Matairesinol blunts adverse cardiac remodeling and heart failure induced by pressure overload by regulating Prdx1 and PI3K/AKT/FOXO1 signaling.

BACKGROUND: Pathological cardiac remodeling is a critical process leading to heart failure, characterized primarily by inflammation and apoptosis. Matairesinol (Mat), a key chemical component of Podocarpus macrophyllus resin, exhibits a wide range of pharmacological activities, including anti-hydatid, antioxidant, antitumor, and anti-inflammatory effects. PURPOSE: This study aims to investigate whether Matairesinol alleviate cardiac hypertrophy and remodeling caused by pressure overload and to elucidate its mechanism of action. METHODS: An in vitro pressure loading model was established using neonatal rat cardiomyocytes treated with angiotensin Ⅱ, while an in vivo model was created using C57 mice subjected to transverse aortic constriction (TAC). To activate the PI3K/Akt/FoxO1 pathway, Ys-49 was employed. Moreover, small interfering RNA (siRNA) and short hairpin RNA (shRNA) were utilized to silence Prdx1 expression both in vitro and in vivo. Various techniques, including echocardiography, wheat germ agglutinin (WGA) staining, HE staining, PSR staining, and Masson trichrome staining, were used to assess cardiac function, cardiomyocyte cross-sectional area, and fibrosis levels in rats. Apoptosis in myocardial tissue and in vitro was detected by TUNEL assay, while reactive oxygen species (ROS) content in tissues and cells was measured using DHE staining. Furthermore, the affinity of Prdx1 with Mat and PI3K was analyzed using computer-simulated molecular docking. Western blotting and RT-PCR were utilized to evaluate Prdx1 levels and proteins related to apoptosis and oxidative stress, as well as the mRNA levels of cardiac hypertrophy and fibrosis-related indicators. RESULTS: Mat significantly alleviated cardiac hypertrophy and fibrosis induced by TAC, preserved cardiac function, and markedly reduced cardiomyocyte apoptosis and oxidative damage. In vitro, mat attenuated ang Ⅱ - induced hypertrophy of nrvms and activation of neonatal rat fibroblasts. Notably, activation of the PI3K/Akt/FoxO1 pathway and downregulation of Prdx1 expression were observed in TAC mice; however, these effects were reversed by Mat treatment. Furthermore, Prdx1 knockdown activated the PI3K/Akt/FoxO1 pathway, leading to exacerbation of the disease. Molecular docking indicated that Molecular docking indicated that Mat upregulated Prdx1 expression by binding to it, thereby inhibiting the PI3K/Akt/FoxO1 pathway and protecting the heart by restoring Prdx1 expression levels. CONCLUSION: Matairesinol alleviates pressure overload-induced cardiac remodeling both in vivo and in vitro by upregulating Prdx1 expression and inhibiting the PI3K/Akt/FoxO1 pathway. This study highlights the therapeutic potential of Matairesinol in the treatment of cardiac hypertrophy and remodeling, providing a promising avenue for future research and clinical application.

Pericardial Fluid Accumulates microRNAs That Regulate Heart Fibrosis after Myocardial Infarction.

Pericardial fluid (PF) has been suggested as a reservoir of molecular targets that can be modulated for efficient repair after myocardial infarction (MI). Here, we set out to address the content of this biofluid after MI, namely in terms of microRNAs (miRs) that are important modulators of the cardiac pathological response. PF was collected during coronary artery bypass grafting (CABG) from two MI cohorts, patients with non-ST-segment elevation MI (NSTEMI) and patients with ST-segment elevation MI (STEMI), and a control group composed of patients with stable angina and without previous history of MI. The PF miR content was analyzed by small RNA sequencing, and its biological effect was assessed on human cardiac fibroblasts. PF accumulates fibrotic and inflammatory molecules in STEMI patients, namely causing the soluble suppression of tumorigenicity 2 (ST-2), which inversely correlates with the left ventricle ejection fraction. Although the PF of the three patient groups induce similar levels of fibroblast-to-myofibroblast activation in vitro, RNA sequencing revealed that PF from STEMI patients is particularly enriched not only in pro-fibrotic miRs but also anti-fibrotic miRs. Among those, miR-22-3p was herein found to inhibit TGF-ß-induced human cardiac fibroblast activation in vitro. PF constitutes an attractive source for screening diagnostic/prognostic miRs and for unveiling novel therapeutic targets in cardiac fibrosis.

Mechanism of multifunctional adaptor protein SHARPIN regulating myocardial fibrosis and how SNP mutation affect the prognosis of myocardial infarction.

Myocardial fibrosis (MF) is characterized by the excessive deposition of extracellular matrix within the heart, often following a cardiovascular insult. SHARPIN, a protein implicated in fibrosis, has emerged as a potential therapeutic target. This study aimed to elucidate the molecular mechanisms of SHARPIN in MF and to investigate the influence of its single nucleotide polymorphism (SNP), rs117299156, on myocardial infarction (MI) patients. A mouse model of Angiotensin II (AngII)-induced MF was established in SHARPIN heterozygous (SHARPIN+/-) and wild-type mice. Adult mouse cardiac fibroblasts (CFs) were isolated and subjected to adenovirus-encapsulated SHARPIN short hairpin RNA (shRNA) infection. Transcriptomic analysis was performed on CFs from SHARPIN+/- and wild-type (WT) mice, complemented by single-cell sequencing data from human cardiac tissues. Additionally, the association between the rs117299156 mutation and cardiovascular events in MI patients was assessed. Our findings indicate that SHARPIN is predominantly expressed in CFs and is upregulated in fibrotic myocardium. Partial knockdown of SHARPIN in murine hearts mitigated AngII-induced cardiac dysfunction and MF. Furthermore, reduced SHARPIN expression in CFs attenuated TGF-ß1-induced collagen synthesis, cell proliferation, and myofibroblast transformation. Notably, MI patients carrying the rs117299156_C allele exhibited a reduced incidence of stroke events compared to those without the mutation.

Fibroblasts activation by embryonic signal switching: A novel mechanism of placental growth factor-induced cardiac remodeling.

INTRODUCTION: Cardiac remodeling is defined as cellular interstitial changes that lead dysfunction of the heart after injury. Placental growth factor (PlGF), a member of the VEGF family, has been reported to regulate cardiac hypertrophy in hemodynamic state. We therefore analyze the function of PlGF during cardiac remodeling using cardiac cells and fibroblasts, under Angiotensin II (AngII) stimulation. METHODS: PlGF overexpressed mouse embryonic fibroblasts derived from C57BL/6 mice, were made by deficient retrovirus vector, designated as C57/PlGF. Only retrovirus vector introduced C57 cells (C57/EV) were used as control. After AngII stimulation, wound scratching assay and MTT proliferation assay with or without p38 MAPK inhibitor, SB205580 were performed in retrovirally-introduced C57 cells. Reactive oxygen species (ROS) production, NF-kB activation, IL-6 and TNF-α production were also measured. Then we assessed AngII-induced cell proliferation of mouse cardiac fibroblasts (CFs) and rat primary cardiomyocytes incubating with C57/PlGF conditioned-medium. RESULTS: The PlGF production in C57/PlGF were confirmed by ELISA (1093.48 ± 3.5 pg/ml, ±SE). AngII-induced cell migration, proliferation and H2O2 production were increased in C57/PlGF compared with C57/EV. SB205580 inhibited the AngII-induced cell proliferation in C57/PlGF. In C57/PlGF cells, NF-kB activation was higher, followed by up-regulation of IL-6 and TNF-α production. CFs and cardiomyocytes proliferation increased when stimulated with C57/PlGF conditioned-medium. DISCUSSION: The activation of fibroblast is stimulated by PlGF signaling via p38 MAPK/NF-kB pathway accompanied by elevation of ROS and inflammatory response. Furthermore, these signals stimulate the activation of CFs and cardiomyocytes, indicating that high circulating level of PlGF have a potential to regulate cardiac remodeling.

Fowl adenovirus serotype 4 (FAdV-4) infection induces inflammatory responses in chicken embryonic cardiac fibroblasts via PI3K/Akt and IκBα/NF-κB signaling pathways.

Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of the acute infectious disease hepatitis-hydropericardium syndrome (HHS). Previous studies have focused on the mechanisms of FAdV-4 caused liver injury, while studies revealing potential mechanisms of inflammatory injury in FAdV-4-infected chicken cardiac cells remain scare. Here we found that FAdV-4 successfully infected chicken embryonic cardiac fibroblasts (CECF) cells in vitro and significantly upregulated production of inflammatory cytokines including IL-1ß, IL-6, IL-8, and TNF-α, suggesting induction of a strong inflammatory response. Mechanistically, FAdV-4 infection increased expression of phosphorylated Akt in a time-dependent manner, while phosphorylation of Akt and production of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α were greatly reduced in FAdV-4-infected CECF cells after treatment with LY294002, a potent inhibitor of PI3K, indicating that the inflammatory response induced by FAdV-4 infection is mediated by the PI3K/Akt signaling pathway. Furthermore, FAdV-4 infection increased expression of phosphorylated IκBα, a recognized indicator of NF-κB activation, and treatment with the BAY11-7082, a selective IκBα phosphorylation and NF-κB inhibitor, significantly reduced IκBα phosphorylation and inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α) production in FAdV-4-infected CECF cells, suggesting a critical role of IκBα/NF-κB signaling in FAdV-4-induced inflammatory responses in CECF cells. Taken together, our results suggest that FAdV-4 infection induces inflammatory responses through activation of PI3K/Akt and IκBα/NF-κB signaling pathways in CECF cells. These results reveal potential mechanisms of inflammatory damage in chicken cardiac cells caused by FAdV-4 infection, which sheds new insight into clarification of the pathogenic mechanism of FAdV-4 infection and development of new strategies for HHS prevention and control.

WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts' Activation and Collagen Processing.

Hypertension induces cardiac fibrotic remodelling characterised by the phenotypic switching of cardiac fibroblasts (CFs) and collagen deposition. We tested the hypothesis that Wnt1-inducible signalling pathway protein-1 (WISP-1) promotes CFs' phenotypic switch, type I collagen synthesis, and in vivo fibrotic remodelling. The treatment of human CFs (HCFs, n = 16) with WISP-1 (500 ng/mL) induced a phenotypic switch (α-smooth muscle actin-positive) and type I procollagen cleavage to an intermediate form of collagen (pC-collagen) in conditioned media after 24h, facilitating collagen maturation. WISP-1-induced collagen processing was mediated by Akt phosphorylation via integrin ß1, and disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2). WISP-1 wild-type (WISP-1+/+) mice and WISP-1 knockout (WISP-1-/-) mice (n = 5-7) were subcutaneously infused with angiotensin II (AngII, 1000 ng/kg/min) for 28 days. Immunohistochemistry revealed the deletion of WISP-1 attenuated type I collagen deposition in the coronary artery perivascular area compared to WISP-1+/+ mice after a 28-day AngII infusion, and therefore, the deletion of WISP-1 attenuated AngII-induced cardiac fibrosis in vivo. Collectively, our findings demonstrated WISP-1 is a critical mediator in cardiac fibrotic remodelling, by promoting CFs' activation via the integrin ß1-Akt signalling pathway, and induced collagen processing and maturation via ADAMTS-2. Thereby, the modulation of WISP-1 levels could provide potential therapeutic targets in clinical treatment.

Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) leads to enhanced activity of cardiac fibroblasts (CFs) and abnormal deposition of extracellular matrix proteins, resulting in cardiac fibrosis. Tartrate-resistant acid phosphatase 5 (ACP5) has been shown to promote cell proliferation and phenotypic transition. However, it remains unclear whether ACP5 is involved in the development of cardiac fibrosis after MI. The present study aimed to investigate the role of ACP5 in post-MI fibrosis and its potential underlying mechanisms. METHODS: Clinical blood samples were collected to detect ACP5 concentration. Myocardial fibrosis was induced by ligation of the left anterior descending coronary artery. The ACP5 inhibitor, AubipyOMe, was administered by intraperitoneal injection. Cardiac function and morphological changes were observed on Day 28 after injury. Cardiac CFs from neonatal mice were extracted to elucidate the underlying mechanism in vitro. The expression of ACP5 was silenced by small interfering RNA (siRNA) and overexpressed by adeno-associated viruses to evaluate its effect on CF activation. RESULTS: The expression of ACP5 was increased in patients with MI, mice with MI, and mice with Ang II-induced fibrosis in vitro. AubipyOMe inhibited cardiac fibrosis and improved cardiac function in mice after MI. ACP5 inhibition reduced cell proliferation, migration, and phenotypic changes in CFs in vitro, while adenovirus-mediated ACP5 overexpression had the opposite effect. Mechanistically, the classical profibrotic pathway of glycogen synthase kinase-3ß (GSK3ß)/ß-catenin was changed with ACP5 modulation, which indicated that ACP5 had a positive regulatory effect. Furthermore, the inhibitory effect of ACP5 deficiency on the GSK3ß/ß-catenin pathway was counteracted by an ERK activator, which indicated that ACP5 regulated GSK3ß activity through ERK-mediated phosphorylation, thereby affecting ß-catenin degradation. CONCLUSION: ACP5 may influence the proliferation, migration, and phenotypic transition of CFs, leading to the development of myocardial fibrosis after MI through modulating the ERK/GSK3ß/ß-catenin signaling pathway.

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.

Mechanism of sodium butyrate, a metabolite of gut microbiota, regulating cardiac fibroblast transdifferentiation via the NLRP3/Caspase-1 pyroptosis pathway.

BACKGROUND: Cardiac fibroblasts (CFs) are activated after initial injury, and then differentiate into myofibroblasts (MFs), which play a pivotal role as the primary mediator cells in pathological remodeling. Sodium butyrate (NaB), being a metabolite of gut microbiota, exhibits anti-inflammatory property in local therapies on sites other than the intestine. Thus, this study aimed to probe the mechanism by which NaB regulates CFs transdifferentiation through the NLRP3/Caspase-1 pyroptosis pathway. METHODS: CFs were cultured in vitro and induced into MFs by TGFß1. CFs were identified by immunofluorescence labelling technique of vimentin and α-SMA, followed by treatment with NaB or NLRP3 inflammasome inhibitor (CY-09) and its activator [nigericin sodium salt (NSS)]. The expression levels of α-SMA, GSDMD-N/NLRP3/cleaved Caspase-1 proteins, and inflammatory factors IL-1ß/IL-18/IL-6/IL-10 were determined using immunofluorescence, Western blot and ELISA. Cell proliferation and migration were evaluated using the CCK-8 assay and the cell scratch test, respectively. RESULTS: Following the induction of TGFß1, CFs exhibited increased expression levels of α-SMA proteins and IL-6/IL-10, as well as cell proliferative and migratory abilities. TGFß1 induced CFs to differentiate into MFs, while NaB inhibited this differentiation. NaB inactivated the NLRP3/Caspase-1 pyroptosis pathway. CY-09 demonstrated inhibitory effects on the NLRP3/Caspase-1 pyroptosis pathway, leading to a reduction in TGFß1-induced CFs transdifferentiation. NSS activated the NLRP3/Caspase-1 pyroptosis pathway, and thus partially counteracting the inhibitory effect of intestinal microbiota metabolite NaB on CFs transdifferentiation. CONCLUSION: NaB, a metabolite of the gut microbiota, inhibited the activation of the NLRP3/Caspase-1 pyroptosis pathway in TGFß1-induced CFs, repressed the transdifferentiation of CFs into MFs.

M2 macrophage-derived paracrine factor TNFSF13 affects the fibrogenic alterations in endothelial cells and cardiac fibroblasts by mediating the NF-κB and Akt pathway.

Heart failure remains a global threaten to public health, cardiac fibrosis being a crucial event during the development and progression of heart failure. Reportedly, M2 macrophages might affect endothelial cell (ECs) and fibroblast proliferation and functions through paracrine signaling, participating in myocardial fibrosis. In this study, differentially expressed paracrine factors between M0/1 and M2 macrophages were analyzed and the expression of TNFSF13 was most significant in M2 macrophages. Culture medium (CM) of M2 (M2 CM) coculture to ECs and cardiac fibroblasts (CFbs) significantly promoted the cell proliferation of ECs and CFbs, respectively, and elevated α-smooth muscle actin (α-SMA), collagen I, and vimentin levels within both cell lines; moreover, M2 CM-induced changes in ECs and CFbs were partially abolished by TNFSF13 knockdown in M2 macrophages. Lastly, the NF-κB and Akt signaling pathways were proved to participate in TNFSF13-mediated M2 CM effects on ECs and CFbs. In conclusion, TNFSF13, a paracrine factor upregulated in M2 macrophages, could mediate the promotive effects of M2 CM on EC and CFb proliferation and fibrogenic alterations.

Adeno-associated virus-based approach for genetic modification of cardiac fibroblasts in adult rat hearts.

Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the key to develop successful therapies for treating the pressure-overloaded heart. CF-specific knockout (KO) mouse lines with a Cre recombinase under the control of human TCF21 (hTCF21) promoter and/or an adeno-associated virus serotype 9 (AAV9)-hTCF21 system provide a powerful tool for understanding CF biology in vivo. Although a variety of rat disease models are vital for the research of cardiac fibrosis similar to mouse models, there are few rat models that employ cardiac cell-specific conditional gene modification, which has hindered the development and translational relevance of cardiac disease models. In addition, to date, there are no reports of gene manipulation specifically in rat CFs in vivo. Here, we report a simplified CF-specific rat transgenic model using an AAV9-hTCF21 system that achieved a CF-specific expression of transgene in adult rat hearts. Moreover, we successfully applied this approach to specifically manipulate mitochondrial morphology in quiescent CFs. In summary, this model will allow us to develop fast and simple rat CF-specific transgenic models for studying cardiovascular diseases in vivo.

TAX1BP1 downregulation by STAT3 in cardiac fibroblasts contributes to diabetes-induced heart failure with preserved ejection fraction.

Heart failure (HF) with preserved ejection fraction (HFpEF) is now the most common form of HF and has been reported to be closely related to diabetes. Accumulating evidence suggests that HFpEF patients exhibit cardiac fibrosis. This study investigates whether direct targeted inhibition of the activation of cardiac fibroblasts (CFs), the main effector cells in cardiac fibrosis, improves diabetes-induced HFpEF and elucidates the underlying mechanisms. Twenty-week-old db/db mice exhibited HFpEF, as confirmed by echocardiography and hemodynamic measurements. Proteomics was performed on CFs isolated from the hearts of 20-week-old C57BL/6 and db/db mice. Bioinformatic prediction was used to identify target proteins. Experimental validation was performed in both high glucose (HG)-treated neonatal mouse CFs (NMCFs) and diabetic hearts. TAX1 binding protein 1 (TAX1BP1) was identified as the most significantly differentially expressed protein between 20-week-old C57BL/6 and db/db mice. TAX1BP1 mRNA and protein were markedly downregulated in CFs from diabetic hearts and HG-cultured NMCFs. Overexpression of TAX1BP1 profoundly inhibited HG/diabetes-induced NF-κB nuclear translocation and collagen synthesis in CFs, improved cardiac fibrosis, hypertrophy, inflammation and HFpEF in diabetic mice. Mechanistically, signal transducer and activator of transcription 3 (STAT3), which is phosphorylated and translocated from the cytoplasm into the nucleus under hyperglycemic conditions, bound to TAX1BP1 promoter and blocked TAX1BP1 transcriptional activity, consequently promoting NF-κB nuclear translocation and collagen synthesis in CFs, aggravating cardiac fibrosis, hypertrophy and inflammation, leading to HFpEF in db/db mice. Taken together, our findings demonstrate that targeting regulation of STAT3-TAX1BP1-NF-κB signaling in CFs may be a promising therapeutic approach for diabetes-induced HFpEF.

Blockage of the adenosine A2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension.

Sustained pressure overload and fibrosis of the right ventricle (RV) are the leading causes of mortality in pulmonary arterial hypertension (PAH). Although the role of adenosine in PAH has been attributed to the control of pulmonary vascular tone, cardiac reserve, and inflammatory processes, the involvement of the nucleoside in RV remodelling remains poorly understood. Conflicting results exist on targeting the low-affinity adenosine A2B receptor (A2BAR) for the treatment of PAH mostly because it displays dual roles in acute vs. chronic lung diseases. Herein, we investigated the role of the A2BAR in the viability/proliferation and collagen production by cardiac fibroblasts (CFs) isolated from RVs of rats with monocrotaline (MCT)-induced PAH. CFs from MCT-treated rats display higher cell viability/proliferation capacity and overexpress A2BAR compared to the cells from healthy littermates. The enzymatically stable adenosine analogue, 5'-N-ethylcarboxamidoadenosine (NECA, 1-30 µM), concentration-dependently increased growth, and type I collagen production by CFs originated from control and PAH rats, but its effects were more prominent in cells from rats with PAH. Blockage of the A2BAR with PSB603 (100 nM), but not of the A2AAR with SCH442416 (100 nM), attenuated the proliferative effect of NECA in CFs from PAH rats. The A2AAR agonist, CGS21680 (3 and 10 nM), was virtually devoid of effect. Overall, data suggest that adenosine signalling via A2BAR may contribute to RV overgrowth secondary to PAH. Therefore, blockage of the A2AAR may be a valuable therapeutic alternative to mitigate cardiac remodelling and prevent right heart failure in PAH patients.

Effects of Doxorubicin on Extracellular Matrix Regulation in Primary Cardiac Fibroblasts from Mice.

OBJECTIVE: Doxorubicin (DOX) is a highly effective chemotherapeutic used to treat many adult and pediatric cancers. However, its use is limited due to a dose-dependent cardiotoxicity, which can lead to lethal cardiomyopathy. In contrast to the extensive research efforts on toxic effects of DOX in cardiomyocytes, its effects and mechanisms on cardiac extracellular matrix (ECM) homeostasis and remodeling are poorly understood. In this study, we examined the potential effects of DOX on cardiac ECM to further our mechanistic understanding of DOX-induced cardiotoxicity. RESULTS: DOX-induced significant down-regulation of several ECM related genes in primary cardiac fibroblasts, including Adamts1, Adamts5, Col4a1, Col4a2, Col5a1, Fbln1, Lama2, Mmp11, Mmp14, Postn, and TGFß. Quantitative proteomics analysis revealed significant global changes in the fibroblast proteome following DOX treatment. A pathway analysis using iPathwayGuide of the differentially expressed proteins revealed changes in a list of biological pathways that involve cell adhesion, cytotoxicity, and inflammation. An apparent increase in Picrosirius red staining indicated that DOX-induced an increase in collagen production in cardiac primary fibroblasts after 3-day treatment. No significant changes in collagen organization nor glycoprotein production were observed.

Voltage-dependent anion channel 1 (VDAC1) overexpression alleviates cardiac fibroblast activation in cardiac fibrosis via regulating fatty acid metabolism.

Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix in the myocardium with cardiac fibroblast activation, leading to chronic cardiac remodeling and dysfunction. However, little is known about metabolic alterations in fibroblasts during cardiac fibrosis, and there is a lack of pharmaceutical treatments that target metabolic dysregulation. Here, we provided evidence that fatty acid ß-oxidation (FAO) dysregulation contributes to fibroblast activation and cardiac fibrosis. With transcriptome, metabolome, and functional assays, we demonstrated that FAO was downregulated during fibroblast activation and cardiac fibrosis, and that perturbation of FAO reversely affected the fibroblast-to-myofibroblast transition. The decrease in FAO may be attributed to reduced long-chain fatty acid (LCFA) uptake. Voltage-dependent anion channel 1 (VDAC1), the main gatekeeper of the outer mitochondrial membrane (OMM), serves as the transporter of LCFA into the mitochondria for further utilization and has been shown to be decreased in myofibroblasts. In vitro, the addition of exogenous VDAC1 was shown to ameliorate cardiac fibroblast activation initiated by transforming growth factor beta 1 (TGF-ß1) stimuli, and silencing of VDAC1 displayed the opposite effect. A mechanistic study revealed that VDAC1 exerts a protective effect by regulating LCFA uptake into the mitochondria, which is impaired by an inhibitor of carnitine palmitoyltransferase 1A. In vivo, AAV9-mediated overexpression of VDAC1 in myofibroblasts significantly alleviated transverse aortic constriction (TAC)-induced cardiac fibrosis and rescued cardiac function in mice. Finally, we treated mice with the VDAC1-derived R-Tf-D-LP4 peptide, and the results showed that R-Tf-D-LP4 prevented TAC-induced cardiac fibrosis and dysfunction in mice. In conclusion, this study provides evidence that VDAC1 maintains FAO metabolism in cardiac fibroblasts to repress fibroblast activation and cardiac fibrosis and suggests that the VDAC1 peptide is a promising drug for rescuing fibroblast metabolism and repressing cardiac fibrosis.

miR-214 could promote myocardial fibrosis and cardiac mesenchymal transition in VMC mice through regulation of the p53 or PTEN-PI3K-Akt signali pathway, promoting CF proliferation and inhibiting its ng pathway.

INTRODUCTION: This study aimed to investigate the role of miR-214 in the bidirectional regulation of p53 and PTEN and its influence on myocardial fibrosis and cardiac mesenchymal transformation in mice with viral myocarditis (VMC). METHODS: The study established a VMC model in BALB/c mice by injecting them with the CVB3 virus intraperitoneally. Techniques such as ELISA, H&E staining, Masson staining, immunohistochemical staining, RT-qPCR, western blot, and dual-luciferase reporter gene assay were used to detect the expression levels of relevant factors in tissues and cells. Isolation and culture of cardiac fibroblasts (CFs) were also conducted. RESULTS: The study found that miR-214 bidirectional regulation of p53 and PTEN promotes myocardial fibrosis and cardiac mesenchymal transformation in mice with VMC. The expression levels of collagen-related peptides, inflammatory-related factors, miR-214, mesenchymal transformation-related factors, and fibrosis-related factors were significantly increased, while the expression levels of p53, PTEN, and epithelial/endothelial cell phenotype marker factors were significantly decreased. Downregulation of miR-214 or upregulation of p53 and PTEN expression inhibited inflammatory cell and fibroblast infiltration in VMC mouse myocardial tissue. It reduced the proliferation ability while increasing the apoptosis of cardiac fibroblasts. CONCLUSION: miR-214 plays a significant role in the bidirectional inhibition of p53 and PTEN, which leads to myocardial fibrosis and cardiac mesenchymal transformation in mice with VMC. Downregulation of miR-214 or upregulation of p53 and PTEN expression may provide potential therapeutic targets for treating VMC-induced cardiac fibrosis and mesenchymal transformation.

Tissue kallikrein-related peptidase8 accentuates cardiac fibrosis after myocardial ischemia-reperfusion injury via regulation of cardiac fibroblasts.

AIMS: Tissue kallikrein-related peptidase8 (KLK8) has been found to mitigate acute myocardial ischemia-reperfusion (IR) injury. However, the effect of KLK8 on cardiac remodeling in response to IR injury has not been determined. MATERIALS AND METHODS: KLK8 overexpressing transgenic rat (KLK8-TG) was used as the animal model. IR injury was induced by ligating the left anterior descending coronary artery for 1 h and subsequent reperfusion. The functional and morphological changes of the heart were examined 14 days after the injury. Neonatal rat cardiac fibroblasts (CFs) were used to investigate the molecular mechanisms in vitro. KEY FINDINGS: KLK8 overexpression enhanced cardiac diastolic dysfunction, fibrosis, and hypertrophy after IR injury, indicating that KLK8 accentuated cardiac remodeling in response to IR injury. Moreover, KLK8 overexpression increased epidermal growth factor (EGF) release and promoted the phosphorylation of EGF receptor (EGFR) and ERK1/2 in the heart after IR injury. It was interesting to find that both EGFR antagonist (AG 1478) and MEK inhibitor (PD98059) attenuated the KLK8-induced proliferation and activation of CFs in vitro, indicating that EGFR signaling might mediate the pro-fibrotic action of KLK8. SIGNIFICANCE: KLK8 plays a crucial role in cardiac remodeling after myocardial infarction. KLK8 accentuates cardiac fibrosis after IR injury, possibly mediated by EGFR signaling in CFs.

mRNA sequencing reveals the distinct gene expression and biological functions in cardiac fibroblasts regulated by recombinant fibroblast growth factor 2.

After myocardial injury, cardiac fibroblasts (CFs) differentiate into myofibroblasts, which express and secrete extracellular matrix (ECM) components for myocardial repair, but also promote myocardial fibrosis. Recombinant fibroblast growth factor 2 (FGF2) protein drug with low molecular weight can promote cell survival and angiogenesis, and it was found that FGF2 could inhibit the activation of CFs, suggesting FGF2 has great potential in myocardial repair. However, the regulatory role of FGF2 on CFs has not been fully elucidated. Here, we found that recombinant FGF2 significantly suppressed the expression of alpha smooth muscle actin (α-SMA) in CFs. Through RNA sequencing, we analyzed mRNA expression in CFs and the differently expressed genes regulated by FGF2, including 430 up-regulated genes and 391 down-regulated genes. Gene ontology analysis revealed that the differentially expressed genes were strongly enriched in multiple biological functions, including ECM organization, cell adhesion, actin filament organization and axon guidance. The results of gene set enrichment analysis (GSEA) show that ECM organization and actin filament organization are down-regulated, while axon guidance is up-regulated. Further cellular experiments indicate that the regulatory functions of FGF2 are consistent with the findings of the gene enrichment analysis. This study provides valuable insights into the potential therapeutic role of FGF2 in treating cardiac fibrosis and establishes a foundation for further research to uncover the underlying mechanisms of CFs gene expression regulated by FGF2.

EphrinB2 drives osteogenic fate of adult cardiac fibroblasts in a calcium influx dependent manner.

Cardiac calcification is a crucial but underrecognized pathological process, greatly increasing the risk of cardiovascular diseases. Little is known about how cardiac fibroblasts, as a central mediator, facilitate abnormal mineralization. Erythropoietin-producing hepatoma interactor B2 (EphrinB2), previously identified as an angiogenic regulator, is involved in fibroblast activation, while its role in the osteogenic differentiation of cardiac fibroblasts is unknown. Bioinformatics analysis was conducted to characterize the expression of the Ephrin family in human calcified aortic valves and calcific mouse hearts. The effects of EphrinB2 on cardiac fibroblasts to adopt osteogenic fate was determined by gain- and loss-of-function. EphrinB2 mRNA level was downregulated in calcified aortic valves and mouse hearts. Knockdown of EphrinB2 attenuated mineral deposits in adult cardiac fibroblasts, whereas overexpression of EphrinB2 promoted their osteogenic differentiation. RNA sequencing data implied that Ca2+-related S100/receptor for advanced glycation end products (RAGE) signaling may mediate EphrinB2-induced mineralization in cardiac fibroblasts. Moreover, L-type calcium channel blockers inhibited osteogenic differentiation of cardiac fibroblasts, implying a critical role in Ca2+ influx. In conclusion, our data illustrated an unrecognized role of EphrinB2, which functions as a novel osteogenic regulator in the heart through Ca2+ signaling and could be a potential therapeutic target in cardiovascular calcification.NEW & NOTEWORTHY In this study, we observed that adult cardiac fibroblasts but not neonatal cardiac fibroblasts exhibit the ability of osteogenic differentiation. EphrinB2 promoted osteogenic differentiation of cardiac fibroblasts through activating Ca2+-related S100/RAGE signaling. Inhibition of Ca2+ influx using L-type calcium channel blockers inhibited EphrinB2-mediated calcification of cardiac fibroblasts. Our data implied an unrecognized role of EphrinB2 in regulating cardiac calcification though Ca2+-related signaling, suggesting a potential therapeutic target of cardiovascular calcification.

sST2: A Bridge Between Sirt1/p53/p21 Signal-Induced Senescence and TGF-ß1/Smad2/3 Regulation of Cardiac Fibrosis in Mouse Viral Myocarditis.

Soluble interleukin 1 receptor-like 1 (sST2) is a novel predictor of poor outcomes, which is involved in inflammatory response and fibrosis of myocarditis. Cellular senescence is a state of irreversible cell cycle arrest. Studies have shown that senescence of myofibroblasts can limit or reduce cardiac fibrosis. However, the molecular mechanism of sST2 regulating cellular senescence is still unclear. Here, we investigate the role of sST2 on cellular senescence in cardiac fibrosis. Our results found that sST2 was upregulated in coxsackievirus group B type 3 (CVB3)-induced viral myocarditis (VMC), which correlated with the expression of senescence markers. In vitro, sST2 activated TGFß signaling through the phosphorylation of the SMAD complex to induce mouse cardiac fibroblast (MCF) activation and inhibit cellular senescence by the Sirt1/p53/p21 signaling pathway. In vivo, anti-ST2 mAb attenuated CVB3-induced cardiac fibrosis. Our findings elucidate a crucial mechanism underlying through which sST2 inhibits cellular senescence and regulates MCF activation, providing a potential treatment strategy for cardiac fibrosis.