MicroRNA-378 suppresses myocardial fibrosis through a paracrine mechanism at the early stage of cardiac hypertrophy following mechanical stress.
Jie YuanHaibo LiuWei GaoLi ZhangYong YeLingyan YuanZhiwen DingJian WuLe KangXiaoyi ZhangXiaoyan WangGuoping ZhangHui GongAijun SunXiangdong YangRuizhen ChenZhaoqiang CuiJunbo GeYunzeng Zou
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Abstract:
Rationale: Excessive myocardial fibrosis is the main pathological process in the development of cardiac remodeling and heart failure; therefore, it is important to prevent excessive myocardial fibrosis. We determined that microRNA-378 (miR-378) is cardiac-enriched and highly repressed during cardiac remodeling. We therefore proposed that miR-378 has a critical role in regulation of cardiac fibrosis, and examined the effects of miR-378 on cardiac fibrosis after mechanical stress. Methods: Mechanical stress was respectively imposed on mice through a transverse aortic constriction (TAC) procedure and on cardiac fibroblasts by stretching silicon dishes. A chemically modified miR-378 mimic (Agomir) or an inhibitor (Antagomir) was administrated to mice by intravenous injection and to cells by direct addition to the culture medium. MiR-378 knockout mouse was constructed. Cardiac fibroblasts were cultured in the conditioned media from the cardiomyocytes with either miR-378 depletion or treatment with sphingomyelinase inhibitor GW4869. Quantitative real-time polymerase chain reaction analysis of gene and miRNA expression, Western blot analysis, immunochemistry and electron microscopy were performed to elucidate the mechanisms. Results: Mechanical stress induced significant increases in fibrotic responses, including myocardial fibrosis, fibroblast hyperplasia, and protein and gene expression of collagen and matrix metalloproteinases (MMPs) both in vivo and in vitro. All these fibrotic responses were attenuated by treatment with a chemically modified miR-378 mimic (Agomir) but were exaggerated by treatment with an inhibitor (Antagomir). MiR-378 knockout mouse models exhibited aggravated cardiac fibrosis after TAC. Media from the cardiomyocytes with either miR-378 depletion or treatment with sphingomyelinase inhibitor GW4869 enhanced the fibrotic responses of stimulated cardiac fibroblasts, confirming that miR-378 inhibits fibrosis in an extracellular vesicles-dependent secretory manner. Mechanistically, the miR-378-induced anti-fibrotic effects manifested partially through the suppression of p38 MAP kinase phosphorylation by targeting MKK6 in cardiac fibroblasts. Conclusions: miR-378 is secreted from cardiomyocytes following mechanical stress and acts as an inhibitor of excessive cardiac fibrosis through a paracrine mechanism.Keywords:
Cardiac Fibrosis
Myocardial fibrosis
Antagomir
Pressure overload
Cardiac fibrosis, characterized by an unbalanced production and degradation of extracellular matrix components, is a common pathophysiology of multiple cardiovascular diseases. Recent studies suggested that endothelial to mesenchymal transition (EndMT) could be a source of activated fibroblasts and contribute to cardiac fibrosis. Here, the role of pioglitazone (PIO) in cardiac fibrosis and EndMT was elaborated.Male C57BL/6 mice were subjected to aortic banding (AB), which was used to construct a model of pressure overload-induced cardiac hypertrophy. PIO and GW9662 was given for 4 weeks to detect the effects of PIO on EndMT.Our results showed PIO treatment attenuated cardiac hypertrophy, dysfunction and fibrosis response to pressure overload. Mechanistically, PIO suppressed the TGF-β/Smad signaling pathway activated by 4-week AB surgery. Moreover, PIO dramatically inhibited EndMT in vivo and in vitro stimulated by pressure overload or TGF-β. A selective antagonist of PPAR-γ, GW9662, neutralized the anti-fibrotic effect and abolished the inhibitory effect of EndMT during the treatment of PIO.Our data implied that PIO exerts an alleviative effect on cardiac fibrosis via inhibition of the TGF-β/Smad signaling pathway and EndMT by activating PPAR-γ.
Pressure overload
Cardiac Fibrosis
Pioglitazone
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Myocardial Fibrosis,caused by the imbalance of collagen metabolism,is crucial for the transformation from viral myocarditis to dilated cardiomyopathy.Recently,it is demonstrated that matrix metalloproteinases and their tissue inhibitors(MMPs/TIMP),as a collagen degeneration system,are related to the imbalance of collagen metabolism to the formation of myocardial fibrosis.
Myocardial fibrosis
Viral Myocarditis
Dilated Cardiomyopathy
Degeneration (medical)
Endomyocardial fibrosis
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Background — Excessive myocardial fibrosis impairs cardiac function in hypertensive hearts. Roles of transforming growth factor (TGF)-β in myocardial remodeling and cardiac dysfunction were examined in pressure-overloaded rats. Methods and Results — Pressure overload was induced by a suprarenal aortic constriction in Wistar rats. Fibroblast activation (proliferation and phenotype transition to myofibroblasts) was observed after day 3 and peaked at days 3 to 7. Thereafter, myocyte hypertrophy and myocardial fibrosis developed by day 28. At day 28, echocardiography showed normal left ventricular fractional shortening, but the decreased ratio of early to late filling velocity of the transmitral Doppler velocity and hemodynamic measurement revealed left ventricular end-diastolic pressure elevation, indicating normal systolic but abnormal diastolic function. Myocardial TGF-β mRNA expression was induced after day 3, peaked at day 7, and remained modestly increased at day 28. An anti–TGF-β neutralizing antibody, which was administered intraperitoneally daily from 1 day before operation, inhibited fibroblast activation and subsequently prevented collagen mRNA induction and myocardial fibrosis, but not myocyte hypertrophy. Neutralizing antibody reversed diastolic dysfunction without affecting blood pressure and systolic function. Conclusions — TGF-β plays a causal role in myocardial fibrosis and diastolic dysfunction through fibroblast activation in pressure-overloaded hearts. Our findings may provide an insight into a new therapeutic strategy to prevent myocardial fibrosis and diastolic dysfunction in pressure-overloaded hearts.
Pressure overload
Myocardial fibrosis
Cardiac Fibrosis
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Abstract Background: Endothelial-to-mesenchymal transition (Endo-MT) is associated with myocardial fibrosis in dilated cardiomyopathy (DCM). Endothelial-to-mesenchymal transition (Endo-MT) is induced by coxsackievirus B3 (CVB3) in cardiac microvascular endothelial cells (CMVECs). Bone morphogenetic protein 7 (BMP7) significantly inhibits Endo-MT and the progression of cardiac fibrosis. The study was aimed to investigate the effect and the underlying mechanism of BMP7 on Endo-MT in myocardial fibrosis induce by CVB3 infection in vivo. Methods: BALB/c mice were intraperitoneally injected by CVB3 to induce viral myocarditis (VMC). Mice were treated with BMP7 after CVB3 infection. Subsequently, all groups of mice were determined by echocardiography, histopathologic and molecular detection. Results: We found that the ratio of BMP7/TGF-β1 in mRNA levels was decreased obviously at different time points after CVB3 injection. BMP7 facilitated the recovery of cardiac function after CVB3 infection via inhibition of myocardial damage, collagen deposition. Double immunofluorescence staining indicated that Endo-MT was implicated in CVB3-induced myocardial fibrosis, which was attenuated by BMP7. The protein levels of pSmad3 and Smad4 were significantly upregulated in VMC group, as well as Wnt/β-catenin and the transcription factor snail. BMP7 treatment reversed the changes of these protein levels. Moreover, CO-IP demonstrated the crosstalk between β-catenin and Smad3 in VMC mice, which was downregulated by BMP7 treatment. Conclusions: These results indicated that BMP7 obviously ameliorated myocardial fibrosis in CVB3-infected mice via Endo-MT, which was involved in the TGF-β/Smad and Wnt/β-catenin pathway. β-Catenin/Smad3 interaction may be associated with Endo-MT in the development of viral myocardial fibrosis.
Myocardial fibrosis
Cardiac Fibrosis
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Background— Excessive myocardial fibrosis impairs cardiac function in hypertensive hearts. Roles of transforming growth factor (TGF)-β in myocardial remodeling and cardiac dysfunction were examined in pressure-overloaded rats. Methods and Results— Pressure overload was induced by a suprarenal aortic constriction in Wistar rats. Fibroblast activation (proliferation and phenotype transition to myofibroblasts) was observed after day 3 and peaked at days 3 to 7. Thereafter, myocyte hypertrophy and myocardial fibrosis developed by day 28. At day 28, echocardiography showed normal left ventricular fractional shortening, but the decreased ratio of early to late filling velocity of the transmitral Doppler velocity and hemodynamic measurement revealed left ventricular end-diastolic pressure elevation, indicating normal systolic but abnormal diastolic function. Myocardial TGF-β mRNA expression was induced after day 3, peaked at day 7, and remained modestly increased at day 28. An anti–TGF-β neutralizing antibody, w...
Pressure overload
Myocardial fibrosis
Cardiac Fibrosis
Myofibroblast
Constriction
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Objectives
MicroRNAs, as one of key regulators for post-transcriptional gene control, have shown to be involved in cardiovascular diseases. This study aimed to investigate the roles of miR-378 in regulation of cardiac hypertrophy and cardiac fibrosis stimulated by pressure overload.Methods
To construct cardiac remodelling mice models, pressure overload was imposed on the heart of C57B/L6 mice (8 weeks) by constriction of transverse aortic banding (TAC). Chemically modified oligonucleotides miRNA mimics (miR-378-Agomir) and inhibitors (miR-378-Antagomir) treatment started 24 h after TAC by intravenous injections on two consecutive days. Mice were randomly divided into four groups: 1) the TAC group; 2) the TAC-Agomir group; 3) the TAC- Antagomir group; 4) the Sham operation group. Mice after sham or 2 weeks TAC operation were subjected to hemodynamic and echocardiographic measurement. Tissue and serum miR-378 expressions were detected by TaqMan MicroRNA Assay Kit. ANP, BNP, β-MHC, collagen I and collagen III was measured by Real-time PCR. ERK1/2, p-ERK1/2, MMPs and α-SMA expression were analysed by western blotting. Collagen was assessed by immunohistochemistry.Results
We found that miR-378 has a high expression pattern in the heart. After two-week acute pressure overload, the TAC group exhibit prominent cardiac hypertrophic responses and decreased expression of miR-378(p < 0.05) comparing with the sham group. In vivo overexpression of miR-378 in TAC-Agomir group markerly inhibited cardiac hypertrophy, accompanied with downregulation of fetal gene ANP, BNP and β-MHC compared to that of TAC group. Howerver, inhibition of endogenous miR-378 by an infusion of an antagomir caused sustained cardiac hypertrophy, associated with a reinduction of fetal gene expression to a less extent. The p-ERK level was also downregulated in TAC-Agomir group and upregulated in TAC- Antagomir group. In part of cardiac fibrosis, we found that the over-expression of miR-378 of TAC-Agomir group reduced the expression of col I, col III, MMP2, MMP9 and α-SMA(p < 0.05) compared to that of TAC group, whereas down regulation of endogenous miR-378 with anti-miRs promoted collagens and MMPs expression.Conclusions
These findings reveal orchestrating regulatory roles for miR-378 in pressure-overload induced cardiac remodeling. It suggests that miR-378 not only suppresses cardiac hypertrophy by regulating fetal genes expression and ERK1/2 signaling pathway but also controls fibrosis-related genes expression in the heart.Pressure overload
Antagomir
Atrial natriuretic peptide
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Background: Although few microRNAs (miRNAs) have been involved in the regulation of post-ischemic cardiac fibrosis, the exact effect and underlying mechanism of miRNAs in cardiac fibrosis remains unclear. Here, we sought to investigate whether microRNA-34 (miR-34) plays a role in the pathogenic development of myocardial fibrosis.Methods: The myocardial infarction (MI) mice model was induced and cardiac fibroblasts were cultured. Histological analyses, quantitative real-time polymerase chain reaction and Western blotting analysis were used.Results: We found that the miR-34 cluster, especially miR-34a, was upregulated in the MI heart. In vivo, inhibition of miR-34a reduces the severity of experimental cardiac fibrosis in mice. TGF-β1 increased miR-34a expression in cardiac fibroblasts. Overexpressing miR-34a levels increased the profibrogenic activity of TGF-β1 in cardiac fibroblast, whereas inhibition miR-34a levels weakened the activity. Finally, we showed that miR-34a's underlying mechanism during cardiac fibrosis occurs through the targeting of Smad4 expression.Conclusions: Our findings provide evidence that miR-34a plays a critical role in the progression of cardiac tissue fibrosis by directly targeting Smad4, which suggests that miR-34a may be new marker for cardiac fibrosis progression and that inhibition of miR-34a may be a promising strategy in the treatment of cardiac fibrosis.
Cardiac Fibrosis
Myocardial fibrosis
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Cardiac fibrosis stiffens the ventricular wall, predisposes to cardiac arrhythmias and contributes to the development of heart failure. In the present study, our aim was to identify novel miRNAs that regulate the development of cardiac fibrosis and could serve as potential therapeutic targets for myocardial fibrosis.Analysis for cardiac samples from sudden cardiac death victims with extensive myocardial fibrosis as the primary cause of death identified dysregulation of miR-185-5p. Analysis of resident cardiac cells from mice subjected to experimental cardiac fibrosis model showed induction of miR-185-5p expression specifically in cardiac fibroblasts. In vitro, augmenting miR-185-5p induced collagen production and profibrotic activation in cardiac fibroblasts, whereas inhibition of miR-185-5p attenuated collagen production. In vivo, targeting miR-185-5p in mice abolished pressure overload induced cardiac interstitial fibrosis. Mechanistically, miR-185-5p targets apelin receptor and inhibits the anti-fibrotic effects of apelin. Finally, analysis of left ventricular tissue from patients with severe cardiomyopathy showed an increase in miR-185-5p expression together with pro-fibrotic TGF-β1 and collagen I.Our data show that miR-185-5p targets apelin receptor and promotes myocardial fibrosis.
Cardiac Fibrosis
Myocardial fibrosis
Apelin
Pressure overload
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Pressure overload
Myocardial fibrosis
Ventricular pressure
Ventricular remodeling
Cardiac Fibrosis
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Abstract Cardiac fibrosis critically injured the cardiac structure and function of the hypertensive patients. However, the anti‐fibrotic strategy is still far from satisfaction. This study aims to determine the effect and mechanism of Pirfenidone (PFD), an anti‐lung fibrosis medicine, in the treatment of cardiac fibrosis and heart failure induced by pressure overload. Male C57BL/6 mice were subjected to thoracic aorta constriction (TAC) or sham surgery with the vehicle, PFD (300 mg/kg/day) or Captopril (CAP, 20 mg/kg/day). After 8 weeks of surgery, mice were tested by echocardiography, and then sacrificed followed by morphological and molecular biological analysis. Compared to the sham mice, TAC mice showed a remarkable cardiac hypertrophy, interstitial and perivascular fibrosis and resultant heart failure, which were reversed by PFD and CAP significantly. The enhanced cardiac expression of TGF‐β1 and phosphorylation of Smad3 in TAC mice were both restrained by PFD. Cardiac fibroblasts isolated from adult C57BL/6 mice were treated by Angiotensin II, which led to significant increases in cellular proliferation and levels of α‐SMA, vimentin, TGF‐β1 and phosphorylated TGF‐β receptor and Smad3. These changes were markedly inhibited by pre‐treatment of PFD. Collectively, PFD attenuates myocardial fibrosis and dysfunction induced by pressure overload via inhibiting the activation of TGF‐β1/Smad3 signalling pathway.
Pressure overload
Cardiac Fibrosis
Pirfenidone
Myocardial fibrosis
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