Pathway In Cardiac Fibroblasts Research Articles

The Senescent Heart-"Age Doth Wither Its Infinite Variety".

Cardiovascular diseases are a leading cause of morbidity and mortality world-wide. While many factors like smoking, hypertension, diabetes, dyslipidaemia, a sedentary lifestyle, and genetic factors can predispose to cardiovascular diseases, the natural process of aging is by itself a major determinant of the risk. Cardiac aging is marked by a conglomerate of cellular and molecular changes, exacerbated by age-driven decline in cardiac regeneration capacity. Although the phenotypes of cardiac aging are well characterised, the underlying molecular mechanisms are far less explored. Recent advances unequivocally link cardiovascular aging to the dysregulation of critical signalling pathways in cardiac fibroblasts, which compromises the critical role of these cells in maintaining the structural and functional integrity of the myocardium. Clearly, the identification of cardiac fibroblast-specific factors and mechanisms that regulate cardiac fibroblast function in the senescent myocardium is of immense importance. In this regard, recent studies show that Discoidin domain receptor 2 (DDR2), a collagen-activated receptor tyrosine kinase predominantly located in cardiac fibroblasts, has an obligate role in cardiac fibroblast function and cardiovascular fibrosis. Incisive studies on the molecular basis of cardiovascular aging and dysregulated fibroblast function in the senescent heart would pave the way for effective strategies to mitigate cardiovascular diseases in a rapidly growing elderly population.

Cyclic strain has antifibrotic effects on the human cardiac fibroblast transcriptome in a human cardiac fibrosis-on-a-chip platform

In cardiac fibrosis, in response to stress or injury, cardiac fibroblasts deposit excessive amounts of collagens which contribute to the development of heart failure. The biochemical stimuli in this process have been extensively studied, but the influence of cyclic deformation on the fibrogenic behavior of cardiac fibroblasts in the ever-beating heart is not fully understood. In fact, most investigated mechanotransduction pathways in cardiac fibroblasts seem to ultimately have profibrotic effects, which leaves an important question in cardiac fibrosis research unanswered: how do cardiac fibroblasts stay quiescent in the ever-beating human heart? In this study, we developed a human cardiac fibrosis-on-a-chip platform and utilized it to investigate if and how cyclic strain affects fibrogenic signaling. The pneumatically actuated platform can expose engineered tissues to controlled strain magnitudes of 0–25% – which covers the entire physiological and pathological strain range in the human heart – and to biochemical stimuli and enables high-throughput screening of multiple samples. Microtissues of human fetal cardiac fibroblasts (hfCF) embedded in gelatin methacryloyl (GelMA) were 3D-cultured on this platform and exposed to strain conditions which mimic the healthy human heart. The results provide evidence of an antifibrotic effect of the applied strain conditions on cardiac fibroblast behavior, emphasizing the influence of biomechanical stimuli on the fibrogenic process and giving a detailed overview of the mechanosensitive pathways and genes involved, which can be used in the development of novel therapies against cardiac fibrosis.

IL-27 promotes cardiac fibroblast activation and aggravates cardiac remodeling post myocardial infarction

Excessive and chronic inflammation post myocardial infarction (MI) causes cardiac fibrosis and progressive ventricular remodeling, which leads to heart failure. We previously found high levels of IL-27 in the heart and serum until day 14 in murine cardiac ischemia‒reperfusion injury models. However, whether IL-27 is involved in chronic inflammation-mediated ventricular remodeling remains unclear. In the present study, we found that MI triggered high IL-27 expression in murine cardiac macrophages. The increased expression of IL-27 in serum is correlated with cardiac dysfunction and aggravated fibrosis after MI. Furthermore, the addition of IL-27 significantly activated the JAK/STAT signaling pathway in cardiac fibroblasts (CFs). Meanwhile, IL-27 treatment promoted the proliferation, migration and extracellular matrix (ECM) production of CFs induced by angiotensin II (Ang II). Collectively, high levels of IL-27 mainly produced by cardiac macrophages post MI contribute to the activation of CFs and aggravate cardiac fibrosis.

Abstract 15359: Multi-Omics Integration Reveals Mechanosensitive Regulatory Pathways in Cardiac Fibroblasts

Introduction: Regionalized myocardial fibrosis exists in the inferobasal myocardium of patients undergoing surgical repair for Mitral Valve Prolapse, and supports a process in which cells within mechanically stressed myocardial regions alter their phenotype in order to increase tissue stiffness, eventually causing establishment of a fibrotic scar. How cells behave in different fibromechanical environments is not currently well understood. Hypothesis: Integrating RNA Seq and ATAC Seq datasets will identify mechanosensitive regulatory pathways in cardiac fibroblasts. Methods: Primary human cardiac fibroblasts (HCFs) were cultured on stiff (TC Plastic, ~2GPa), as well as soft (2kPa) substrates coated with type 1 collagen. Following 48 hours in serum-free conditions, HCFs were lysed and subjected to immunofluorescence (IF), Western blotting (WB), RNA Seq, and ATAC Seq. Differential analysis of RNA Seq, and ATAC Seq data was performed, followed by an integration analysis which linked differentially expressed genes to chromatin that opened/closed within 10 kB. Finally, motif enrichment analysis was performed using the online bioinformatics package Homer. Results: IF and WB revealed that HCFs cultured on a stiff substrate expressed markers of fibroblast activation (alpha SMA, P<0.0001), as well as increased amounts of Connexin 43 (P<0.0001). Additionally, there were higher numbers of Ki-67 positive cells (7.2% vs 2.1% P=0.001). RNA Seq revealed differential changes in 4324 genes (FDR adjusted P<0.05), several of which were subsequently validated using western blots. ATAC Seq identified 5855 chromatin regions which opened/closed in response to different mechanical environments in fibroblasts (adjusted P<0.05). Integrating RNA Seq and ATAC Seq datasets revealed 803 Chromatin changes that fell within 10kb of differentially expressed genes, with a predicted enrichment of TEAD binding sites in differentially opened chromatin (P=1.0*10-40). Conclusions: The mechanical environment plays a major role in cellular phenotype and directly regulates chromatin organization and expression of pro-fibrotic genes, with a possible role for TEAD as a driver of fibroblast activation.

Potential role of the apelin-APJ pathway in sex-related differential cardiotoxicity induced by doxorubicin in mice.

Preclinical and clinical findings suggest sexual dimorphism in cardiotoxicity induced by a chemotherapeutic drug, doxorubicin (DOX). However, molecular alterations leading to sex-related differential vulnerability of heart to DOX toxicity are not fully explored. In the present study, RNA sequencing in hearts of B6C3F1 mice indicated more differentially expressed genes in males than females (224 vs. 19; ≥1.5-fold, False Discovery Rate [FDR] < 0.05) at 1week after receiving 24 mg/kg total cumulative DOX dose that induced cardiac lesions only in males. Pathway analysis further revealed probable inactivation of cardiac apelin fibroblast signaling pathway (p = 0.00004) only in DOX-treated male mice that showed ≥1.25-fold downregulation in the transcript and protein levels of the apelin receptor, APJ. In hearts of DOX-treated females, the transcript levels of apelin (1.24-fold) and APJ (1.47-fold) were significantly (p < 0.05) increased compared to saline-treated controls. Sex-related differential DOX effect was also observed on molecular targets downstream of the apelin-APJ pathway in cardiac fibroblasts and cardiomyocytes. In cardiac fibroblasts, upregulation of Tgf-β2, Ctgf, Sphk1, Serpine1, and Timp1 (fibrosis; FDR < 0.05) in DOX-treated males and upregulation of only Tgf-β2 and Timp1 (p < 0.05) in females suggested a greater DOX toxicity in hearts of males than females. Additionally, Ryr2 and Serca2 (calcium handling; FDR < 0.05) were downregulated in conjunction with 1.35-fold upregulation of Casp12 (sarcoplasmic reticulum-mediated apoptosis; FDR < 0.05) in DOX-treated male mice. Drug effect on the transcript level of these genes was less severe in female hearts. Collectively, these data suggest a likely role of the apelin-APJ axis in sex-related differential DOX-induced cardiotoxicity in our mouse model.

Abstract P2025: BET Bromodomain Proteins Are Necessary For TGFβ2-Mediated Myofibroblast Activation

Fibrosis is a key feature of dilated cardiomyopathy (DCM). Stress signaling triggers the activation of quiescent fibroblasts to myofibroblasts. Bromodomain and extraterminal (BET) epigenetic reader proteins integrate stress-responsive signaling. We explored the role of BETs on distinct inflammatory signaling pathways in cardiac fibroblasts. Fibroblasts were isolated from wild type hearts using the Langendorff procedure. Cell cultures were treated with vehicle, the pan-BET inhibitor JQ1, or siRNA targeting Smad3 or Rela (to inhibit TGFβ and NFκB pathways, respectively) followed by TGFβ2 stimulation. Gene expression was assessed by QPCR, α-smooth muscle actin (αSMA) was quantified by western blot, and NFκB transcription was evaluated using a dual luciferase reporter assay. The interaction between BRD4 and SMAD3 was assessed by coimmunoprecipitation (coIP) in immortalized adult mouse cardiac fibroblast cell cultures treated with TGFβ2, JQ1, or vehicle. TGFβ2-induced fibroblast activation was ablated by JQ1 and Smad3 siRNA (reduced expression of Acta2, Col3a1, Postn and αSMA). CoIP revealed a direct interaction between BRD4 and SMAD3 in fibroblasts, which increased 2.2-fold (p&lt;0.05) after TGFβ2 stimulation. By contrast, Rela siRNA alone induced myofibroblast gene expression ( Acta2 , 6.9-fold; Col3a1 , 7.0-fold; Postn , 11.2-fold; p&lt;0.05 vs. vehicle for all), and increased αSMA levels (2.2-fold; p&lt;0.05). This effect of Rela siRNA was similar to that observed with TGFβ2 stimulation. Further, TGFβ2 inhibited NFκB-driven gene transcription (-2.9-fold; p&lt;0.05 vs. vehicle). These data demonstrate that BRD4 is a critical co-activator of TGFβ2-SMAD3 signaling and cardiac fibroblast activation. We also found that NFκB signaling is downregulated by TGFβ2, suggesting that NFκB may modulate inflammatory gene network activation.

Epicardial adipose tissie-derived leptin induce MMPs/TIMPs imbalance and promotes myocardial remodeling in high fat diet-induced obese rats

Abstract Background and aims Epicardial adipose tissue (EAT)-derived leptin contributes to myocardial remodeling in metabolic syndrome. However, the precise mechanisms remain to be determined. The present study was designed to elucidate the adverse effects of EAT-derived leptin on obesity-related myocardial remodeling. Methods and results Eight-week-old male Wistar rats were divided into two groups that received either a normal diet (control, n=10) or a high-fat diet (obese, n=10) for 12 weeks. Obese rats exhibited abnormal myocardial structure, diastolic dysfunction and abundant collagen deposition. Local leptin expression in obese rats EAT upregulated along with adipocyte hypertrophy, accompanied by renin-angiotensin-aldosterone system (RAAS) activation and increased oxidative stress level. Leptin receptor (ObR) and angiotensin II type 1 receptor (AT1R) expression in obese EAT were significantly higher than that in control. In vitro, mature adipocytes treated with angiotensin II (Ang II) exhibited pronounced leptin synthesis and secretion by promoting AP-1 nuclear translocation via the AT1R-ROS-ERK1/2 pathway. Moreover, cardiac fibroblasts were incubated with obese rat EAT-conditioned medium (EAT-CM), plus various inhibitors. EAT-derived leptin promoted proliferation of cardiac fibroblasts associated with increased ERK1/2 phosphorylation and induced MMPs/TIMPs imbalance, stimulating upregulation of type I collagen via the JAK2/STAT3-TGF-β1/Smad3 pathway in cardiac fibroblasts of obese rats. Conclusions The paracrine effect of EAT-derived leptin on myocardial remodeling, inducing MMPs/TIMPs imbalance and promoting the proliferation of cardiac fibroblasts via activating ERK1/2 and JAK2/STAT3-TGF-β1/Smad3 pathway in obesity. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): the Nature Science Foundation of China (grant no. 81873513, 81600574, and 30871042)

A Catecholaldehyde Metabolite of Norepinephrine Induces Myofibroblast Activation and Toxicity via the Receptor for Advanced Glycation Endproducts: Mitigating Role of l-Carnosine.

Monoamine oxidase (MAO) is rapidly gaining appreciation for its pathophysiologic role in cardiac injury and failure. Oxidative deamination of norepinephrine by MAO generates H2O2 and the catecholaldehyde 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), the latter of which is a highly potent and reactive electrophile that has been linked to cardiotoxicity. However, many questions remain as to whether catecholaldehydes regulate basic physiological processes in the myocardium and the pathways involved. Here, we examined the role of MAO-derived oxidative metabolites in mediating the activation of cardiac fibroblasts in response to norepinephrine. In neonatal murine cardiac fibroblasts, norepinephrine increased reactive oxygen species (ROS), accumulation of catechol-modified protein adducts, expression and secretion of collagens I/III, and other markers of profibrotic activation including STAT3 phosphorylation. These effects were attenuated with MAO inhibitors, the aldehyde-scavenging dipeptide l-carnosine, and FPS-ZM1, an antagonist for the receptor for advanced glycation endproducts (RAGE). Interestingly, treatment of cardiac fibroblasts with a low dose (1 μM) of DOPEGAL-modified albumin phenocopied many of the effects of norepinephrine and also induced an increase in RAGE expression. Higher doses (>10 μM) of DOPEGAL-modified albumin were determined to be toxic to cardiac fibroblasts in a RAGE-dependent manner, which was mitigated by l-carnosine. Collectively, these findings suggest that norepinephrine may influence extracellular matrix remodeling via an adrenergic-independent redox pathway in cardiac fibroblasts involving the MAO-mediated generation of ROS, catecholaldehydes, and RAGE. Furthermore, since elevations in the catecholaminergic tone and oxidative stress in heart disease are linked with cardiac fibrosis, this study illustrates novel drug targets that could potentially mitigate this serious disorder.

Chlorogenic Acid Alleviates Hyperglycemia-Induced Cardiac Fibrosis through Activation of the NO/cGMP/PKG Pathway in Cardiac Fibroblasts.

Hyperglycemia-induced cardiac fibrosis is one of the main causes of diabetic cardiomyopathy (DM). Chlorogenic acid (CGA) found in many foods has excellent hypoglycemic effectiveness, but it is not known whether CGA can improve DM by inhibiting cardiac fibrosis caused by hyperglycemia. Type I diabetic mice are induced by streptozotocin, and after treatment with CGA for 12 weeks, cardiac functions and fibrosis are determined. CGA significantly attenuates hyperglycemia-induced cardiac fibrosis and improves cardiac functions. The mechanism of CGA on fibrotic inhibition is further studied by immunofluorescence, western blot and RNA interference technology in vivo and in vitro. The results show CGA exerted its anti-fibrotic effects through activating the cyclic GMP/protein kinase G pathway (cGMP/PKG) to block hyperglycemia-induced nuclear translocation of p-Smad2/3, and then inhibiting pro-fibrotic gene expression in cardiac fibroblasts without depending on its hypoglycemic function. Moreover, the data also revealed that CGA increased cGMP level and activated PKG in cardiac fibroblasts by enhancing endothelial nitric oxide synthase (eNOS) activity and NO production. Besides lowering blood glucose, CGA also has an independent ability to inhibit cardiac fibrosis. Therefore, long-term consumption of foods rich in CGA for diabetic patients will have great benefits to improve diabetic cardiomyopathy.

β2-adrenergic stimulation induces interleukin-6 by increasing Arid5a, a stabilizer of mRNA, through cAMP/PKA/CREB pathway in cardiac fibroblasts.

Background and PurposeIn cardiovascular diseases, cardiac fibroblasts (CFs) participate in the myocardial inflammation by producing pro‐inflammatory cytokines, worsening the prognosis. β2‐adrenergic receptor (AR) and β3AR are expressed in CFs, and β‐adrenergic stimulation promotes CFs to produce pro‐inflammatory cytokines. However, the mechanism of the expression of pro‐inflammatory cytokines in response to β‐adrenergic stimulation remains to be fully elucidated.Experimental ApproachCFs were isolated from adult wild‐type or AT‐rich interactive domain‐containing protein 5A (Arid5a) knockout mice. The expression of mRNA was measured by real‐time RT‐PCR. Interleukin (IL)‐6 protein was measured by ELISA. The activity of nuclear factor‐κB (NF‐κB) and cyclic AMP (cAMP) response element binding protein (CREB) was assessed by ELISA‐like assay or Western blotting.Key ResultsThe β‐adrenergic stimulation remarkably induced IL‐6 mRNA and protein through β2AR in CFs. The activation of adenylate cyclase and the enhancement of intracellular cAMP resulted in the upregulation of IL‐6 mRNA expression. The induction of IL‐6 transcript by β2AR signaling was independent of NF‐κB. Concomitant with IL‐6, the expression of Arid5a, an IL‐6 mRNA stabilizing factor, was enhanced by β2‐adrenergic stimulation and by cAMP increase. Importantly, β2AR signaling‐mediated IL‐6 induction was suppressed in Arid5a knockout CFs. Finally, β2AR stimulation phosphorylated CREB via PKA pathway, and the activation of CREB was essential for the induction of Arid5a and IL‐6 mRNA.Conclusion and Implicationsβ2‐adrenergic stimulation post‐transcriptionally upregulates the expression of IL‐6 by the induction of Arid5a through cAMP/PKA/CREB pathway in adult CFs. β2AR/Arid5a/IL‐6 axis could be a therapeutic target against cardiac inflammation.

Cardiac Fibroblast p38 MAPK: A Critical Regulator of Myocardial Remodeling.

The cardiac fibroblast is a remarkably versatile cell type that coordinates inflammatory, fibrotic and hypertrophic responses in the heart through a complex array of intracellular and intercellular signaling mechanisms. One important signaling node that has been identified involves p38 MAPK; a family of kinases activated in response to stress and inflammatory stimuli that modulates multiple aspects of cardiac fibroblast function, including inflammatory responses, myofibroblast differentiation, extracellular matrix turnover and the paracrine induction of cardiomyocyte hypertrophy. This review explores the emerging importance of the p38 MAPK pathway in cardiac fibroblasts, describes the molecular mechanisms by which it regulates the expression of key genes, and highlights its potential as a therapeutic target for reducing adverse myocardial remodeling.

Calcium‑sensing receptor promotes high glucose‑induced myocardial fibrosis via upregulation of the TGF‑β1/Smads pathway in cardiac fibroblasts.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes and myocardial fibrosis is its major pathological feature. Calcium-sensing receptor (CaSR) is a G protein-coupled receptor and participates in the regulation of calcium homeostasis; it is implicated in a range of diseases, including myocardial ischemia/reperfusion injury, myocardial infarction and pulmonary hypertension. However, whether CaSR is associated with myocardial fibrosis in DCM has remained elusive. In the present study, type 1 diabetic (T1D) rats and primary neonatal rat cardiac fibroblasts (CFs) were used to observe changes in CaSR to assess its potential as an indicator of myocardial fibrosis. The in vivo experiments revealed that in the T1D and CaSR agonist (R568) groups, evident collagen (Col)-I and -III deposition was present after 12 weeks. Furthermore, the in vitro experiment indicated that the levels of transforming growth factor (TGF)-β1, phosphorylated (p-) protein kinase C, p-p38, p-Smad2, TβRI, TβRII, along with the intracellular Ca2+ levels and the content of TGF-β1 in the culture medium were significantly increased in a high-glucose (HG) group and an R568-treated group. Treatment with the CaSR inhibitor Calhex231 significantly inhibited the abovementioned changes. Collectively, the results indicated that the increase of CaSR expression in CFs may induce intracellular Ca2+ increases and the activation of TGF-β1/Smads, and enhance the proliferation of CFs, along with the excessive deposition of Col, resulting in myocardial fibrosis. The present results indicate an important novel mechanism for HG-induced myocardial fibrosis and suggest that CaSR may be a promising potential therapeutic target for DCM.

The orphan receptor GPRC5B modulates inflammatory and fibrotic pathways in cardiac fibroblasts and mice hearts

Inflammation is a major driver of cardiac remodeling. Cardiac fibroblasts play an integral role in cardiac inflammation, fibrosis and remodeling. The orphan G-protein-coupled-receptor 5B of family C (GPRC5B) has recently been shown to have pro-inflammatory effects in adipocytes via the NFκB-signaling-pathway. Here, we investigated whether GPRC5B is involved in myocardial inflammation and fibrosis.Using neonatal rat cardiac fibroblasts (NRCF) we show that the transcription and the expression of endogenous GPRC5B is induced by stimulation with TNFα and LPS as well as through cyclic mechanical stretch, while the principle pro-fibrotic factor TGFβ has no effect on the GPRC5B expression. Furthermore, we demonstrate that adenoviral overexpression and siRNA-mediated knockdown of GPRC5B in NRCF significantly alters the transcription level of the pro-inflammatory and pro-fibrotic cytokines TNFα, IL-1β, IL-6 and MCP-1, and extracellular matrix-degrading MMP-9 in vitro. Additionally, in adult GPRC5B-transgenic mice the protein expression of collagen-1A1 is decreased and the production of MMP-9 is increased, indicating remodeling of the extracellular matrix in vivo.Our data show that GPRC5B is up-regulated by inflammatory signals and mechanical stress in NRCF, while GPRC5B modulates the inflammatory response of cardiac fibroblasts and the degradation of extracellular matrix-proteins in the mice heart. Thus, our findings are the first to report a novel role of the orphan receptor GPRC5B in fibroblast-driven myocardial inflammation and cardiac remodeling.

Qishen granule attenuates cardiac fibrosis by regulating TGF-β /Smad3 and GSK-3β pathway

BackgroundCardiac fibrosis is a common pathological progress of cardiovascular disease resulting from the excessive accumulation of extracellular matrix (ECM). Transforming growth factor (TGF)-β/SMADs pathway is a canonical signaling pathway which directly induces expressions of ECM related genes. Qishen Granule (QSG), a traditional Chinese formula developed from Zhen-Wu Decoration for heart failure (HF), has been proven to have definite therapeutic effects on cardiac fibrosis. However, its underlying mechanisms remain unclear. PurposeTo investigate the effects of QSG on TGF-β pathway and the downstream mediators including Smad3 and Glycogen synthase kinase (GSK)-3β. MethodsHF model was induced by ligation of left coronary artery on male Sprague-Dawley (SD) rats. Rat were randomly divided into four groups including sham group, model group, QSG group and Fosinopril control group. Rats in each group were treated for 28 days, and 2D echocardiography was adopted to evaluate the heart function. The degree of cardiac fibrosis was assessed by Hematoxylin-Eosin (HE), Masson's trichrome and Picrosirius red (PSR) staining. Contents of collagen Ⅰ and Ⅲ were assessed by immunohistochemical method. Expressions of genes and proteins in TGF-β/SMADs and PI3K-GSK-3 signaling pathways were detected by Real-time Fluorescence Quantitative PCR (RT-qPCR) and Western blot respectively. TGF-β1-treated cardiac fibroblasts of neonatal SD rats were adopted for in vitro studies. Results28 days after the surgery, cardiac ejection fraction (EF) and fractional shortening (FS) values in the model group showed a remarkable decrease, indicating the induction of HF model. QSG and Fosinopril elevated the EF and FS values, demonstrating cardio-protective effects. Pathological staining and immunohistochemistry showed that the contents of collagen I and III dramatically increased in the cardiac tissue of the model group compared with the sham group while QSG treatment reduced collagen contents. Furthermore, expressions of TGF-β1, p-Smad3 and p-GSK-3β were significantly decreased in the QSG treatment group compared with the model group, suggesting that the QSG may attenuate cardiac fibrosis through regulating TGF-β/Smad3 pathway. In vitro study further showed that the productions of type Ⅰ and Ⅲ collagen and α-smooth muscle actin (α-SMA) of cardiac fibroblasts were significantly increased by incubation with TGF-β1. QSG could markedly reduce the secretion of collagen Ⅰ and Ⅲ and α-SMA expression. Protein expressions of p-Smad3, PI3K, p-Akt and p-GSK-3β were significantly up-regulated by stimulation of TGF-β1. Treatment with QSG could suppress the activity of Smad3 and PI3K-GSK-3β signaling pathway in cardiac fibroblasts. ConclusionQSG improves cardiac function through inhibiting cardiac fibrosis. The anti-fibrotic effects are potentially mediated by the inhibition of the TGF-β/Smad3 pathway and the phosphorylation of GSK-3β.

Collagen‐derived matricryptin reduces cardiac dysfunction post‐MI via integrin alpha 4

Adverse cardiac remodeling post myocardial infarction (MI) triggers a compensatory maladaptive cardiac response that contributes to heart failure. The infarcted area is replaced by a fibrotic scar that favors collagen deposition instead of its degradation. The evolution of the scar formation depends on the interaction between cardiac cells, particularly fibroblasts, and the extracellular matrix (ECM). Matricryptins are biologically active peptides, generated from ECM proteolysis, able to regulate cell function and survival. We recently identified a collagen‐derived matricryptin (p1158/59) that gradually forms post‐MI. p1158/59 plasma levels negatively correlate with left ventricle (LV) filling pressure, suggesting a beneficial effect for this matricryptin. p1158/59 stimulates LV fibroblast migration in vitro, and mice treated with p1158/59 for 7 days post‐MI displayed less collagen deposition and reduced LV dilation. Accordingly, our goal was to identify the fibroblast receptor(s) involved in p1158/59 actions on cell migration and ECM secretion responsible for the beneficial effects observed on LV remodeling. Additionally, we aim to establish the therapeutic potential of p1158/59 in long term cardiac remodeling.MethodsUsing LV and NIH 3T3 fibroblasts, we performed automatic migration assays with increasing doses of p1158/59 to evaluate changes in receptor gene expression. Blocking antibodies against integrins were used to validate the receptor's findings. We used a murine permanent occlusion MI model (male and female C57Bl/6 mice 3–5 months old, n=4–6/group) to assess effects of p1158/59 treatment (950 μg/day for 14 days), and cardiac function was assessed by serial echocardiography (D0, D7, D14).ResultsCardiac fibroblasts incubated for 24h with 100nM p1158/59 displayed &gt;2‐fold increase of several integrins, with the highest increase on integrin a4 (Itga4), suggesting it as a receptor for p1158/59. Using a migration assay, fibroblasts were incubated with p1158/59 100nM ± Itga4 blocking antibody (15μg/mL) for 2h before wound. The antibody abolished the pro‐migratory effects of the matricryptin, indicating Itga4 as the receptor for p1158/59. Echocardiography analysis demonstrated that mice treated with p1158/59 post‐MI had an increase of 42% in ejection fraction, compared to controls. Additionally, cardiac output exceeded 50% when compared to vehicle controls. These results indicate that p1158/59 attenuated cardiac dysfunction post‐MI.ConclusionOur data suggest that matricryptin p11/58/59 treatment reduces maladaptive LV remodeling post‐MI. Noteworthy, p1158/59 beneficial effects occur through activation of signaling pathways in cardiac fibroblasts via the receptor Itga4.Support or Funding InformationWe acknowledge support from the American Heart Association (18AIREA33960311 and 19PRE34450066) and the Research and Graduate School at East Carolina University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calreticulin promotes proliferation and extracellular matrix expression through Notch pathway in cardiac fibroblasts.

Cardiac fibrosis is one of the most important underlying causes of several cardiac diseases. The role of calreticulin (CRT) in cardiac diseases has already been established. The overor under-expression of CRT can lead to cardiac diseases. This study was aimed to explore the effect of CRT on cardiac fibrosis and also to investigate the possible underlying molecular mechanism. Human cardiac fibroblast cells (HCF) were used in the experiment. The cells were transfected with the CRT expression vector constructed by sub-cloning the full-length wild-type CRT coding sequence into pcDNA3.1 (pc-CRT group), empty construct pcDNA3.1 (pcDNA3.1 group), CRT-specific siRNA (si-CRT), and si-NC (negative control). The Cell Counting Kit-8 (CCK-8) assay, apoptosis assay and invasion assay were performed. Quantitative real time polymerase chain reaction (qRT PCR) and western blot analysis were performed to measure the expressions of different mRNAs and proteins. The CRT expression was significantly increased (p < 0.01) and decreased (p < 0.01) in the pc-CRT and si-CRT groups, respectively. The CRT over-expression led to increased cell viability and invasiveness (p < 0.05) and a decreased percentage of apoptotic cells. The over-expression of CRT led to a significant increase in the expressions of collagen (I and III) (p < 0.01) and matrix metalloproteinases (MMP-2 and 9) (p < 0.05). The Notch pathway was also significantly activated (p < 0.05) by the over-expression of CRT and vice versa when suppressed. The results showed that the CRT over-expression was associated with increased cell viability and invasiveness and decreased apoptosis, and the activation of the Notch pathway in HCF, which suggests its possible implication in CRT-induced cardiac fibrosis.

Featured Article: TGF-β1 dominates extracellular matrix rigidity for inducing differentiation of human cardiac fibroblasts to myofibroblasts.

Cardiac fibroblasts and their activated derivatives, myofibroblasts, play a critical role in wound healing after myocardial injury and often contribute to long-term pathological outcomes, such as excessive fibrosis. Thus, defining the microenvironmental factors that regulate the phenotype of cardiac fibroblasts and myofibroblasts could lead to new therapeutic strategies. Both chemical and biomechanical cues have previously been shown to induce myofibroblast differentiation in many organs and species. For example, transforming growth factor beta 1, a cytokine secreted by neutrophils, and rigid extracellular matrix environments have both been shown to promote differentiation. However, the relative contributions of transforming growth factor beta 1 and extracellular matrix rigidity, two hallmark cues in many pathological myocardial microenvironments, to the phenotype of human cardiac fibroblasts are unclear. We hypothesized that transforming growth factor beta 1 and rigid extracellular matrix environments would potentially have a synergistic effect on the differentiation of human cardiac fibroblasts to myofibroblasts. To test this, we seeded primary human adult cardiac fibroblasts onto coverslips coated with polydimethylsiloxane of various elastic moduli, introduced transforming growth factor beta 1, and longitudinally quantified cell phenotype by measuring expression of α-smooth muscle actin, the most robust indicator of myofibroblasts. Our data indicate that, although extracellular matrix rigidity influenced differentiation after one day of transforming growth factor beta 1 treatment, ultimately transforming growth factor beta 1 superseded extracellular matrix rigidity as the primary regulator of myofibroblast differentiation. We also measured expression of POSTN, FAP, and FSP1, proposed secondary indicators of fibroblast/myofibroblast phenotypes. Although these genes partially trended with α-smooth muscle actin expression, they were relatively inconsistent. Finally, we demonstrated that activated myofibroblasts incompletely revert to a fibroblast phenotype after they are re-plated onto new surfaces without transforming growth factor beta 1, suggesting differentiation is partially reversible. Our results provide new insights into how microenvironmental cues affect human cardiac fibroblast differentiation in the context of myocardial pathology, which is important for identifying effective therapeutic targets and dictating supporting cell phenotypes for engineered human cardiac disease models. Impact statement Heart disease is the leading cause of death worldwide. Many forms of heart disease are associated with fibrosis, which increases extracellular matrix (ECM) rigidity and compromises cardiac output. Fibrotic tissue is synthesized primarily by myofibroblasts differentiated from fibroblasts. Thus, defining the cues that regulate myofibroblast differentiation is important for understanding the mechanisms of fibrosis. However, previous studies have focused on non-human cardiac fibroblasts and have not tested combinations of chemical and mechanical cues. We tested the effects of TGF-β1, a cytokine secreted by immune cells after injury, and ECM rigidity on the differentiation of human cardiac fibroblasts to myofibroblasts. Our results indicate that differentiation is initially influenced by ECM rigidity, but is ultimately superseded by TGF-β1. This suggests that targeting TGF-β signaling pathways in cardiac fibroblasts may have therapeutic potential for attenuating fibrosis, even in rigid microenvironments. Additionally, our approach can be leveraged to engineer more precise multi-cellular human cardiac tissue models.

Cyclosporin A induces autophagy in cardiac fibroblasts through the NRP-2/WDFY-1 axis

Cyclosporin A (CsA) is an effective immunosuppressive agent, but its myocardial toxicity limits its widespread and long-term clinical application. In this study, CsA treatment led to damages in myocardial fiber structure, an increase in myocardial fibrosis, and changes in heart size and shape; moreover, the degree of damage was exacerbated with prolonged drug application and increases in dose. However, the mechanism is not clear; therefore, the purpose of this study was to reveal the mechanism of CsA-induced myocardial fibrosis and identify a new target for the prevention and treatment of CsA-induced myocardial injury. Cardiac fibroblasts were treated with CsA (5, 10, or 20 μg/mL) for 24 h. Autophagy was observed by electron microscopy and immunofluorescence. The expression of NRP-2/WDFY-1, autophagy-related proteins (Beclin1 and LC3B), fibrosis-related proteins (MMP2/9), and fibroblast phenotype conversion factor (α-SMA) was evaluated by Western blot. The expression of collagen I was determined by ELISA. Then, we used the gene interference technique to alter WDFY-1 expression with or without CsA or 3-MA treatment for 24 h, and the effects on autophagy and the expression of autophagy-related proteins, fibrosis-associated proteins, IFN-α, TNF-α, and IL-6 were determined. The results showed the following: (1) CsA induced fibrosis-related protein (MMP2/9), fibroblast phenotype conversion factor (α-SMA), and collagen I up-regulation in a dose-dependent manner. (2) CsA induced the formation of autophagosomes and up-regulated the expression of Beclin1, LC3B, and the ERK/MAPK pathway in cardiac fibroblasts. (3) CsA induced NRP-2 down-regulation and WDFY-1 up-regulation. (4) Depletion of WDFY-1 inhibited CsA-induced autophagy, TNF-α and IFN-α up-regulation, and fibrosis. (5) The autophagy inhibitor 3-MA inhibited CsA-induced TNF-α and IFN-α up-regulation and fibrosis. Overall, cyclosporin A induces autophagy in cardiac fibroblasts through the NRP-2/WDFY-1 axis, which promotes the progression of myocardial fibrosis.

Role of microRNA-1-mediated AMP-activated protein kinase pathway in cardiac fibroblasts induced by high glucose in rats

To investigate the role of microRNA-1 (miR-1) in cardiac fibroblasts induced by high glucose in rats. The primary fibroblasts were cultured from the apical tissue of 1-3 day-old Sprague-Dawley (SD) rats. The cells which were passaged to generation 3 or 4, were randomly divided into normal glucose+lentivector-vehicle group (CON+Lv-Vehicle group), normal glucose+lentivector-miR-1 group (CON+Lv-miR1 group), high glucose+lentivector-vehicle group (HG+Lv-Vehicle group), high glucose+lentivector-miR-1 group (HG+Lv-miR1 group), high glucose+Lv-Vehicle+inhibitor group (HG+Lv-Vehicle+CC group), and high glucose+lentivector-miR-1+inhibitor group (HG+Lv-miR1+CC group). The myocardial fibroblasts were cultured in the concentration of 5.5 mmol/L glucose (normal glucose) or 25.0 mmol/L (high glucose) DMEM medium. Then lentiviral vector containing miR-1 silent sequence or the same volume of lentiviral vector was inoculated into the cells. The AMP activated protein kinase (AMPK) inhibitor Compound C (20 μmol/L) was added to the medium at 12 hours before sampling in inhibitor groups. The expression of phosphorylation of AMPK (p-AMPK), collagenIandIII, matrix metalloproteinase (MMP-2, MMP-9), and autophagy flux related protein LC3B-II and p62/SQSTM1 were measured by Western Blot. The purity of rat myocardial fibroblasts in vitro was 97%. Compared with CON+Lv-Vehicle group, there was no significant difference in the expression of p-AMPK in CON+Lv-miR1 group, the expression of p-AMPK in HG+Lv-Vehicle group was significantly decreased (p-AMPK/t-AMPK: 44.72±3.29 vs. 100.00±7.77, P < 0.01). The expression of p-AMPK in HG+Lv-miR1 group was higher than that in HG+Lv-Vehicle group (p-AMPK/t-AMPK: 60.52±5.16 vs. 44.72±3.29, P < 0.05). Compared with HG+Lv-Vehicle group, the expressions of collagen, MMP, LC3B-II and p62/SQSTM1 in HG+Lv-miR1 group were significantly decreased; after the treatment with AMPK inhibitor, the expressions of collagen, MMP, LC3B-II, p62/SQSTM1 were significantly increased (HG+Lv-Vehicle+CC group vs. HG+Lv-Vehicle group: collagen I/β-actin: 158.74±13.21 vs. 100.00±7.64, collagen III/β-actin: 177.38±17.31 vs. 100.00±5.18, MMP-2/β-actin: 130.09±14.31 vs. 100.00±10.47, MMP-9/β-actin: 215.54±20.92 vs. 100.00±11.28, LC3B-II/β-actin: 159.34±13.83 vs. 100.00±6.44, p62/SQSTM1/β-actin: 201.01±24.02 vs. 100.00±8.62; HG+Lv-miR1+CC group vs. HG+Lv-miR1 group: collagen I/β-actin: 108.69±9.93 vs. 80.83±7.24, collagen III/β-actin: 127.68±10.46 vs. 81.56±9.97, MMP-2/β-actin: 106.66±10.21 vs. 74.80±7.43, MMP-9/ β-actin: 145.65±11.56 vs. 74.63±10.55, LC3B-II/β-actin: 150.15±13.28 vs. 22.98±2.87, p62/SQSTM1/β-actin: 130.48±10.74 vs. 49.90±2.27, all P < 0.05). miR-1 gene silencing inhibits myocardial fibrosis induced by high glucose, its mechanism may be related to the up-regulation of p-AMPK, which can recover autophagy flux.

MiR-26a-5p regulates cardiac fibroblasts collagen expression by targeting ULK1

MiRNA is a class of small non-coding RNA which has an important effect on posttranscriptional gene regulation. It can regulate the expression of the target gene at the mRNA level and further influence the protein level of the target gene. We found that ULK1 may be the target gene of miR-26a-5p, and ULK1 (unc-51 like autophagy activating kinase 1) is a key component in autophagy pathway. In this study, we overexpressed miR-26a-5p by transfecting miR-26a-5p mimic into cells and simultaneously inhibited miR-26a-5p by transfecting miR-26a-5p inhibitor into cells. We demonstrated that overexpression of miR-26a-5p can reduce the expression of ULK1 and collagen I, and decrease the activation of LC3-I to LC3-II. In contrast, inhibition of miR-26a-5p can increase the expression of ULK1 and collagen I, and increase the activation of LC3-I to LC3-II. The Dual-luciferase reporter assay showed that miR-26a-5p directly acted on the 3′UTR of ULK1 and thus affected the expression of ULK1. As such, our study demonstrated that miR-26a-5p might regulate the autophagy in cardiac fibroblasts by targeting ULK1, which may have an effect on cardiac fibrosis. To our knowledge, this is the first study that shows miR-26a-5p regulates the autophagic pathway in cardiac fibroblasts.