Migration Of Cardiac Fibroblasts Research Articles

Reprogramming cardiac macrophages with circular RNAs to attenuate post-MI adverse remodeling

Cardiac macrophages (cMφ) play a critical role in cardiac remodeling following myocardial infarction (MI). Reprogramming cMφ towards a favorable wound-healing phenotype is emerging as a promising therapeutic strategy for MI patients. Circular RNAs (circRNAs) are a unique class of non-coding RNAs with high stability and abundance, making them attractive therapeutic targets. However, the role of circRNAs in cMφ physiology during heart failure (HF) progression is poorly understood. To identify circRNAs as key regulators of cMφ dynamics, we performed RNA sequencing and bioinformatics analysis on heart biopsies from healthy donors and patients with ischemic HF. We identified a promising circRNA, circMJ1, as a regulator of Mφ polarization and inflammatory response. CircMJ1 was highly expressed in cMφ and its expression was associated with cMφ polarization. Knockdown of circMJ1 significantly inhibited inflammatory signaling pathways in cMφ and reduced the secretion of pro-inflammatory cytokines. The secretome from circMJ1-silenced cMφ (circMJ1-KD cMφ) also significantly suppressed the migration of cardiac fibroblasts and apoptosis of cardiomyocytes. To further assess the therapeutic effcacy of circMJ1-KD Mφ in cardiac remodeling in the context of MI, we employed a novel multicellular 3D ex vivo model, called living myocardial slices (LMS). We applied circMJ1-KD Mφ to both rat LMS subjected to cryo-injury mimicking MI and HF patient-derived LMS. Interestingly, co-culture with circMJ1-KD Mφ demonstrated a remarkable reduction in inflammation and fibrosis, alongside notable improvement in cardiac function in both cryo-injured rat LMS and HF patient-derived LMS. Overall, our findings reveal circMJ1 as a crucial "switch" for Mφ dynamics and suggest the therapeutic potential of engineered circMJ1-silenced Mφ to improve post-MI healing. Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) –465131031. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

FBXL8 inhibits post-myocardial infarction cardiac fibrosis by targeting Snail1 for ubiquitin-proteasome degradation

Abnormal cardiac fibrosis is the main pathological change of post-myocardial infarction (MI) heart failure. Although the E3 ubiquitin ligase FBXL8 is a key regulator in the cell cycle, cell proliferation, and inflammation, its role in post-MI ventricular fibrosis and heart failure remains unknown. FBXL8 was primarily expressed in cardiac fibroblasts (CFs) and remarkably decreased in CFs treated by TGFβ and heart subjected to MI. The echocardiography and histology data suggested that adeno-associated viruses (AAV9)-mediated FBXL8 overexpression had improved cardiac function and ameliorated post-MI cardiac fibrosis. In vitro, FBXL8 overexpression prevented TGFβ-induced proliferation, migration, contraction, and collagen secretion in CFs, while knockdown of FBXL8 demonstrated opposite effects. Mechanistically, FBXL8 interacted with Snail1 to promote Snail1 degradation through the ubiquitin–proteasome system and decreased the activation of RhoA. Moreover, the FBXL8ΔC3 binding domain was indispensable for Snail1 interaction and degradation. Ectopic Snail1 expression partly abolished the effects mediated by FBXL8 overexpression in CFs treated by TGFβ. These results characterized the role of FBXL8 in regulating the ubiquitin-mediated degradation of Snail1 and revealed the underlying molecular mechanism of how MI up-regulated the myofibroblasts differentiation-inducer Snail1 and suggested that FBXL8 may be a potential curative target for improving post-MI cardiac function.

Morroniside improves AngII-induced cardiac fibroblast proliferation, migration, and extracellular matrix deposition by blocking p38/JNK signaling pathway through the downregulation of KLF5.

Myocardial fibrosis (MF), which is an inevitable pathological manifestation of many cardiovascular diseases in the terminal stage, often contributes to severe cardiac dysfunction and sudden death. Morroniside (MOR) is the main active component of Cornus officinalis with a variety of biological activities. This study was designed to explore the efficacy of MOR in MF and to investigate its pharmacological mechanism. The viability of MOR-treated human cardiac fibroblast (HCF) cells with or without Angiotensin II (AngII) induction was assessed with Cell Counting Kit-8 (CCK-8). The migration of AngII-induced HCF cells was appraised with a transwell assay. Gelatin zymography analysis was adopted to evaluate the activities of MMP2 and MMP9, while immunofluorescence assay was applied for the estimation of Collagen I and Collagen III. By means of western blot, the expressions of migration-, fibrosis-, and p38/c-Jun N-terminal kinase (JNK) signal pathway-related proteins were resolved. The transfection efficacy of oe-Kruppel-like factor 5 (KLF5) was examined with reverse transcription-quantitative PCR (RT-qPCR) and western blot. In this study, it was found that MOR treatment inhibited AngII-induced hyperproliferation, migration, and fibrosis of HCF cells, accompanied with decreased activities of matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), connective tissue growth factor (CTGF), Fibronectin, and α-SMA, which were all reversed by KLF5 overexpression. Collectively, MOR exerted protective effects on MF by blocking p38/JNK signal pathway through the downregulation of KLF5.

PARP9 affects myocardial function through TGF-β/Smad axis and pirfenidone

Cardiac arrhythmias are often linked to the overactivity of cardiac fibroblasts (CFs). Investigating the impact of poly (ADP-ribose) polymerase 9 (PARP9) on Angiotensin II (Ang II)-induced fibroblast activation and the therapeutic effects of pirfenidone (PFD) offers valuable insights into cardiac arrhythmias. This study utilized weighted gene co-expression network analysis (WGCNA), differential gene expression (DEG) analysis, protein–protein interaction (PPI), and receiver operating characteristic (ROC) analysis on the GSE42955 dataset to identify the hub gene with a significant diagnostic value. The ImmuCellAI tool revealed an association between PARP9 and immune cell infiltration. Our in vitro assessments focused on the influence of PFD on myofibroblast differentiation, transforming growth factor-beta (TGF-β) expression, and Ang II-induced proliferation and migration in CFs. Additionally, we explored the impact on fibrosis markers and the TGF-β/Smad signaling pathway in the context of PARP9 overexpression. Analysis of the GSE42955 dataset revealed PARP9 as a central gene with high clinical diagnostic value, linked to seven types of immune cells. The in vitro studies demonstrated that PFD significantly mitigates Ang II-induced CF proliferation, migration, and fibrosis. It also reduces Ang II-induced PARP9 expression and decreases fibrosis markers, including TGF-β, collagen I, collagen III, and α-SMA. Notably, PARP9 overexpression can partially counteract PFD’s inhibitory effects on CFs and modify the expression of fibronectin, CTGF, α-SMA, collagen I, collagen III, MMP2, MMP9, TGF-β, and p-Smad2/3 in the TGF-β/Smad signaling pathway. In summary, our findings suggest that PFD effectively counteracts the adverse effects of Ang II-induced CF proliferation and fibrosis, and modulates the TGF-β/Smad signaling pathway and PARP9 expression. This identifies a potential therapeutic approach for managing myocardial fibrosis.

Diabetes Promotes Myocardial Fibrosis via AMPK/EZH2/PPAR-γ Signaling Pathway.

Diabetes-induced cardiac fibrosis is one of the main mechanisms of diabetic cardiomyopathy. As a common histone methyltransferase, enhancer of zeste homolog 2 (EZH2) has been implicated in fibrosis progression in multiple organs. However, the mechanism of EZH2 in diabetic myocardial fibrosis has not been clarified. In the current study, rat and mouse diabetic model were established, the left ventricular function of rat and mouse were evaluated by echocardiography and the fibrosis of rat ventricle was evaluated by Masson staining. Primary rat ventricular fibroblasts were cultured and stimulated with high glucose (HG) in vitro. The expression of histone H3 lysine 27 (H3K27) trimethylation, EZH2, and myocardial fibrosis proteins were assayed. In STZ-induced diabetic ventricular tissues and HG-induced primary ventricular fibroblasts in vitro, H3K27 trimethylation was increased and the phosphorylation of EZH2 was reduced. Inhibition of EZH2 with GSK126 suppressed the activation, differentiation, and migration of cardiac fibroblasts as well as the overexpression of the fibrotic proteins induced by HG. Mechanical study demonstrated that HG reduced phosphorylation of EZH2 on Thr311 by inactivating AMP-activated protein kinase (AMPK), which transcriptionally inhibited peroxisome proliferator-activated receptor γ (PPAR-γ) expression to promote the fibroblasts activation and differentiation. Our data revealed an AMPK/EZH2/PPAR-γ signal pathway is involved in HG-induced cardiac fibrosis.

Characterization of cardiac fibroblast-extracellular matrix crosstalk across developmental ages provides insight into age-related changes in cardiac repair.

Heart failure afflicts an estimated 6.5 million people in the United States, driven largely by incidents of coronary heart disease (CHD). CHD leads to heart failure due to the inability of adult myocardial tissue to regenerate after myocardial infarction (MI). Instead, immune cells and resident cardiac fibroblasts (CFs), the cells responsible for the maintenance of the cardiac extracellular matrix (cECM), drive an inflammatory wound healing response, which leads to fibrotic scar tissue. However, fibrosis is reduced in fetal and early (<1-week-old) neonatal mammals, which exhibit a transient capability for regenerative tissue remodeling. Recent work by our laboratory and others suggests this is in part due to compositional differences in the cECM and functional differences in CFs with respect to developmental age. Specifically, fetal cECM and CFs appear to mitigate functional loss in MI models and engineered cardiac tissues, compared to adult CFs and cECM. We conducted 2D studies of CFs on solubilized fetal and adult cECM to investigate whether these age-specific functional differences are synergistic with respect to their impact on CF phenotype and, therefore, cardiac wound healing. We found that the CF migration rate and stiffness vary with respect to cell and cECM developmental age and that CF transition to a fibrotic phenotype can be partially attenuated in the fetal cECM. However, this effect was not observed when cells were treated with cytokine TGF-β1, suggesting that inflammatory signaling factors are the dominant driver of the fibroblast phenotype. This information may be valuable for targeted therapies aimed at modifying the CF wound healing response and is broadly applicable to age-related studies of cardiac remodeling.

Downregulation of the CD151 protects the cardiac function by the crosstalk between the endothelial cells and cardiomyocytes via exosomes.

Heart failure (HF) is the last stage in the progression of various cardiovascular diseases. Although it is documented that CD151 contributes to regulate the myocardial infarction, the function of CD151 on HF and involved mechanisms are still unclear. In the present study, we found that the recombinant adeno-associated virus (rAAV)-mediated endothelial cell-specific knockdown of CD151-transfected mice improved transverse aortic constriction (TAC)-induced cardiac function, attenuated myocardial hypertrophy and fibrosis, and increased coronary perfusion, whereas overexpression of the CD151 protein aggravated cardiac dysfunction and showed the opposite effects. In vitro, the cardiomyocytes hypertrophy induced by PE were significantly improved, while the proliferation and migration of cardiac fibroblasts (CFs) were significantly reduced, when co-cultured with the CD151-silenced endothelial cells (ECs). To further explore the mechanisms, the exosomes from the CD151-silenced ECs were taken by cardiomyocyte (CMs) and CFs, verified the intercellular communication. And the protective effects of CD151-silenced ECs were inhibited when exosome inhibitor (GW4869) was added. Additionally, a quantitative proteomics method was used to identify potential proteins in CD151-silenced EC exosomes. We found that the suppression of CD151 could regulate the PPAR signaling pathway via exosomes. Our observations suggest that the downregulation of CD151 is an important positive regulator of cardiac function of heart failure, which can regulate exosome-stored proteins to play a role in the cellular interaction on the CMs and CFs. Modulating the exosome levels of ECs by reducing CD151 expression may offer novel therapeutic strategies and targets for HF treatment.

Protective Effect of CD137 Deficiency against Post-infarction Cardiac Fibrosis and Adverse Cardiac Remodeling via ERK1/2 Signaling Pathways.

Myocardial fibrosis, a common complication of myocardial infarction (MI), is characterized by excessive collagen deposition and can result in impaired cardiac function. The specific role of CD137 in the development of post-MI myocardial fibrosis remains unclear. Thus, this study aimed to elucidate the effects of CD137 signaling using CD137 knockout mice and in vitro experiments. CD137 expression levels progressively increased in the heart following MI, particularly in myofibroblast, which play a key role in fibrosis. Remarkably, CD137 knockout mice exhibited improved cardiac function and reduced fibrosis compared to wild-type mice at day 28 post-MI. The use of Masson's trichrome and picrosirius red staining demonstrated a reduction in the infarct area and collagen volume fraction in CD137 knockout mice. Furthermore, the expression of alpha-smooth muscle actin (α-SMA) and collagen I, key markers of fibrosis, was decreased in heart tissues lacking CD137. In vitro experiments supported these findings, as CD137 depletion attenuated cardiac fibroblast differentiation, and migration, and collagen I synthesis. Additionally, the administration of CD137L recombinant protein further promoted α-SMA expression and collagen I synthesis, suggesting a pro-fibrotic effect. Notably, the application of an inhibitor targeting the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway attenuated the pro-fibrotic effects of CD137L. To conclude, this study provides evidence that CD137 plays a significant role in promoting myocardial fibrosis after MI. Inhibition of CD137 signaling pathways may hold therapeutic potential for mitigating pathological cardiac remodeling and improving post-MI cardiac function.

FOXO3a Induces Myocardial Fibrosis by Upregulating Mitophagy.

Atrial fibrillation is one of the most common cardiac arrhythmias. Myocardial fibrosis is closely associated with atrial remodeling, which leads to heightened risk of atrial fibrillation. This study aimed to explore whether forkhead box protein O3 (FOXO3a) impacts myocardial fibrosis incidence by regulating mitophagy. Cell viability was assessed by cell counting kit-8 (CCK-8) assays. The expression of vimentin and cytochrome C was detected by immunofluorescence assays. Quantitative real-time polymerase chain reaction (PCR) was used to analyze the relative mRNA level of FOXO3a. Expression of FOXO3a, phosphorylated FOXO3a, Collagen I, Collagen III, alpha-smooth muscle actin (α-SMA), matrix metalloprotease 9 (MMP9), light chain 3 (LC3), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), Parkin, and sequestosome-1 (p62) proteins were determined by western blotting. 5-ethynyl-2'-deoxyuridine (EDU) incorporation was employed to measure cell proliferation. Changes in mitochondrial membrane potential were determined by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) staining. A wound healing assay was used to examine cell migration, and the levels of reactive oxygen species were determined by flow cytometry. The expression of FOXO3a was upregulated in cardiac fibroblasts treated with angiotensin II (AngII), while the expression of phosphorylated FOXO3a was downregulated under these conditions. FOXO3a knockdown significantly inhibited the proliferation, migration and collagen secretion of cardiac fibroblasts treated with AngII. The ratio of LC3 II/I as well as expression of PINK1 and Parkin was increased, and the expression of p62 was decreased, in cardiac fibroblasts treated with AngII. Moreover, these effects were limited by FOXO3a knockdown. Finally, the mitophagy inducer everolimus (RAD001) attenuated the suppressive effect of FOXO3a knockdown on cardiac fibroblast activation. FOXO3a promotes the progress of myocardial fibrosis by triggering mitophagy in cardiac fibroblasts.

Carbonic Anhydrase 3 is required for cardiac repair post myocardial infarction via Smad7-Smad2/3 signaling pathway.

Appropriate fibrosis is required to prevent subsequent adverse remodeling and heart failure post myocardial infarction (MI), and cardiac fibroblasts (CFs) play a critical role during the process. Carbonic anhydrase 3 (CAR3) is an important mediator in multiple biological processes besides its CO2 hydration activity; however, the role and underlying mechanism of CAR3 on cardiac repair post MI injury remains unknown. Here, we found that CAR3 expression was up-regulated in cardiac tissue in infarct area at the reparative phase of MI, with a peak at 7 days post MI. The upregulation was detected mainly on fibroblast instead of cardiomyocyte, and primary cardiac fibroblasts treated with TGF-β1 recaptured our observation. While CAR3 deficiency leads to weakened collagen density, enlarged infarct size and aggravated cardiac dysfunction post-MI. In fibroblast, we observed that CAR3 deficiency restrains collagen synthesis, cell migration and gel contraction of cardiac fibroblasts, whereas overexpression of CAR3 in CFs improves wound healing and cardiac fibroblast activation. Mechanistically, CAR3 stabilizes Smad7 protein via modulating its acetylation, which dampens phosphorylation of Smad2 and Smad3, thus inhibiting fibroblast transformation. In contrast, inhibition of Smad7 acetylation with C646 blunts CAR3 deficiency induced suppression of fibroblast activation and impaired cardiac healing. Our data demonstrate a protective role of CAR3 in cardiac wound repair post MI via promoting fibroblasts activation through Smad7-TGF-β/Smad2/3 signaling pathway.

SLIT3 promotes cardiac fibrosis and differentiation of cardiac fibroblasts by RhoA/ROCK1 signaling pathway.

Slit guidance ligand 3 (SLIT3) has been identified as a potential therapeutic regulator against fibroblast activity and fibrillary collagen production in an autocrine manner. However, this research aims to investigate the potential role of SLIT3 in cardiac fibrosis and fibroblast differentiation and its underlying mechanism. C57BL/6 mice (male, 8-10 weeks, n=47) were subcutaneously infused with Ang II (2.0 mg/kg/day) for 4 weeks. One to two-day-old Sprague-Dawley (SD) rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (60 mg/kg) and ketamine (50 mg/kg) and the cardiac fibroblast was isolated aseptically. The mRNA and protein expression were analyzed using RT-qPCR and Western blotting. The SLIT3 expression level was increased in Ang II-induced mice models and cardiac fibroblasts. SLIT3 significantly increased migrated cells and α-smooth muscle actin (α-SMA) expression in cardiac fibroblasts. Ang II-induced increases in mRNA expression of collagen I (COL1A1), and collagen III (COL3A1) was attenuated by SLIT3 inhibition. SLIT3 knockdown attenuated the Ang II-induced increase in mRNA expression of ACTA2 (α-SMA), Fibronectin, and CTGF. SLIT3 suppression potentially reduced DHE expression and decreased malondialdehyde (MDA) content, and the superoxide dismutase (SOD) and catalase (CAT) levels were significantly increased in cardiac fibroblasts. Additionally, SLIT3 inhibition markedly decreased RhoA and ROCK1 protein expression, whereas ROCK inhibitor Y-27632 (10 μM) markedly attenuated the migration of cardiac fibroblasts stimulated by Ang II and SLIT3. The results speculate that SLIT3 could significantly regulate cardiac fibrosis and fibroblast differentiation via the RhoA/ROCK1 signaling pathway.

The inhibition of FTO attenuates the antifibrotic effect of leonurine in rat cardiac fibroblasts

BackgroundMyocardial fibrosis (MF) is a common pathological condition in cardiovascular diseases that often causes severe cardiac dysfunction. MF is characterized by changes in cardiomyocytes, cardiac fibroblasts (CFs), levels of collagen (Col) -1, -3, and overdeposition of the extracellular matrix. Our previous research showed that leonurine (LE) effectively inhibits collagen synthesis and differentiation of CFs, but the mechanism is not fully elucidated. Recent evidence indicates that fat mass and obesity-associated proteins (FTO) regulates the occurrence and development of MF. This study aimed to explore the role of FTO in the antifibrotic effects of LE. MethodsNeonatal rat CFs were isolated, and induced using angiotensin II (Ang II) to establish a cell model of MF. Cell viability, wound healing and transwell assays were used to detect cell activity and migration ability. The protein and mRNA levels of MF-related factors were measured following stimulation with Ang II and LE under normal conditions or after FTO knockdown. The RNA methylation level was measured by dot blot assay. ResultsThe results showed that LE (20, 40 μM) was not toxic to normal CFs. LE reduced the proliferation, migration and collagen synthesis of Ang II-induced CFs. Further investigation showed that FTO was downregulated by Ang II stimulation, whereas LE reversed this effect. FTO knockdown facilitated the migration of CFs, upregulated the protein levels of Col-3, α-SMA and Col-1 in Ang II and LE-stimulated CFs, and enhanced the fluorescence intensity of α-SMA. Furthermore, LE reduced N6-methyladenosine (m6A) RNA methylation, which was partially blocked by FTO knockdown. FTO knockdown also reduced the expression levels of p53 protein in Ang II and LE-stimulated CFs. ConclusionsOur findings suggest that the inhibition of FTO may attenuate the antifibrotic effect of LE in CFs, suggesting that FTO may serve as a key protein for anti-MF of LE.

Biochanin A inhibits cardiac hypertrophy and fibrosis in vivo and in vitro

The heart undergoes pathological cardiac hypertrophy as an adaptive response to prolonged pathological stimulation, leading to cardiomyocyte hypertrophy, fibroblast proliferation, and an increase in extracellular matrix. Chinese medicine monomers are now receiving much attention for the treatment of cardiac hypertrophy and myocardial remodeling. Biochanin A (BCA) is a kind of flavonoid structural monomer, which has a certain therapeutic effect on bone thinning disease, aging syndrome, lung cancer, etc. Moreover, it exhibits hypoglycemic, anti-inflammatory, anti-oxidation, anti-bacteria and other pharmacological properties. It is still unknown whether BCA has an impact on the mechanism of TAC-induced cardiac hypertrophy. Here, cardiac remodeling was induced by TAC. BCA was injected intraperitoneally at 25 and 50 mg/kg/day one week in advance. Masson, WGA, DHE and other pathological staining and serum were used to detect the inhibitory effect of BCA on cardiac hypertrophy in mice. The anti-hypertrophic effect of BCA was demonstrated by studying the pathological manifestations of Neonatal rat cardiomyocytes (NRCMs) and cardiac fibroblasts (CFs) in vitro. The results showed that BCA significantly reduced TAC-induced fibrosis, inflammation, oxidative stress, and myocardial hypertrophy. BCA inhibited Ang II-induced cell hypertrophy and oxidative stress in NRCMs in vitro and Ang II-induced CF migration, proliferation, and collagen secretion. This suggests that BCA plays a key role in inhibiting the progression of myocardial remodeling, suggesting that BCA may be a promising agent for the treatment of myocardial hypertrophy and fibrosis.

Salvianolic acid B ameliorates myocardial fibrosis in diabetic cardiomyopathy by deubiquitinating Smad7

BackgroundSalvianolic acid B (Sal B), a water-soluble phenolic compound derived from Salvia miltiorrhiza Bunge, is commonly used in Traditional Chinese Medicine to treat cardiovascular disease. In our previous study, Sal B protected against myocardial fibrosis induced by diabetic cardiomyopathy (DCM). This study aimed to investigate the ameliorative effects and potential mechanisms of Sal B in mitigating myocardial fibrosis induced by DCM.MethodsVarious methods were used to investigate the effects of Sal B on myocardial fibrosis induced by DCM in vivo and in vitro. These methods included blood glucose measurement, echocardiography, HE staining, Masson’s trichrome staining, Sirius red staining, cell proliferation assessment, determination of hydroxyproline levels, immunohistochemical staining, evaluation of fibrosis-related protein expression (Collagen-I, Collagen-III, TGF-β1, p-Smad3, Smad3, Smad7, and α-smooth muscle actin), analysis of Smad7 gene expression, and analysis of Smad7 ubiquitin modification.ResultsThe animal test results indicated that Sal B significantly improved cardiac function, inhibited collagen deposition and phenotypic transformation, and ameliorated myocardial fibrosis in DCM by upregulating Smad7, thereby inhibiting the TGF-β1 signaling pathway. In addition, cell experiments demonstrated that Sal B significantly inhibited the proliferation, migration, phenotypic transformation, and collagen secretion of cardiac fibroblasts (CFs) induced by high glucose (HG). Sal B significantly decreased the ubiquitination of Smad7 and stabilized the protein expression of Smad7, thereby increasing the protein expression of Smad7 in CFs and inhibiting the TGF-β1 signaling pathway, which may be the potential mechanism by which Sal B mitigates myocardial fibrosis induced by DCM.ConclusionThis study revealed that Sal B can improve myocardial fibrosis in DCM by deubiquitinating Smad7, stabilizing the protein expression of Smad7, and blocking the TGF-β1 signaling pathway.

Orphan nuclear receptor 4 A1 involvement in transforming growth factor beta1-induced myocardial fibrosis in diabetic mice.

We explored the involvement of orphan nuclear receptor 4 A1 (NR4A1) in myocardial fibrosis mediated by transforming growth factor-beta1 (TGF-β1) and its response to cytosporone B (Csn-B). We developed a diabetic cardiomyopathy mouse model by administering a high-fat diet in conjunction with a low-dose streptozotocin injection. Our analysis involved monitoring alterations in blood glucose and lipid levels, cardiac function and structure, as well as profibrotic factors such as α smooth muscle actin (α-SMA), collagen I, collagen III, TGF-β1, connective tissue growth factor, and fibronectin. These assessments were conducted using biochemical techniques, Doppler ultrasound, histopathology, and real-time quantitative polymerase chain reaction. Cardiac fibroblasts (CFs) were extracted from suckling mice and cultivated in a high-glucose medium to simulate diabetes-induced myocardial fibrosis in vitro. These CFs were then subjected to coculture experiments with TGF-β1 or Csn-B. The proliferation and migration of CFs were assessed using cell counting kit 8 (CCK-8) assays and Transwell assays, respectively. Western blotting and immunofluorescence assays were employed to evaluate the expression levels of NR4A1, p-NR4A1, and α-SMA in CFs treated with TGF-β1 after NR4A1 knockdown or Csn-B administration, respectively. In diabetic heart tissue, the expression of p-NR4A1 was notably elevated. Furthermore, CFs exhibited enhanced proliferative capabilities and increased p-NR4A1 expression following high glucose exposure. Interestingly, NR4A1 knockdown resulted in a significant increase in the expression of fibrosis-related proteins in CFs following treatment with TGF-β1. Moreover, our observations revealed a marked decrease in p-NR4A1 levels and a reduction in the expression of fibrosis-related proteins after Csn-B treatment. In diabetic mice treated with Csn-B, we noted diminished NR4A1 phosphorylation and a mitigation of myocardial fibrosis. We concluded that in the mouse model, Csn-B played a pivotal role in inhibiting diabetes-induced myocardial fibrosis by activating NR4A1.

Protein tyrosine phosphatase sigma and zeta1 regulate cardiac fibroblast contractile function through TGF-beta dependent and independent pathways

Abstract Background Heart failure is a frequently fatal cardiovascular disease with limited therapeutic options. To improve our understanding of signaling pathways in affected cells is therefore important. Protein tyrosine phosphatase sigma (PTPRσ) and zeta 1 (PTPRz1) have been shown to be involved in cardiac development and disease [1,2]. Both phosphatases are upregulated in patients with end stage heart failure. We demonstrated that PTPRz1 inhibits while PTPRσ is required for migration of neonatal cardiac fibroblasts (nCF) [3]. Purpose To further evaluate the influence of PTPRσ and PTPRz1 on fibroblast phenotype with regard to stress fiber formation and collagen contraction and their interaction with the TGFβ pathway. Methods nCF were transfected with either scrambled-, PTPRσ-, PTPRz1-, or both siRNAs. mRNA lysates were harvested 24h after transfection with subsequent rt-PCR analysis for the phosphatases. Transfected cells were seeded and stained with labeled phalloidin to measure the formation of stress fibers or in collagen gel matrices to evaluate their contractile capacities. To evaluate the nuclear translocation of SMAD3 immunohistochemical colocalization of SMAD3 and DAPI was quantified. Data were analyzed by Kruskal-Wallis and Mann-Whitney-U or ANOVA followed by t-test with Welsh correction using GraphPad prism. Results PTPRz1 and -σ knockdown (kd) was highly efficient (&amp;gt;85%). PTPRz1 mRNA was upregulated by 48% after PTPRσ kd (p &amp;lt; 0.05, n=4) but not vice versa and not after combined kd. Compared to control, nCF showed a 26% increase in the number of stress fibers in response to the PTPRz1 kd (p = 0.057, n=3-4) and an increase of 29% after PTPRσ/-z1 kd (p &amp;lt; 0.05, n=4). Collagen gels were contracted to about 70% of their surface by TGFβ in control (p&amp;lt;0.05, n=4) or any kd condition. They were also contracted by 24,5% after PTPRz1 knockdown (p &amp;lt; 0.01, n=4), whereas the additional kd of PTPRσ obliterated the effect. Nuclear translocation of SMAD3 was roughly doubled upon TGFβ stimulation in controls and all kd conditions. In unstimulated nCF, PTPRσ kd induced a 28%, PTPRz1 kd a 45%, and their combination a 114% increase of nuclei positive for SMAD3 (p &amp;lt; 0.05). Conclusion SMAD3 nuclear translocation is additively induced by PTPRz1 and PTPRσ kd independent of TGFβ. Stress fiber formation, however, seems to be inhibited by PTPRz1 independent of PTPRσ, while PTPRz1-kd-induced collagen contraction does depend on the presence of PTPRσ. PTPRσ and PTPRz1 thus have separate, overlapping and antagonistic effects that are TGFβ dependent and independent. The elucidation of the involvement of the phosphatases in fibroblast signaling and function could improve our understanding of adverse remodeling during heart failure and deliver novel options for therapeutic interventions.

CircMACF1 alleviates myocardial fibrosis after acute myocardial infarction by suppressing cardiac fibroblast activation via the miR-16-5p/SMAD7 axis.

Circular RNAs (circRNAs) played a pivotal role in myocardial fibrosis after acute myocardial infarction (AMI). The activation of cardiac fibroblasts (CFs) and accumulation of extracellular matrix are the main characteristics of myocardial fibrosis. In our research, we aimed to elucidate the functional roles of circMACF1 in CF activation after AMI as well as the underlying mechanism. Human CFs were activated by TGF-β1 treatment. qPCR and western blotting were performed to investigate gene and protein expression. CCK-8 and transwell assays were carried out to measure cell proliferation, and migration. Immunofluorescence was used to investigate α-SMA level. The interaction between miR-16-5p and circMACF1 or SMAD7 was revealed by RIP or dual luciferase reporter gene assays. CircMACF1 and SMAD7 were repressed in AMI patients and CFs treated with TGF-β1, and miR-16-5p was increased. In addition, circMACF1 was resistant to RNase R and abundantly expressed in the cytoplasm. Overexpression of circMACF1 inhibited cell proliferation and migration and reduced the expression levels of fibrosis-related proteins, including Collagen I, Collagen III, and α-SMA. Furthermore, circMCAF1 could directly bind to miR-16-5p, and SMAD7 was a target gene of miR-16-5p. Knockdown of miR-16-5p suppressed the activation, proliferation, and migration of TGF-β1-treated CFs, but silencing circMACF1 or SMAD7 partially reversed this phenomenon. CircMACF1 attenuated the TGF-β1-induced activation, proliferation and migration of CFs via the miR-16-5p/SMAD7 signaling pathway, indicating that circMACF1 might be a new therapeutic target for AMI.

WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation

Dysregulated lipid metabolism occurs in pathological processes characterized by cell proliferation and migration. Nonetheless, the mechanism of increased mitochondrial lipid oxidation is poorly appreciated in diabetic cardiac fibrosis, which is accompanied by enhanced fibroblast proliferation and migration. Herein, increased WTAP expression promotes cardiac fibroblast proliferation and migration, contributing to diabetic cardiac fibrosis. Knockdown of WTAP suppresses mitochondrial lipid oxidation, fibroblast proliferation and migration to ameliorate diabetic cardiac fibrosis. Mechanistically, WTAP-mediated m6A methylation of AR induced its degradation, dependent on YTHDF2. Additionally, AR directly interacts with mitochondrial lipid oxidation enzyme Decr1; overexpression of AR-suppressed Decr1-mediates mitochondrial lipid oxidation, inhibiting cardiac fibroblast proliferation and migration. Knockdown of AR produced the opposite effect. Clinically, increased WTAP and YTHDF2 levels correlate with decreased AR expression in human DCM heart tissue. We describe a mechanism wherein WTAP boosts higher mitochondrial lipid oxidation, cardiac fibroblast proliferation, and migration by enhancing AR methylation in a YTHDF2-dependent manner.

Exogenous Spermidine Alleviates Diabetic Myocardial Fibrosis Via Suppressing Inflammation and Pyroptosis in db/db Mice

The main pathological feature of diabetic cardiomyopathy (DCM) caused by diabetes mellitus is myocardial fibrosis. According to recent studies in cardiology, it has been suggested that spermidine (SPD) has cardioprotective properties. To explore the role and mechanism of SPD in alleviating myocardial fibrosis of DCM. In vivo and in vitro study. Type 2 diabetic mice and primary neonatal mouse cardiac fibroblasts (CFs) were selected. Measurements of serum-related markers, echocardiographic analysis, and immunohistochemistry were used to evaluate myocardial fibrosis injury and the effects of SPD. The proliferation and migration of CFs undergoing different treatments were studied. Immunoblotting and real-time quantitative reverse transcription polymerase chain reaction were used to demonstrate molecular mechanisms. In vivo immunoblotting analysis indicated a downregulation of ornithine decarboxylase and an upregulation of SPD/spermine N1-acetyltransferase. We observed cardiac dysfunction in diabetic mice after 12 weeks. However, the administration of exogenous SPD improved cardiac function, decreased collagen deposition, and reduced myocardial tissue damage. mRNA expression levels of NLRP3, Caspase-1, GSDMD-N, interleukin (IL)-1β, IL-17A, and IL-18 were increased and suppressed in the myocardium of db/db mice upon treatment with SPD. SPD inhibited the proliferation, migration, and collagen secretion of high-glucose-treated fibroblasts in vitro. SPD inhibits the activation of the TGF-β1/Smad signaling pathway and decreases collagen deposition by reducing pyroptosis and Smad-7 ubiquitination levels. Based on our findings, SPD may have potential applications in protecting against the deterioration of cardiac function in patients with DCM due to a significant new mechanism for diabetic myocardial fibrosis that we discovered.

Neuroepithelial cell-transforming 1 promotes cardiac fibrosis via the Wnt/β-catenin signaling pathway

This study found that the level of neuroepithelial cell-transforming gene 1 protein (NET1) was significantly increased in a mouse cardiac fibrosis model. Moreover, the expression level of NET1 was increased in cardiac fibrosis induced by TGF-β1, suggesting that NET1 was involved in the pathological process of cardiac fibrosis. Overexpression of NET1 promoted β-catenin expression in the nucleus and significantly increased the proliferation and migration of cardiac fibroblasts. NET1 may form a complex with β-catenin through GSK3β. Knockdown of β-catenin alleviated the effects of NET1 overexpression on collagen production and cell migration. In the heart of NET1 knockout mice, NET1 knockout can reduce the expression of β-catenin, α-SMA, and collagen content induced by MI. In conclusion, NET1 may regulate the activation of Wnt/β-catenin and TGF/Smads signaling pathway, promote collagen synthesis in fibroblasts, and participate in cardiac fibrosis. Thus, NET1 may be a potential therapeutic target in cardiac fibrosis.