Differentiation Of Cardiac Fibroblasts Research Articles

Mutation in NPPA causes atrial fibrillation by activating inflammation and cardiac fibrosis in a knock‐in rat model

Atrial fibrillation (AF) affects >30 million individuals worldwide. However, no genetic mutation from human patients with AF has been linked to inflammation. Here, we show that AF-associated human variant p.Ile138Thr in natriuretic peptide A (NPPA) encoding the atrial natriuretic peptide (ANP) causes inflammation, fibroblast activation, atrial fibrosis, and AF in knock-in (KI) rats. Variant p.Ile138Thr inhibits the interaction between ANP and its receptor natriuretic peptide receptor A and reduces intracellular cGMP levels. RNA sequencing and follow-up analyses showed that mutant ANP (mANP) activates multiple innate immunity pathways, including TNF-α, NF-κB, and IL-1β signaling. mANP induces differentiation of cardiac fibroblasts (CFs) to myofibroblasts and promotes CF proliferation and fibrosis. These results suggest that NPPA variant p.Ile138Thr causes AF by activating TNF-α, NF-κB, and IL-1β signaling, inflammation, and fibrosis. Multiple computational programs suggest that p.Ile138Thr is damaging or deleterious. Based on the 2015 American College of Medical Genetics and Genomics Standards and Guidelines, p.Ile138Thr can be classified as a likely pathogenic variant. Variant p.Ile138Thr was found only in Asian people in the Genome Aggregation Database and Exome Aggregation Consortium database at an averaged frequency of 0.026%. An estimated 1.15 million Asian people carry the variant and might be at risk of AF. The KI rats may provide an inflammation-based, genetic animal model for AF valuable for testing anti-inflammation or other therapies for AF.-Cheng, C., Liu, H., Tan, C., Tong, D., Zhao, Y., Liu, X., Si, W., Wang, L., Liang, L., Li, J., Wang, C., Chen, Q., Du, Y., Wang, Q. K., Ren, X. Mutation in NPPA causes atrial fibrillation by activating inflammation and cardiac fibrosis in a knock-in rat model.

ALDH2 Activation Inhibited Cardiac Fibroblast-to-Myofibroblast Transformation Via the TGF-β1/Smad Signaling Pathway.

Pathological stimulus-triggered differentiation of cardiac fibroblasts plays a major role in the development of myocardial fibrosis. Aldehyde dehydrogenase 2 (ALDH2) was reported to exert a protective role in cardiovascular disease, and whether ALDH2 is involved in cardiac fibroblast differentiation remains unclear. In this study, we used transforming growth factor-β1 (TGF-β1) to induce the differentiation of human cardiac fibroblasts (HCFs) and adopted ALDH2 activator Alda-1 to verify the influence of ALDH2 on HCF differentiation. Results showed that ALDH2 activity was obviously impaired when treating HCFs with TGF-β1. Activation of ALDH2 with Alda-1 inhibited the transformation of HCFs into myofibroblasts, demonstrated by the decreased smooth muscle actin (α-actin) and periostin expression, reduced HCF-derived myofibroblast proliferation, collagen production, and contractility. Moreover, application of Smad2/3 inhibitor alleviated TGF-β1-induced HCF differentiation and improved ALDH2 activity, which was reversed by the application of ALDH2 inhibitor daidzin. Finally, Alda-1-induced HCF alterations alleviated neonatal rat cardiomyocyte hypertrophy, supported by the immunostaining of α-actin. To summarize, activation of ALDH2 enzymatic activity inhibited the differentiation of cardiac fibroblasts via the TGF-β1/Smad signaling pathway, which might be a promising strategy to relieve myocardial fibrosis of various causes.

Vascular peroxidase 1 is a novel regulator of cardiac fibrosis after myocardial infarction

Cardiac fibrosis is the most important mechanism contributing to cardiac remodeling after myocardial infarction (MI). VPO1 is a heme enzyme that uses hydrogen peroxide (H2O2) to produce hypochlorous acid (HOCl). Our previous study has demonstrated that VPO1 regulates myocardial ischemic reperfusion and renal fibrosis. We investigated the role of VPO1 in cardiac fibrosis after MI. The results showed that VPO1 expression was robustly upregulated in the failing human heart with ischemic cardiomyopathy and in a murine model of MI accompanied by severe cardiac fibrosis. Most importantly, knockdown of VPO1 by tail vein injection of VPO1 siRNA significantly reduced cardiac fibrosis and improved cardiac function and survival rate. In VPO1 knockdown mouse model and cardiac fibroblasts cultured with TGF-β1, VPO1 contributes to cardiac fibroblasts differentiation, migration, collagen I synthesis and proliferation. Mechanistically, the fibrotic effects following MI of VPO1 manifested partially through HOCl formation to activate Smad2/3 and ERK1/2. Thus, we conclude that VPO1 is a crucial regulator of cardiac fibrosis after MI by mediating HOCl/Smad2/3 and ERK1/2 signaling pathways, implying a promising therapeutic target in ischemic cardiomyopathy.

LIM kinase 1 acts as a profibrotic mediator in permanent atrial fibrillation patients with valvular heart disease

Atrial fibrillation (AF) is the most frequently diagnosed cardiac arrhythmia worldwide. Patients with permanent atrial fibrillation are at an increased risk of developing valvular heart disease. Atrial fibrosis occurs in this pathophysiological setting. LIM kinase 1 (LIMK1) is a serine/threonine kinase that regulates microtubule stability and actin polymerization in fibroblasts. LIMK1 has been implicated in the pathogenesis of atrial fibrillation. Clinical data and biopsies of the right atrial appendage were collected from 50 patients with valvular heart disease who underwent heart valve replacement surgery. Data from patients with permanent atrial fibrillation (AF) and patients with sinus rhythm (SR) were compared. We found that AF patients had upregulated expression of LIMK1 as well as higher fibrosis. Transforming growth factor-β (TGF-β) stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts as well as upregulated expression of LIMK1. Downregulation of LIMK1 by siRNA inhibited TGF-β induced fibroblast-myofibroblast transition, as evidenced by the downregulation of the expression of several differentiation markers, namely alpha-smooth muscle actin and type I and III collagen. Our findings revealed that increased LIMK1 protein levels may contribute to atrial fibrosis, and suggested that LIMK1 might be involved in AF development by promoting fibrogenesis associated with TGF-β.

Tongguan capsule-derived herb reduces susceptibility to atrial fibrillation by inhibiting left atrial fibrosis via modulating cardiac fibroblasts.

Tongguan capsule is a compound Chinese medicine used to treat ischaemic heart diseases. This study aimed to investigate whether Tongguan capsule‐derived herb (TGD) has a preventive effect on atrial fibrillation (AF) in post‐myocardial infarction (MI) rats and to determine the underlying mechanisms. MI was induced by ligation of the left anterior descending coronary artery. TGD was administered to the post‐MI rats over a 4‐week period. The TGD‐treated rats had lower rates of AF inducibility and shorter AF durations than the MI rats. TGD improved the left atrial (LA) conduction velocity and homogeneity. It reduced the fibrosis‐positive areas and the protein levels of collagen types I and III in the left atrium. In vitro, it inhibited the expression of collagen types I and III by inhibiting the proliferation, migration, differentiation and cytokine secretion of cardiac fibroblasts (CFs). In conclusion, the current study demonstrated that TGD reduces susceptibility to AF and improves LA conduction function in rats with post‐MI by inhibiting left atrial fibrosis and modulating CFs. Targeting the CF population may be a novel antiarrhythmic therapeutic approach.

Angiotensin II confers resistance to apoptosis in cardiac myofibroblasts through the AT1/ERK1/2/RSK1 pathway.

Myofibroblast apoptosis is essential for normal resolution of wound repair, including cardiac infarction repair. Impaired cardiac myofibroblast (CMF) apoptosis is associated with excessive extracellular matrix (ECM) deposition, which could be responsible for pathological cardiac fibrosis. Conventionally, angiotensin II (Ang II), a soluble peptide, is implicated in fibrogenesis because it induces cardiac fibroblast (CFb) proliferation, differentiation, and collagen synthesis. However, the role of Ang II in regulation of CMF survival and apoptosis has not been fully clarified. In this report, we cultured neonatal rat CFbs, which transform into CMFs after passage 3 (6-8 days), and investigated the effects of Ang II on CMFs challenged by TNF-α combined with cycloheximide and the underlying mechanisms. Here, we show that Ang II rapidly activates MAPKs but not AKT in CMFs and confers apoptosis resistance, as evidenced by the inhibition of caspase-3 cleavage, early apoptotic cells and late apoptotic cells. This inhibitory effect of Ang II was reversed by blockade of AT1 or inactivation of ERK1/2 or RSK1 but not AT2, indicating that activation of the prosurvival AT1/ERK1/2/RSK1 signaling pathway mediates apoptosis resistance. TGF-β, a latent fibrotic factor, was found to have no relation to Ang II-induced apoptosis resistance in our study. Furthermore, Ang II-mediated apoptosis resistance, which was conferred by activation of the AT1/ERK1/2/RSK1 signaling pathway, was also confirmed in human adult ventricular cardiac myofibroblasts. Collectively, our findings suggest a novel profibrotic mechanism of Ang II in which it promotes myofibroblast resistance to apoptosis in addition to classical mechanisms, providing a potential novel therapeutic approach by targeting prosurvival signaling pathways. © 2018 IUBMB Life, 71(1):261-276, 2019.

Fibroblast growth factor-2, but not the adipose tissue-derived stromal cells secretome, inhibits TGF-\u03b21-induced differentiation of human cardiac fibroblasts into myofibroblasts

Transforming growth factor-β1 (TGF-β1) is a potent inducer of fibroblast to myofibroblast differentiation and contributes to the pro-fibrotic microenvironment during cardiac remodeling. Fibroblast growth factor-2 (FGF-2) is a growth factor secreted by adipose tissue-derived stromal cells (ASC) which can antagonize TGF-β1 signaling. We hypothesized that TGF-β1-induced cardiac fibroblast to myofibroblast differentiation is abrogated by FGF-2 and ASC conditioned medium (ASC-CMed). Our experiments demonstrated that TGF-β1 treatment-induced cardiac fibroblast differentiation into myofibroblasts, as evidenced by the formation of contractile stress fibers rich in αSMA. FGF-2 blocked the differentiation, as evidenced by the reduction in gene (TAGLN, p < 0.0001; ACTA2, p = 0.0056) and protein (αSMA, p = 0.0338) expression of mesenchymal markers and extracellular matrix components gene expression (COL1A1, p < 0.0001; COL3A1, p = 0.0029). ASC-CMed did not block myofibroblast differentiation. The treatment with FGF-2 increased matrix metalloproteinases gene expression (MMP1, p < 0.0001; MMP14, p = 0.0027) and decreased the expression of tissue inhibitor of metalloproteinase gene TIMP2 (p = 0.0023). ASC-CMed did not influence these genes. The proliferation of TGF-β1-induced human cardiac fibroblasts was restored by both FGF-2 (p = 0.0002) and ASC-CMed (p = 0.0121). The present study supports the anti-fibrotic effects of FGF-2 through the blockage of cardiac fibroblast differentiation into myofibroblasts. ASC-CMed, however, did not replicate the anti-fibrotic effects of FGF-2 in vitro.

DNA methylation regulates α-smooth muscle actin expression during cardiac fibroblast differentiation.

Cardiac fibroblast (CF) differentiation to myofibroblasts expressing α-smooth muscle actin (α-SMA) plays a key role in cardiac fibrosis. Therefore, a study of the mechanism regulating α-SMA expression is a means to understanding the mechanism of fibroblast differentiation and cardiac fibrosis. Previous studies have shown that DNA methylation is associated with gene expression and is related to the development of tissue fibrosis. However, the mechanisms by which CF differentiation is regulated by DNA methylation remain unclear. Here, we explored the epigenetic regulation of α-SMA expression and its relevance in CF differentiation. In this study, we demonstrated that α-SMA was overexpressed and DNMT1 expression was downregulated in the infarct area after myocardial infarction. Treatment of CFs with transforming growth factor-β1 (TGF-β1 ) in vitro upregulated α-SMA expression via epigenetic modifications. TGF-β1 also inhibited DNMT1 expression and activity during CF differentiation. In addition, α-SMA expression was regulated by DNMT1. Conversely, increasing DNMT1 expression levels rescued the TGF-β1 -induced upregulation of α-SMA expression. Finally, TGF-β1 regulated α-SMA expression by inhibiting the DNMT1-mediated DNA methylation of the α-SMA promoter. Taken together, our research showed that inhibition of the DNMT1-mediated DNA methylation of the α-SMA promoter plays an essential role in CF differentiation. In addition, DNMT1 may be a new target for the prevention and treatment of myocardial fibrosis.

AdipoRon, an adiponectin receptor agonist, attenuates cardiac remodeling induced by pressure overload.

AdipoRon, a small-molecule adiponectin receptor (AdipoR) agonist, has been reported to be implicated in cardiovascular diseases. However, its role in pressure-overload-induced cardiac remodeling is still elusive. To elucidate the role of AdipoRon in the pathogenesis of cardiac remodeling in vivo and vitro, in the left ventricle of human end-stage heart failure, the expression of AdipoR2 is upregulated. Meanwhile, increased expression of AdipoR2 was also observed in mice failing hearts. Oral administration of AdipoRon alleviated cardiac hypertrophy and fibrosis induced by pressure overload, as evidenced by the beneficial change of cross-sectional area of cardiomyocytes, heart weight-to-body weight ratio, gene expression of hypertrophic markers, ventricle collagen ratio, and cardiac function. The AMPKα activation mediated by AdipoRon significantly inhibited AngII-induced TGF-β1 expression and cardiac fibroblast differentiation, and these inhibitory effects were abrogated by treatment with the AMPK inhibitor Compound C. Consistent with the above results, AdipoRon abolished the ability to retard AngII-induced TGF-β1 expression in AMPKα2-/- cardiac fibroblasts. In AMPKα2-/- mice subjected to aortic banding, AdipoRon abolished the protective effect, as indicated by increased cross-sectional area, cardiac collagen ratio, and cardiac dysfunction. Our results demonstrated that AdipoR2 expression was markedly increased in the failing hearts. AdipoRon inhibited TGF-β1 expression and myofibroblast differentiation in AMPKα-dependent manner in vitro. In line with the vitro results, AMPKα2-/- mice markedly abrogated the inhibitory effects of AdipoRon in cardiac remodeling. These results indicated AdipoRon may hold promise of an effective therapy against pressure-overload-induced cardiac remodeling. KEY MESSAGES: • The increased expression of AdipoR2 is observed in human and mice failing hearts, the changeable expression of AdipoR suggests the possible role of AdipoR in cardiac remodeling. • Oral administration of AdipoRon alleviates cardiac hypertrophy and fibrosis induced by pressure overload, and AMPKα activation mediated by AdipoRon significantly inhibited AngII-induced TGF-β1 expression and cardiac fibroblast differentiation. • These findings provide new mechanistic insight and open new therapeutic pathways for heart failure.

The Inflammatory Transcription Factor C/EBPβ Plays a Critical Role in Cardiac Fibroblast Differentiation and a Rat Model of Cardiac Fibrosis Induced by Autoimmune Myocarditis

The aim of the present study was to investigate the mechanisms of CCAAT/enhancer-binding protein β (C/EBPβ) in cardiac myofibroblast (CMF) differentiation and in a rat model of cardiac fibrosis induced by experimental autoimmune myocarditis (EAM).In vitro studies performed in primary neonatal rat CMF revealed that silencing of C/EBPβ expression (via lentiviral mediated shRNA strategies) was sufficient to reduce C/EBPβ mRNA and protein levels as well as to decrease the expressions of actin cytoskeletal proteins, cofilin, and filamin A (FLNA). TGFβ increased IL-1β, IL-6 and TNF-a production in cardiac fibroblasts (CF), while C/EBPβ knockdown reduced the secretion of these inflammatory mediators. In vivo studies performed in rats exhibiting EAM revealed that lentiviral-mediated silencing of C/EBPβ was sufficient to reduce the expression of C/EBPβ as well as inflammation and fibrosis in the hearts of EAM rats, when compared to controls. Echocardiography further revealed that C/EBPβ knockdown was sufficient to significantly improve cardiac dimensions and function in EAM rats. Immunohistochemical results showed that C/EBPβ knockdown attenuated the expression of C/EBPβ protein as well as the expressions of collagen I, collagen III, MMP-2, MMP-9, and α-SMA in heart tissue sections from rats in the EAM + Lenti-shC/EBPβ group.Strategies targeted at inhibiting C/EBPβ expression can be potentially exploited to regulate cofilin and FLNA expression, thereby regulating actin polymerization/depolymerization, cytoskeleton rearrangement, and CF differentiation into CMF and the production of inflammatory cytokines. C/EBPβ knock down reduces the degree of inflammation-mediated myocardial fibrosis in a rat model of EAM.

Shensong Yangxin capsule reduces atrial fibrillation susceptibility by inhibiting atrial fibrosis in rats with post-myocardial infarction heart failure

PurposeShensong Yangxin (SSYX) capsule is a traditional Chinese medicine that has been used widely to treat cardiac arrhythmia. This study aimed to assess whether SSYX prevents atrial fibrillation (AF) after chronic myocardial infarction (MI)-induced heart failure and to determine the underlying mechanisms.Materials and methodsThe study included 45 male Sprague Dawley rats. The rats underwent MI induction or sham surgery. One week after MI induction surgery, we performed serial echocardiography and administered SSYX capsule to some rats that experienced MI. After 4 weeks of treatment, AF inducibility was assessed with transesophageal programmed electrical stimulation technology. Additionally, multielectrode array assessment, histological analysis, and Western blot analysis were performed.ResultsAF inducibility was significantly lower in SSYX rats than in MI rats (33.3% vs 73.3%, P<0.05). Additionally, conduction velocities in the left atrium were greater in SSYX rats than in MI rats. Moreover, SSYX decreased left atrial fibrosis, downregulated TGF-β1, MMP-9, TIMP-I, and type I and III collagen expressions, and inhibited the differentiation of cardiac fibroblasts to myofibroblasts.ConclusionSSYX reduces AF inducibility after MI by improving left atrial conduction function via the inhibition of left atrial fibrosis. It prevents the development of an MI-induced vulnerable substrate for AF.

Stevioside inhibits transforming growth factor-β1-induced cardiac fibroblast differentiation and collagen synthesis by modulation of Smad signaling pathway

Stevioside, a low calorie sweetener used in food and beverage, has some beneficial effects. However, the inhibitory effect of stevioside on cardiac fibrosis has not been reported yet. This study aimed to investigate the effect and potential mechanisms in cultured cardiac fibroblasts (CFs). The CFs were stimulated with 10 ng/mL transforming growth factor-β1 (TGF-β1) for 2 h and then incubated with the different concentrations of stevioside for an additional 24 h. The results showed that after treatment of CFs with stevioside, the α-smooth muscle actin, collagen I/III, Smad2/3, P-Smad2/3 and Smad4 expressions were dose-dependently decreased, while the Smad7 expression was increased, especially in the 400 μM group. These findings demonstrated that stevioside could inhibit the differentiation and collagen synthesis in TGF-β1-stimulated CFs, and its mechanisms were associated with the synergic regulatory effects on Smad signaling pathway, including decrease in Smad2/3, P-Smad2/3 and Smad4 expressions and increase in Smad7 expression.

Ectopic overexpression of Kir6.1 in the mouse heart impacts on the life expectancy

We recently reported the reduced ATP-sensitive potassium (KATP) channel activities in the transgenic mouse heart overexpressing the vascular type KATP channel pore-forming subunit (Kir6.1). Although dysfunction of cardiac KATP channel has been nominated as a cause of cardiomyopathy in human, these transgenic mice looked normal as wild-type (WT) during the experiment period (~20 weeks). Extended observation period revealed unexpected deaths beginning from 30 weeks and about 50% of the transgenic mice died by 55 weeks. Surface ECG recordings from the transgenic mice at rest demonstrated the normal sinus rhythm and the regular ECG complex as well as the control WT mice except for prolonged QT interval. However, the stress ECG test with noradrenaline revealed abnormal intraventricular conduction delay and arrhythmogeneity in the transgenic mouse. Fibrotic changes in the heart tissue were remarkable in aged transgenic mice, and the cardiac fibrosis developed progressively at least from the age of 30 weeks. Gene expression analyses revealed the differentiation of cardiac fibroblasts to myofibroblasts with elevated cytokine expressions was initiated way in advance before the fibrotic changes and the upregulation of BNP in the ventricle. In sum, Kir6.1TG mice provide an electro-pathological disease concept originated from KATP channel dysfunction.

Cardiac fibrosis in regenerative medicine: destroy to rebuild

The major limitations for cardiac regeneration in patients after myocardial infarction (MI) are the wide loss of cardiomyocytes and the adverse structural alterations of extracellular matrix (ECM). Cardiac fibroblast differentiation into myofibroblasts (MFB) leads to a huge deposition of ECM and to the subsequent loss of ventricular structural integrity. All these molecular events depict the fundamental features at the basis of the post-MI fibrosis and deserve in depth cellular and molecular studies to fill the gap in the clinical practice. Indeed, to date, there are no effective therapeutic approaches to limit the post-MI massive fibrosis development. In this review we describe the involvement of integrins and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)/ADAMTS-like (ADAMTSL) proteins in cardiac reparative pro-fibrotic response after MI, proposing some of them as novel potential pharmacological tools.

The E3 ubiquitin ligase SMURF1 regulates cell-fate specification and outflow tract septation during mammalian heart development

Smad ubiquitin regulatory factor 1 (SMURF1) is a HECT-type E3 ubiquitin ligase that plays a critical role in vertebrate development by regulating planar cell polarity (PCP) signaling and convergent extension (CE). Here we show that SMURF1 is involved in mammalian heart development. We find that SMURF1 is highly expressed in outflow tract cushion mesenchyme and Smurf1−/− mouse embryos show delayed outflow tract septation. SMURF1 is expressed in smooth muscle cells of the coronary arteries and great vessels. Thickness of the aortic smooth muscle cell layer is reduced in Smurf1−/− mouse embryos. We show that SMURF1 is a negative regulator of cardiomyogenesis and a positive regulator of smooth muscle cell and cardiac fibroblast differentiation, indicating that SMURF1 is important for cell-type specification during heart development. Finally, we provide evidence that SMURF1 localizes at the primary cilium where it may regulate bone morphogenetic protein (BMP) signaling, which controls the initial phase of cardiomyocyte differentiation. In summary, our results demonstrate that SMURF1 is a critical regulator of outflow tract septation and cell-type specification during heart development, and that these effects may in part be mediated via control of cilium-associated BMP signaling.

MicroRNA-495 inhibits the high glucose-induced inflammation, differentiation and extracellular matrix accumulation of cardiac fibroblasts through downregulation of NOD1

BackgroundMicroRNAs (miRNAs) have physiological and pathophysiological functions that are involved in the regulation of cardiac fibrosis. This study aimed to investigate the effects of miR-495 on high glucose-induced cardiac fibrosis in human cardiac fibroblasts (CFs) and to establish the mechanism underlying these effects.MethodsHuman CFs were transfected with an miR-495 inhibitor or mimic and incubated with high glucose. The levels of NOD1 and miR-495 were then determined via quantitative RT-PCR. Pro-inflammatory cytokine levels, cell differentiation and extracellular matrix accumulation were respectively detected using ELISA, quantitative RT-PCR and western blot assays. The luciferase reporter assay, quantitative RT-PCR and western blot were used to explore whether NOD1 was a target of miR-495. The effects of miR-495 on the NF-κB and TGF-β1/Smad signaling pathways were also detected via western blot.ResultsOur results show that high glucose can significantly increase the expression of NOD1 in a time-dependent manner. Upregulation of miR-495 significantly alleviated the high glucose-induced increases in cell differentiation and collagen accumulation of CFs. Moreover, the bioinformatics analysis predicted that NOD1 was a potential target gene for miR-495. The luciferase reporter assay showed that miR-495 can directly target NOD1. The introduction of miR-495 could significantly inhibit the high glucose-activated NF-κB and TGF-β1/Smad signaling pathways.ConclusionUpregulation of miR-495 ameliorates the high glucose-induced inflammatory, cell differentiation and extracellular matrix accumulation of human CFs by modulating both the NF-κB and TGF-β1/Smad signaling pathways through downregulation of NOD1 expression. These results provide further evidence for the protective effect of miR-495 overexpression in cases of high glucose-induced cardiac fibrosis.

Inhibition of microRNA-23b prevents polymicrobial sepsis-induced cardiac dysfunction by modulating TGIF1 and PTEN

Cardiovascular dysfunction is a major complication associated with sepsis induced mortality. Cardiac fibrosis plays a critical role in sepsis induced cardiac dysfunction. The mechanisms of the activation of cardiac fibrosis is unclarified. In this study, we found that microRNA-23b (miR-23b) was up-regulated in heart tissue during cecal ligation and puncture (CLP)-induced sepsis and transfection of miR-23b inhibitor improved survival in late sepsis. Inhibition of miR-23b in the myocardium protected against cardiac output and enhanced left ventricular systolic function. miR-23b inhibitor also alleviated cardiac fibrosis in late sepsis. MiR-23b mediates the activation of TGF-β1/Smad2/3 signaling to promote the differentiation of cardiac fibroblasts through suppression of 5′TG3′-interacting factor 1 (TGIF1). MiR-23b also induces AKT/N-Cadherin signaling to contribute to the deposition of extracellular matrix by inhibiting phosphatase and tensin homologue (PTEN). TGIF1 and PTEN were confirmed as the targets of miR-23b in vitro by Dual-Glo Luciferase assay. miR-23b inhibitor blocked the activation of adhesive molecules and restored the imbalance of pro-fibrotic and anti-fibrotic factors. These data provide direct evidence that miR-23b is a critical contributor to the activation of cardiac fibrosis to mediate the development of myocardial dysfunction in late sepsis. Blockade of miR-23b expression may be an effective approach for prevention sepsis-induced cardiac dysfunction.

TRPV4 channel deletion or pharmacological inhibition protects heart against adverse remodeling post‐myocardial infarction

Ischemic heart disease (IHD) is the major underlying cause of myocardial infarction (MI), scarring, and hypertrophy leading to heart failure. Cardiac remodeling following myocardial infarction involves scar formation by synthesis and reorganization of ECM which is mediated by myofibroblasts. We have previously shown that the mechanosensitive ion channel TRPV4 (transient receptor potential vanilloid channel 4) regulates cardiac fibroblast differentiation into myofibroblasts via integration of soluble and mechanical signaling. However, the physiological or translational significance of TRPV4 in cardiac remodeling following MI is unknown. To determine this, we have subjected WT and TRPV4KO mice to MI (permanent LAD ligation). 2D‐echocardiography revealed that the cardiac function (ejection fraction and fractional shortening) is preserved post‐MI in TRPV4KO mice compared to WT mice. Further, we found reduced fibrosis at infarcted and remote zones in TRPV4KO‐MI hearts compared to WT‐MI and sham hearts. Furthermore, TRPV4KO hearts exhibited decreased cardiomyocyte apoptosis (TUNEL assay) and increased capillary density (CD31 staining) post‐MI compared to WT hearts. To explore the translational significance of these findings, in separate experiments, we have given an orally active TRPV4 antagonist GSK2193874, immediately after MI surgery and followed for 5 weeks. Cardiac function analysis revealed that both ejection fraction and fractional shortening were preserved in GSK2193874‐treated WT mice compared to either WT or vehicle treated mice. Our results thus suggest that targeting TRPV4 protects the heart from myocardial infarction‐induced damage by preserving cardiac structure and function via reduced myocyte apoptosis, diminished fibrosis and increased revascularization, and identifies TRPV4 as a novel therapeutic target for heart failure.Support or Funding InformationNIH (1RO1HL119705), NIH (1R15CA202847‐01).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The lipid kinase PIKfyve in cardiac fibroblasts activation: A potential target to control cardiac fibrosis

Fibrotic remodeling of cardiac tissue is a key determinant in the progression of heart failure, a leading cause of death worldwide. Cardiac fibrosis is characterized by the activation of cardiac fibroblasts and their differentiation to myofibroblasts. Activated fibroblasts express the contractile protein α-SMA and produce and secrete inflammatory cytokines (IL-6), pro-fibrotic factors (TGF-β), and extracellular matrix proteins (collagen I/III, fibronectin), leading to the formation of scar tissue which ultimately impedes cardiac normal functions. The enzyme PIKfyve is a dual-substrate lipid kinase which produces the phosphatidylinositol-5 phosphate (PI5P) and PI(3,5)P2. It is localized on the endosomes and is known to regulate the endosomal maturation and recycling of cargoes to the plasma membrane. In this study, we investigate the involvement of PIKfyve in the control of cardiac fibroblasts activation. We demonstrate that pharmacological inhibition of PIKfyve reduces inflammatory and pro-fibrotic factors production by activated cardiac fibroblasts. We demonstrate here that PIKfyve controls cardiac fibroblasts differentiation by regulating the Smad-dependent transforming growth factor-β (TGF-β) signaling pathway. Taken together, our results demonstrate that PIKfyve could be a potentiator of cardiac fibrosis, therefore opening the way to new therapeutic perspectives to control its development in cardiovascular diseases.

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.