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Phenotype switching of cardiac fibroblasts into myofibroblasts plays important role in cardiac fibrosis following myocardial infarction (MI). Cellular repressor of E1A-stimulated genes (CREG) protects against vascular and cardiac remodeling induced by angiotensin-II. However, the effects and mechanisms of CREG on phenotype switching of cardiac fibroblasts after MI are unknown. This study aimed to investigate the role of CREG on the phenotype switching of cardiac fibroblasts following MI and its mechanism. Our findings demonstrated that, compared with littermate control mice, cardiac function was deteriorated in CREG+/− mice on day 14 post-MI. Fibrosis size, αSMA, and collagen-1 expressions were increased in the border regions of CREG+/− mice on day 14 post-MI. Conversely, exogenous CREG protein significantly improved cardiac function, inhibited fibrosis, and reduced the expressions of αSMA and collagen-1 in the border regions of C57BL/6J mice on day 14. In vitro, CREG recombinant protein inhibited αSMA and collagen-1 expression and blocked the hypoxia-induced proliferation and migration of cardiac fibroblasts, which was mediated through the inhibition of cell division control protein 42 (CDC42) expression. Our findings could help in establishing new strategies based on the clarification of the role of the key molecule CREG in phenotype switching of cardiac fibroblasts following MI.

Cardiac fibrosis after myocardial infarction (MI) is mainly associated with cardiac fibroblasts and its differentiation is the key pathological process. However, the cellular mechanism of fibroblast-to-myofibroblast conversion has not been clarified and a deeper mechanistic understanding is needed. We found that miR-574-5p was up-regulated in TGF-β-induced myofibroblast differentiation. Silencing transiently miR-574-5p in HCFs, we found that suppression of miR-574-5p decreased myofibroblasts differentiation as validated by expression levels of fibrosis related genes, EDU imaging assay, wound healing assay and transwell assays. Conversely, overexpression of miR-574-5p displayed opposite results. ARID3A was verified as a direct target gene of miR-574-5p and decreased level of ARID3A forced fibroblast-to-myofibroblast differentiation of TGF-β-induced HCFs. Our data suggests that miR-574-5p plays a pivotal role in human cardiac fibroblasts (HCFs) myofibroblast differentiation and demonstrates that miR-574-5p and arid3a may be a novel therapeutic target for cardiac fibrosis.

Cardiac fibrosis is a key factor to determine the prognosis in patient with myocardial infarction (MI). The aim of this study is to investigate whether the transcriptional factor paired‐related homeobox 2 (Prrx2) regulates Wnt5a gene expression and the role in myocardial fibrosis following MI. The MI surgery was performed by ligation of left anterior descending coronary artery. Cardiac remodelling was assessed by measuring interstitial fibrosis performed with Masson staining. Cell differentiation was examined by analysis the expression of alpha‐smooth muscle actin (α‐SMA). Both Prrx2 and Wnt5a gene expressions were up‐regulated in mice following MI, accompanied with increased mRNA and protein levels of α‐SMA, collagen I and collagen III, compared to mice with sham surgery. Adenovirus‐mediated gene knock down of Prrx2 increased survival rate, alleviated cardiac fibrosis, decreased infarction sizes and improved cardiac functions in mice with MI. Importantly, inhibition of Prrx2 suppressed ischaemia‐induced Wnt5a gene expression and Wnt5a signalling. In cultured cardiac fibroblasts, TGF‐β increased gene expressions of Prrx2 and Wnt5a, and induced cell differentiations, which were abolished by gene silence of either Prrx2 or Wnt5a. Further, overexpression of Prrx2 or Wnt5a mirrored the effects of TGF‐β on cell differentiations of cardiac fibroblasts. Gene silence of Wnt5a also ablated cell differentiations induced by Prrx2 overexpression in cardiac fibroblasts. Mechanically, Prrx2 was able to bind with Wnt5a gene promoter to up‐regulate Wnt5a gene expression. In conclusions, targeting Prrx2‐Wnt5a signalling should be considered to improve cardiac remodelling in patients with ischaemic heart diseases.

Trimethylamine N-oxide (TMAO), a gut microbe-derived metabolite of dietary choline and other trimethylamine-containing nutrients, has been associated with poor prognosis in coronary heart disease. However, the role and underlying mechanisms of TMAO in the cardiac fibrosis after myocardial infarction (MI) remains unclear.We used mouse MI models and primary cardiac fibroblasts cultures to study the role of TMAO in the heart and in cardiac fibroblasts. C57BL/6 mice were fed a control diet, high choline (1.2%) or/and DMB diet or a diet containing TMAO (0.12%) starting 3 weeks before MI. DMB, a structural analogue of choline, inhibited microbial TMA lyases and reduced the level of TMAO in mice. Cardiac function was measured 7 days after MI using echocardiography. One week post MI, myocardial tissues were collected to evaluate cardiac fibrosis, and blood samples were evaluated for TMAO levels. The expression of TGF-β receptor, P-Smad2, α-SMA or collagen I in myocardial tissues and fibroblasts were analyzed by western blot or immunocytochemistry.We demonstrated that cardiac function and cardiac fibrosis were significantly deteriorated in mice fed either TMAO or high choline diets compared with the control diet, and DMB reversed the cardiac function damage of high choline diet (p .05). Cardiomyocyte necrosis, apoptosis and macrophage infiltration after MI was significantly increased after treatment with TMAO or high choline diets. The size and migration of fibroblasts were increased after TMAO treatment compared with non-treated fibroblasts in vitro. Furthermore, TMAO increased TGF-β receptor I expression, which promoted the phosphorylation of Smad2 and up-regulated the expression of α-SMA and collagen I. The ubiquitination of TGF-βRI was decreased in neonatal mouse fibroblasts after TMAO treatment. TMAO also inhibited the expression of smurf2. Inhibition of TGF-β1 receptor with the small molecule inhibitor SB431542 decreased TGF-β receptor I expression, reduced the phosphorylation of Smad2, down-regulated TMAO-induced α-SMA and collagen I expression in cardiac fibroblasts.Cardiac function and cardiac fibrosis were significantly exacerbated in mice fed diets supplemented with either choline or TMAO, probably through accelerating the transformation of fibroblasts into myofibroblasts, indicating activation of TGF-βRI/Smad2 pathway.

We have previously shown that overexpression of SKI, an endogenous TGF-β1 repressor, deactivates the pro-fibrotic myofibroblast phenotype in the heart. We now show that SKI also functions independently of SMAD/TGF-β signaling, by activating the Hippo tumor-suppressor pathway and inhibiting the Transcriptional co-Activator with PDZ-binding motif (TAZ or WWTR1). The mechanism(s) by which SKI targets TAZ to inhibit cardiac fibroblast activation and fibrogenesis remain undefined. A rat model of post-myocardial infarction was used to examine the expression of TAZ during acute fibrogenesis and chronic heart failure. Results were then corroborated with primary rat cardiac fibroblast cell culture performed both on plastic and on inert elastic substrates, along with the use of siRNA and adenoviral expression vectors for active forms of SKI, YAP, and TAZ. Gene expression was examined by qPCR and luciferase assays, while protein expression was examined by immunoblotting and fluorescence microscopy. Cell phenotype was further assessed by functional assays. Finally, to elucidate SKI’s effects on Hippo signaling, the SKI and TAZ interactomes were captured in human cardiac fibroblasts using BioID2 and mass spectrometry. Potential interactors were investigated in vitro to reveal novel mechanisms of action for SKI. In vitro assays on elastic substrates revealed the ability of TAZ to overcome environmental stimuli and induce the activation of hypersynthetic cardiac myofibroblasts. Further cell-based assays demonstrated that SKI causes specific proteasomal degradation of TAZ, but not YAP, and shifts actin cytoskeleton dynamics to inhibit myofibroblast activation. These findings were supported by identifying the bi-phasic expression of TAZ in vivo during post-MI remodeling and fibrosis. BioID2-based interactomics in human cardiac fibroblasts suggest that SKI interacts with actin-modifying proteins and with LIM Domain-containing protein 1 (LIMD1), a negative regulator of Hippo signaling. Furthermore, we found that LATS2 interacts with TAZ, whereas LATS1 does not, and that LATS2 knockdown prevented TAZ downregulation with SKI overexpression. Our findings indicate that SKI’s capacity to regulate cardiac fibroblast activation is mediated, in part, by Hippo signaling. We postulate that the interaction between SKI and TAZ in cardiac fibroblasts is arbitrated by LIMD1, an important intermediary in focal adhesion-associated signaling pathways. This study contributes to the understanding of the unique physiology of cardiac fibroblasts, and of the relationship between SKI expression and cell phenotype. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00865-9.

Cardiac fibrosis is a significant global health problem that is closely associated with multiple forms of cardiovascular disease, including myocardial infarction, dilated cardiomyopathy, and diabetes. Fibrosis increases myocardial wall stiffness due to excessive extracellular matrix deposition, causing impaired systolic and diastolic function, and facilitating arrhythmogenesis. As a result, patient morbidity and mortality are often dramatically elevated compared with those with cardiovascular disease but without overt fibrosis, demonstrating that fibrosis itself is both a pathologic response to existing disease and a significant risk factor for exacerbation of the underlying condition. The lack of any specific treatment for cardiac fibrosis in patients suffering from cardiovascular disease is a critical gap in our ability to care for these individuals. Here we provide an overview of the development of cardiac fibrosis, and discuss new research directions that have recently emerged and that may lead to the creation of novel treatments for patients with cardiovascular diseases. Such treatments would, ideally, complement existing therapy by specifically focusing on amelioration of fibrosis.

Background/Aims: Cardiac fibrosis is the primary cause of deteriorated cardiac function in various cardiovascular diseases. Numerous studies have demonstrated that microRNAs (miRNAs) are critical regulators of myocardial fibrosis. Specifically, many studies have reported that miR-150 is downregulated in cardiovascular diseases, such as acute myocardial infarction (AMI), myocardial hypertrophy and myocardial fibrosis. However, the exact role of miR-150 in these pathological processes remains unknown. Methods: We used the transverse aortic constriction (TAC) mouse model to study the role of miR-150 in cardiac fibrosis induced by pressure overload. After the TAC operation, qRT-PCR was used to measure the expression profiles of miR-150 in left ventricle tissues and populations of primary heart cell types. Then, we used both miR-150 knockout mice and wild type (WT) mice in the TAC model. Changes in cardiac function and pathology were measured using transthoracic echocardiography and pathological analysis, respectively. Furthermore, we predicted the target of miR-150 in cardiac fibroblasts (CFs) and completed in vitro CF transfection experiments using miR-150 analogs and siRNA corresponding to the predicted target. Results: We observed decreased expression levels of miR-150 in hearts suffering pressure overload, and these levels decreased more sharply in CFs than in cardiomyocytes. In addition, the degrees of cardiac function deterioration and cardiac fibrosis in miR-150-/- mice were more severe than were those in WT mice. By transfecting CFs with an miR-150 analog in vitro, we observed that miR-150 inhibited cardiac fibroblast activation. We predicted that the transcription factor c-Myb was the target of miR-150 in CFs. Transfecting CFs with c-Myb siRNA eliminated the effects of an miR-150 inhibitor, which promoted CF activation. Conclusion: These findings reveal that miR-150 acts as a pivotal regulator of pressure overload-induced cardiac fibrosis by regulating c-Myb.

Notch3 and TGF-β1 signaling play a key role in the pathogenesis and progression of chronic cardiovascular disease. However, whether Notch3 protects against myocardial infarction (MI) and the underlying mechanisms remains unknown. C57BL/6 mice were randomized to be treated with Notch3 siRNA (siNotch3) or lentivirus carrying Notch3 cDNA (Notch3) before coronary artery ligation. Four weeks after constructing MI model, cardiac function and fibrosis were compared between groups. The cardiac fibroblast cells (CFs) were isolated from newborn C57BL/6 mice (1-3 days old) and transfected with lentivirus carrying Notch3 cDNA. TGF-β1 (5 ng/ml), a well-known pro-fibrotic factor, was administered 72 h after Notch3 cDNA administration in CFs. The related proteins of fibrosis such as a-smooth muscle actin (a-SMA), Type I collagen, metalloprotease (MMP)-9 and the tissue inhibitor of metalloproteinases (TIMP)-2 were examined by western blot analysis. Notch3 cDNA treatment attenuated cardiac damage and inhibited fibrosis in mice with MI. Meanwhile, Notch3 siRNA administration aggravated cardiac function damage and markedly enhanced cardiac fibrosis in mice with MI. Overexpression of Notch3 inhibited TGF-β1-induced fibroblast-myofibroblast transition of mouse cardiac fibroblast cells, as evidenced by down-regulating a-SMA and Type I collagen expression. Notch3 cDNA treatment also increased MMP-9 expression and decreased TIMP-2 expression in the TGF-β1-stimulated cells. This study indicates that Notch3 is an important protective factor for cardiac fibrosis in a MI model, and the protective effect of Notch3 is attributable to its action on TGF-β1/Smad3 signaling.

Cardiac fibroblasts are essential for beneficial myocardial healing but also cause detrimental adverse remodeling following myocardial infarction. The mechanical properties of the infarcted myocardium and border regions display temporal and spatial characteristics that regulate different aspects of the profibrotic cardiac fibroblast phenotypes., Cardiac fibrosis is a serious condition currently lacking effective treatments. It occurs as a result of cardiac fibroblast (CFB) activation and differentiation into myofibroblasts, characterized by proliferation, extracellular matrix (ECM) production and stiffening, and contraction due to the expression of smooth muscle α-actin. The mechanical properties of myocardium change regionally and over time after myocardial infarction (MI). Although mechanical cues are known to activate CFBs, it is unclear which specific mechanical stimuli regulate which specific phenotypic trait; thus we investigated these relationships using three in vitro models of CFB mechanical activation and found that 1) paracrine signaling from stretched cardiomyocytes induces CFB proliferation under mechanical conditions similar to those of the infarct border region; 2) direct stretch of CFBs mimicking the mechanical environment of the infarct region induces a synthetic phenotype with elevated ECM production; and 3) progressive matrix stiffening, modeling the mechanical effects of infarct scar maturation, causes smooth muscle α-actin fiber formation, up-regulation of collagen I, and down-regulation of collagen III. These findings suggest that myocyte stretch, fibroblast stretch, and matrix stiffening following MI may separately regulate different profibrotic traits of activated CFBs.

Hypoxic injury to the heart results in cardiac fibrosis that leads to cardiac dysfunction and heart failure. SNAIL1 is a zinc finger transcription factor implicated in fibrosis following organ injury and cancer. To determine if the action of SNAIL1 contributed to cardiac fibrosis following hypoxic injury, we used an endogenous SNAIL1 bioluminescence reporter mice, and SNAIL1 knockout mouse models. Here we report that SNAIL1 expression is upregulated in the infarcted heart, especially in the myofibroblasts. Utilizing primary cardiac fibroblasts in ex vivo cultures we find that pro-fibrotic factors and collagen I increase SNAIL1 protein level. SNAIL1 is required in cardiac fibroblasts for the adoption of myofibroblast fate, collagen I expression and expression of fibrosis-related genes. Taken together this data suggests that SNAIL1 expression is induced in the cardiac fibroblasts after hypoxic injury and contributes to myofibroblast phenotype and a fibrotic scar formation. Resultant collagen deposition in the scar can maintain elevated SNAIL1 expression in the myofibroblasts and help propagate fibrosis.