the current experiment is to explore whether lncRNA-HCG18 adsorbed hsa-miR-133a through sponging, resulting in decreased inhibition of hsa-miR-133a on EGFR and ultimately promoting the proliferation of myocardial fibroblasts. To verify and study the correlation and mechanism between lncRNA-HCG18, hsa-miR-133a, and their target genes. Firstly, after overexpression/silencing of lncRNA-HCG18 in myocardial fibroblasts, the level of hsa-miR-133a expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the EGFR, ERK1/2, and p-ERK1/2 expression levels were assessed by Western blotting to confirm that upregulation of EGFR and p-ERK1/2 protein levels by overexpression of lncRNA-HCG18, siRNA lncRNA-HCG18 (siHCG18) reduced the EGFR and p-ERK1/2 protein levels. Then, the luciferase reporter gene system was used to verify that lncRNA-HCG18 regulated EGFR expression by inhibiting the function of the hsa-miR-133a regulatory target gene. Then, a RAP assay was used to confirm that lncRNA-HCG18 interacted with hsa-miR-133a. Finally, the analysis of CCK-8 results indicated that the cell proliferation of myocardial fibroblasts was significantly reduced by siHCG18 while reversed by overexpression of lncRNA-HCG18. Thus, lncRNA-HCG18 inhibited cell viability of cardiac fibroblasts via the hsa-miR-133a/EGFR axis, which was regarded as a regulator of cell proliferation of cardiac fibroblasts in cardiovascular diseases.

Combined role for YAP-TEAD and YAP-RUNX2 signalling in substrate-stiffness regulation of cardiac fibroblast proliferation

Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM) in part mediated by increased cardiac fibroblast proliferation However, our understanding of the mechanisms regulating cardiac fibroblast proliferation are incomplete. Here we characterise a novel mechanism involving a combined activation of Yes-associated protein (YAP) targets RUNX Family Transcription Factor 2 (RUNX2) and TEA Domain Transcription Factor (TEAD).We demonstrate that cardiac fibroblast proliferation is enhanced by interaction with a stiff ECM compared to a soft ECM. This is associated with activation of the transcriptional co-factor, YAP. We demonstrate that this stiffness induced activation of YAP enhances the transcriptional activity of both TEAD and RUNX2 transcription factors. Inhibition of either TEAD or RUNX2, using gene silencing, expression of dominant-negative mutants or pharmacological inhibition, reduces cardiac fibroblast proliferation. Using mutants of YAP, defective in TEAD or RUNX2 activation ability, we demonstrate a dual role of YAP-mediated activation of TEAD and RUNX2 for substrate stiffness induced cardiac fibroblast proliferation.Our data highlights a previously unrecognised role of YAP mediated RUNX2 activation for cardiac fibroblast proliferation in response to increased ECM stiffness.

Tripartite motif 38 attenuates cardiac fibrosis after myocardial infarction by suppressing TAK1 activation via TAB2/3 degradation

SummaryThe role of tripartite motif (TRIM) 38, a ubiquitin E3 ligase regulating various pathophysiological processes, in cardiac fibrosis remains unclear. Here, a model of angiotensin II and myocardial infarction (MI)-induced fibrosis was established to explore its role in cardiac fibrosis and its underlying mechanisms. Cardiac fibrosis in the mouse MI model was mitigated by TRIM38 overexpression, but aggravated by its depletion. Consistently, in vitro overexpression or knockdown of TRIM38 ameliorated or aggravated the proliferation and secretion of cardiac fibroblasts (CFs) exposed to fibrotic stimulation, respectively. Mechanistically, TRIM38 suppressed cardiac fibrosis progression by attenuating TAK1/MAPK signaling. Inhibiting TAK1/MAPK signaling with a pharmacological inhibitor greatly reversed the effects of TRIM38 knockdown on CF secretion. Specifically, TRIM38 interacted with and “targeted” TAB2 and TAB3 for degradation, subsequently inhibiting TAK1 phosphorylation and negatively regulating MAPK signaling. These findings can help develop therapeutic strategies to treat and prevent cardiac fibrosis.

Silencing of TLR4 Inhibits Atrial Fibrosis and Susceptibility to Atrial Fibrillation via Downregulation of NLRP3-TGF-β in Spontaneously Hypertensive Rats.

Introduction This study was aimed at exploring whether silencing of TLR4 could inhibit atrial fibrosis and susceptibility to atrial fibrillation (AF) by regulating NLRP3-TGF-β in hypertensive rats. Methods Spontaneously hypertensive rats (SHRs) were transfected with either a virus containing TLR4-shRNA to downregulate TLR4 or an empty virus (vehicle) at the age of 14 weeks. Fibrosis of left atrium and susceptibility to AF were detected, and expression of NLRP3-TGF-β in left atrial tissue at 22 weeks of age was measured. Primary cardiac fibroblasts were transfected with TLR4-shRNA or scrambled vehicle and stimulated with angiotensin (Ang) II. Proliferation of cardiac fibroblasts and expression of NLRP3-TGF-β were detected. Results Silencing of TLR4 reduced left atrial fibrosis and susceptibility to AF in SHRs and downregulated expression of NLRP3, TGF-β, and collagen I. In vitro, TLR4 silencing reduced proliferation of cardiac fibroblasts induced by Ang II as well as expression of NLRP3, TGF-β, and collagen I. Conclusion Silencing of TLR4 can downregulate NLRP3-TGF-β to reduce atrial fibrosis and susceptibility to AF in SHRs.

FAM114A1 influences cardiac pathological remodeling by regulating angiotensin II signaling.

Cardiac pathological remodeling, a primary contributor to heart failure (HF) and death, is an important target for HF therapy. However, the signaling pathways that govern cardiac remodeling are not fully elucidated. Here, we found that a functionally unannotated human myocardial infarction–associated (MI-associated) gene, family with sequence similarity 114 member A1 (FAM114A1), is induced in failing human and mouse hearts compared with nonfailing hearts. Homozygous KO of Fam114a1 (Fam114a1–/–) in the mouse genome reduces cardiomyocyte hypertrophy, inflammation, and cardiac fibrosis while restoring cardiac function in angiotensin II–induced (Ang II–induced) and MI-induced HF mouse models. Cardiac fibroblasts (CFs) exhibit the highest FAM114A1 expression among different cardiac cell types. FAM114A1 is a critical autonomous factor for CF proliferation, activation, and migration. Mechanistically, FAM114A1 interacts with angiotensin receptor–associated protein (AGTRAP) and regulates the expression of angiotensin type 1 receptor (AT1R) and downstream Ang II signaling transduction, and it subsequently influences profibrotic response. Our results indicate that FAM114A1 regulates Ang II signaling, thereby activating CFs and other cardiac cells and augmenting pathological cardiac remodeling. These findings provide potentially novel insights into the regulation of cardiac remodeling and identify FAM114A1 as a therapeutic target for the treatment of heart disease.

MicroRNA-338-3p as a therapeutic target in cardiac fibrosis through FGFR2 suppression.

BackgroundThe development of cardiac fibrosis involves the activation of cardiac fibroblasts (CFs) and their differentiation into myofibroblasts, which leads to the disruption of the extracellular matrix network. In the past few years, microRNAs (miRNA) have been described as potential targets for treating cardiac diseases. Although miR‐338‐3p has been shown to participate in the development of carcinoma, whether it affects cardiac fibrosis is unclear.MethodsWe examined the expression profiles of microRNAs in left ventricular samples of heart failure mice established by thoracic aortic constriction (TAC). Real‐time quantitative reverse transcription polymerase chain reaction (qRT‐PCR) was used to detect the expression of miR‐338‐3p. CCK‐8 assay/Transwell migration assay was used to measure the proliferation rate/migration of CFs. Luciferase reporter gene assay was used to test the binding between miR‐338‐3p and FGFR2.ResultsThis study demonstrated that miR‐338‐3p was significantly decreased in thoracic aortic constriction mice. Cardiac miR‐338‐3p amounts were also reduced in patients with dilated cardiomyopathy (DCM). Interestingly, miR‐338‐3p overexpression inhibited α‐SMA, COL1A1, and COL3A1 expression, as well as cell proliferation and migration in CFs. Bioinformatics analysis and dual‐luciferase reporter assays revealed FGFR2 was targeted by miR‐338‐3p, whose antifibrotic effect could be alleviated by overexpression of FGFR2. Moreover, in DCM cases, serum miR‐338‐3p levels were markedly elevated in individuals with worse outcomes.ConclusionsThe present study provides evidence that miR‐338‐3p suppresses cardiac fibroblast activation, proliferation, and migration by directly targeting FGFR2 in mice. Besides, serum miR‐338‐3p might constitute a potential prognostic biomarker of dilated cardiomyopathy.

Liraglutide inhibits AngII-induced cardiac fibroblast proliferation and ECM deposition through regulating miR-21/PTEN/PI3K pathway.

Cardiac fibrosis characterized with the aberrant proliferation of cardiac fibroblasts and extracellular matrix (ECM) deposition is a major pathophysiological feature of atrial fibrillation (AF). Liraglutide has exerted an alleviative role in various cardiovascular diseases, and can also regulate the level of microRNAs (miRNAs). It has been reported that miR-21 modulated cardiac fibrosis in AF. However, the regulative effect of liraglutide on atrial fibrosis via miR-21 and the underlying mechanism are still unclear. The atrial fibroblasts were isolated from the heart of C57BL/6 mice, and treated with Angiotensin II (AngII) and liraglutide. The proliferation, migration, and ECM deposition were determined by cell counting Kit-8 (CCK-8), Brdu, transwell assay, cell scratch, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot and immunofluorescence. The underlying mechanism was explored after transfection of miR-21 mimics into cells. Liraglutide inhibited proliferation, migration, invasion of fibroblast cell and ECM deposition in AngII-stimulated cardiac fibroblasts. Additionally, liraglutide decreased the AngII-induced increase in the expression level of miR-21, but enhanced the expression of phosphatase and tensin homolog (PTEN), a target of miR-21, thereby suppressing the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Rescue assay confirmed that overexpression of miR-21 counteracted the ameliorative effect of liraglutide on the proliferation, migration, invasion and ECM deposition in fibroblasts stimulated by AngII. Liraglutide dampened AngII-induced proliferation and migration, and ECM deposition of cardiac fibroblast via modulating miR-21/PTEN/PI3K pathway.

HDAC1 Promotes Myocardial Fibrosis in Diabetic Cardiomyopathy by Inhibiting BMP-7 Transcription Through Histone Deacetylation.

Diabetic cardiomyopathy (DCM) constitutes a primary cause of mortality in diabetic patients. Histone deacetylase (HDAC) inhibition can alleviate diabetes-associated myocardial injury. This study investigated the mechanism of HDAC1 on myocardial fibrosis (MF) in DCM. A murine model of DCM was established by a high-fat diet and streptozotocin injection. The bodyweight, blood glucose, serum insulin, and cardiac function of mice were analyzed. Lentivirus-packaged sh-HDAC1 was injected into DCM mice and high glucose (HG)-induced cardiac fibroblasts (CFs). The pathological structure of the myocardium and the level of myocardial fibrosis were observed by histological staining. HDAC1 expression in mouse myocardial tissues and CFs was determined. Collagen I, collagen III, alpha-smooth muscle actin (α-SMA), and vimentin levels in CFs were detected, and CF proliferation was tested. HDAC activity and histone acetylation levels in tissues and cells were measured. Bone morphogenetic protein-7 (BMP-7) expression in myocardial tissues and CFs was determined. Functional rescue experiments were conducted to confirm the effects of histone acetylation and BMP-7 on myocardial fibrosis. DCM mice showed decreased bodyweight, elevated blood glucose and serum insulin, and cardiac dysfunction. Elevated HDAC1 and reduced BMP-7 expressions were detected in DCM mice and HG-induced CFs. HDAC1 repressed BMP-7 transcription through deacetylation. HDAC1 silencing alleviated MF, reduced CF proliferation and decreased collagen I, -III, α-SMA, and vimentin levels. However, reducing histone acetylation level or BMP-7 downregulation reversed the effects of HDAC1 silencing on CF fibrosis. HDAC1 repressed BMP-7 transcription by enhancing histone deacetylation, thereby promoting MF and aggravating DCM.

Calycosin reduces myocardial fibrosis and improves cardiac function in post-myocardial infarction mice by suppressing TGFBR1 signaling pathways

BackgroundExcessive myocardial fibrosis is the pathological basis of heart failure following myocardial infarction (MI). Although calycosin improves cardiac function, its effect on cardiac fibrosis and cardiac function after MI in mice and its precise mechanism remain unclear. PurposeHere, we firstly investigated the effects of calycosin on cardiac fibrosis and ventricular function in mice after MI and the role of transforming growth factor-beta receptor 1 (TGFBR1) signaling in the amelioration of cardiac fibrosis and ventricular function. MethodsIn vivo effects of calycosin on cardiac structure and function in mice with MI induced by left anterior descending coronary artery ligation were determined by hematoxylin and eosin staining, Masson trichrome staining, and echocardiography. The molecular mechanism of the interaction between TGFBR1 and calycosin was investigated using molecular docking, molecular dynamics (MD) simulation, surface plasmon resonance imaging (SPRi), immunohistochemistry, and western blotting (WB). Subsequently, cardiac-specific Tgfbr1 knockout mice were used to verify the effects of calycosin. The effect of calycosin on primary cardiac fibroblasts (CFs) proliferation and collagen deposition was detected using cell counting (CCK-8), EdU assay, and WB in vitro. CFs infected with an adenovirus that encodes TGFBR1 were used to verify the effects of calycosin. ResultsIn vivo, calycosin attenuated myocardial fibrosis and cardiac dysfunction following MI in a dose-dependent pattern. Calycosin-TGFBR1 complex was found to have a binding energy of -9.04 kcal/mol based on molecular docking. In addition, calycosin bound steadily in the cavity of TGFBR1 during the MD simulation. Based on SPRi results, the solution equilibrium dissociation constant for calycosin and TGFBR1 was 5.11 × 10−5 M. Calycosin inhibited the expression of TGFBR1, Smad2/3, collagen I, and collagen III. The deletion of TGFBR1 partially counteracted these effects. In vitro, calycosin suppressed CFs proliferation and collagen deposition after TGF-β1 stimulation by suppressing the TGFBR1 signaling pathway. The suppressive effects of calycosin were partially rescued by overexpression of TGFBR1. ConclusionCalycosin attenuates myocardial fibrosis and cardiac dysfunction following MI in mice in vivo via suppressing the TGFBR1 signaling pathway. Calycosin suppresses CFs proliferation and collagen deposition induced by TGF-β1 via inhibition of the TGFBR1 signaling pathway in vitro.

Dihydro-artemisinin inhibits angiotensin II-induced cardiac fibroblasts proliferation and induces apoptosis through phosphoinositide 3-kinase/akt pathway

Cardiac fibroblasts (CFs) abnormal proliferation can trigger myocardial fibrosis, which is closely related to a variety of cardiovascular diseases. This study investigated the effect of dihydroartemisinin (DHA) on CF growth through PI3K/AKT with angiotensin II (AngII). CCK-8 method and EdU were used detect cell proliferation, and flow cytometry assessed apoptosis of CFs. The PI3K and AKT expressions were determined, whilst immunofluorescence staining and RT-qPCR measured α-smooth muscle actin (α-SMA) level. With rat CFs exhibiting typical fibroblast morphology and nuclei in spindle, oval or polygonal shape, these cells were positive for the fibroblast marker vimentin. Treatment with AngII resulted in increased proliferation of CFs, but the proliferation declined in the presence of DHA (p < 0.05). Meanwhile, the AngII group had lower apoptosis of CFs than blank and DHA groups with highest apoptosis observed in the DHA group. α-SMA increased at the mRNA and protein levels after Ang II treatment and decreased after DHA treatment (p < 0.05). Importantly, the Ang II upregulated PI3K and AKT in CFs (p < 0.05) and the increase was abrogated by DHA administration with lowest levels (p < 0.05). Ang II activates PI3K/Akt in normal CFs, as administration of DHA can inhibit the effect of Ang II, thereby inhibiting proliferation of CFs induced by AngII and inducing apoptosis.

Kallistatin/Serpina3c inhibits cardiac fibrosis after myocardial infarction by regulating glycolysis via Nr4a1 activation

BackgroundFibrotic remodeling is an essential aspect of heart failure. Human kallistatin (KS, mouse Serpina3c homologs) inhibits fibrosis after myocardial infarction (MI) but the specific underlying mechanism is unknown. MethodsA total of 40 heart failure patients (HFPs) were enrolled and their plasma KS was measured using ELISA. Serpina3c−/− and C57BL/6 mice were used to construct the MI model. TGF-β1 or a hypoxic condition was established to interfere with the functioning of cardiac fibroblasts (CFs). RNA-seq was performed to assess the effect of Serpina3c on the transcriptome. FindingsThe levels of KS were used as a predictor of readmission among the HFPs. Serpina3c expression decreased in MI hearts and CFs. Serpina3c−/− led to the aggravation of MI fibrosis, and increased the proliferation of CFs. The overexpression of Serpina3c in CFs had the opposite effect. Glycolysis-related genes were significantly increased in Serpina3c−/− group by RNA-seq. Enolase (ENO1), which is a key enzyme in glycolysis, increased most significantly. Inhibition of ENO1 could antagonize the promotion of Serpina3c−/− on the proliferation of CFs. Co-IP was performed to verify the interaction between Serpina3c and Nr4a1. Serpina3c−/− inhibited the acetylation of Nr4a1 and increased the degradation of Nr4a1. Activation of Nr4a1 could negatively regulate the expression of ENO1 and inhibited the proliferation of Serpina3c−/− CFs in Serpina3c−/− MI mice. InterpretationSerpina3c inhibits the transcriptional activation of ENO1 by regulating the acetylation of Nr4a1, thereby reducing the fibrosis after MI by inhibiting glycolysis. Serpina3c is a potential target for prevention and treatment of heart failure after MI.

Knockdown of HIPK2 Attenuates Angiotensin II-Induced Cardiac Fibrosis in Cardiac Fibroblasts.

Homeodomain-interacting protein kinase-2 (HIPK2), a member of an evolutionary conserved family of serine/threonine kinases, has been observed to be involved in the pathogenesis of fibrotic diseases. However, its role in cardiac fibrosis remains unclear. In this study, we assessed the effect of HIPK2 on cardiac fibroblasts (CFs) in response to angiotensin II (Ang II) stimulation. The results indicated that HIPK2 expression was significantly increased in Ang II-induced CFs in a dose-dependent manner. Then, HIPK2 was knocked down in CFs to evaluate the roles of HIPK2. Knockdown of HIPK2 suppressed cell proliferation and migration in Ang II-induced CFs. The Ang II-caused increase in expression of α-smooth muscle actin, a hallmark of myofibroblast differentiation, was decreased by knockdown of HIPK2. HIPK2 knockdown also reduced extracellular matrix production including type I collagen and connective tissue growth factor. Furthermore, knockdown of HIPK2 blocked the activation of TGF-β1/Smad pathway in Ang II-induced CFs. These data suggested that HIPK2 knockdown prevented the Ang II-induced activation of CFs through inhibiting TGF-β1/Smad pathway, indicating HIPK2 might be an antifibrosis target for the treatment of cardiac fibrosis.