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Long-chain noncoding RNA (lncRNA) is a new class of molecular regulators in heart development and disease. However, the role of specific lncRNA in cardiac fibrosis remains to be fully explored. This study aimed to investigate the role and potential mechanism of lncRNA MHRT in myocardial fibrosis after myocardial infarction (MI).Cardiac fibroblasts (CFs) were isolated from a mouse model of MI. The expression levels of MHRT and miR-3185 in the hearts of MI and CFs mice treated with transforming growth factor beta 1 (TGF-β1) were analyzed by qRT-PCR. The collagen expression was assessed using qRT-PCR and Western blot. Cell proliferation was assessed by performing MTT and EdU assays. The direct interaction between lncRNA and miRNA was analyzed by luciferase assay, RNA-binding protein immunoprecipitation (RIP) assay, and RNA pull-down assay.The expression levels of MHRT were raised in MI and CFs mice treated with TGF-β1. Overexpression of MHRT promoted collagen production and CF proliferation, while silencing of MHRT showed the opposite effect. MiR-3185 was a target gene of MHRT. In addition, overexpression of MHRT reduced the expression levels of miR-3185, and siMHRT reversed the inhibitory effect of TGF-β1 on the expression of miR-3185. Overexpression of miR-3185 inhibited the upregulation of Col I and Col III induced by TGF-β1.MHRT promoted cardiac fibrosis after MI through miR-3185 and increased myocardial collagen deposition and promoted myocardial fibrosis.

Background Myocardial fibrosis is caused by the adverse and powerful remodeling of the heart secondary to the death of cardiomyocytes after myocardial infarction. Regulators of G protein Signaling (RGS) 4 is involved in cardiac diseases through regulating G protein-coupled receptors (GPCRs). Methods Cardiac fibrosis models were established through cardiac fibroblasts (CFs) treatment with transforming growth factor (TGF)-β1 in vitro and mice subjected to myocardial infarction in vivo. The mRNA expression of RGS4, collagen I/III and α-SMA detected by qRT-PCR. Protein level of RGS4, collagen I, CTGF and α-SMA detected by Western blot. The ejection fraction (EF%) and fractional shortening (FS%) of mice were measured by echocardiography. Collagen deposition of mice was tested by Masson staining. Results The expression of RGS4 increased in CFs treatment with TGF-β1 and in MI mice. The model of cardiac fibrosis detected by qRT-PCR and Western blot. It was demonstrated that inhibition of RGS4 expression improved cardiac fibrosis by transfection with small interfering RNA in CFs and injection with lentivirus shRNA in mice. The protective effect of choline against cardiac fibrosis was counteracted by overexpression of RGS4 in vitro and in vivo. Moreover, choline inhibited the protein level of TGF-β1, p-Smad2/3, p-p38 and p-ERK1/2 in CFs treated with TGF-β1, which were restored by RGS4 overexpression. Conclusion This study demonstrated that RGS4 promoted cardiac fibrosis and attenuated the anti-cardiac fibrosis of choline. RGS4 may weaken anti-cardiac fibrosis of choline through TGF-β1/Smad and MAPK signaling pathways.

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.

Increased matrix metalloprotease 9 (MMP9) after myocardial infarction (MI) exacerbates ischemia-induced chronic heart failure (CHF). Autophagy is cardioprotective during CHF; however, whether increased MMP9 suppresses autophagic activity in CHF is unknown. This study aimed to determine whether increased MMP9 suppressed autophagic flux and MMP9 inhibition increased autophagic flux in the heart of rats with post-MI CHF. Sprague-Dawley rats underwent either sham surgery or coronary artery ligation 6–8 wk before being treated with MMP9 inhibitor for 7 days, followed by cardiac autophagic flux measurement with lysosomal inhibitor bafilomycin A1. Furthermore, autophagic flux was measured in vitro by treating H9c2 cardiomyocytes with two independent pharmacological MMP9 inhibitors, salvianolic acid B (SalB) and MMP9 inhibitor-I, and CRISPR/cas9-mediated MMP9 genetic ablation. CHF rats showed cardiac infarct, significantly increased left ventricular end-diastolic pressure (LVEDP), and increased MMP9 activity and fibrosis in the peri-infarct areas of left ventricular myocardium. Measurement of the autophagic markers LC3B-II and p62 with lysosomal inhibition showed decreased autophagic flux in the peri-infarct myocardium. Treatment with SalB for 7 days in CHF rats decreased MMP9 activity and cardiac fibrosis but increased autophagic flux in the peri-infarct myocardium. As an in vitro corollary study, measurement of autophagic flux in H9c2 cardiomyocytes and fibroblasts showed that pharmacological inhibition or genetic ablation of MMP9 upregulates autophagic flux. These data are consistent with our observations that MMP9 inhibition upregulates autophagic flux in the heart of rats with CHF. In conclusion, the results in this study suggest that the beneficial outcome of MMP9 inhibition in pathological cardiac remodeling is in part mediated by improved autophagic flux. NEW & NOTEWORTHY This study elucidates that the improved cardiac extracellular matrix (ECM) remodeling and cardioprotective effect of matrix metalloprotease 9 (MMP9) inhibition in chronic heart failure (CHF) are via increased autophagic flux. Autophagy is cardioprotective; however, the mechanism of autophagy suppression in CHF is unknown. We for the first time demonstrated here that increased MMP9 suppressed cardiac autophagy and ablation of MMP9 increased cardiac autophagic flux in CHF rats. Restoring the physiological level of autophagy in the failing heart is a challenge, and our study addressed this challenge. The novelty and highlights of this report are as follows: 1) MMP9 regulates cardiomyocyte and fibroblast autophagy, 2) MMP9 inhibition protects CHF after myocardial infarction (MI) via increased cardiac autophagic flux, 3) MMP9 inhibition increased cardiac autophagy via activation of AMP-activated protein kinase (AMPK)α, Beclin-1, Atg7 pathway and suppressed mechanistic target of rapamycin (mTOR) pathway.

Although increased predisposition to cardiac fibrosis and cardiac dysfunction has been demonstrated in the perinatally nicotine-exposed heart, the underlying mechanisms remain unclear. With the use of a well-established rat model and cultured primary neonatal rat cardiac fibroblasts, the effect of perinatal nicotine exposure on offspring heart extracellular matrix deposition and the likely underlying mechanisms were investigated. Perinatal nicotine exposure resulted in increased collagen type I (COL1A1) and III (COL3A1) deposition along with a decrease in miR-29 family and an increase in long noncoding RNA myocardial infarction-associated transcript (MIAT) levels in offspring heart. Nicotine treatment of isolated primary neonatal rat cardiac fibroblasts suggested that these effects were mediated via nicotinic acetylcholine receptors including α7 and the induced collagens accumulation was reversed by a gain-of function of miR-29 family. Knockdown of MIAT resulted in increased miR-29 family and decreased COL1A1 and COL3A1 levels, suggesting nicotine-mediated MIAT induction as the underlying mechanism for nicotine-induced collagen deposition. Luciferase reporter assay and RNA immunoprecipitation studies showed an intense physical interaction between MIAT, miR-29 family, and argonaute 2, corroborating the mechanistic link between perinatal nicotine exposure and increased extracellular matrix deposition. Overall, perinatal nicotine exposure resulted in lower miR-29 family levels in offspring heart, while it elevated cardiac MIAT and collagen type I and III levels. These findings provide mechanistic basis for cardiac dysfunction in perinatal nicotine-exposed offspring and offer multiple novel potential therapeutic targets. NEW & NOTEWORTHY Using an established rat model and cultured primary neonatal cardiac fibroblasts, we show that nicotine mediated MIAT induction as the underlying mechanism for the excessive cardiac collagen deposition. These observations provide mechanistic basis for the increased predisposition to cardiac dysfunction following perinatal cigarette/nicotine exposure and offer novel potential therapeutic targets.

Inflammation contributes to the development of heart failure (HF) through multiple mechanisms including regulating extracellular matrix (ECM) degradation and deposition. Interactions between cells in the myocardium orchestrates the magnitude and duration of inflammatory cell recruitment and ECM remodeling events associated with HF. More recently, studies have shown T-cells have signficant roles in post-MI wound healing. T-cell biology in HF illustrates the complexity of cross-talk between inflammatory cell types and resident fibroblasts. This review will focus on T-cell recruitment to the myocardium and T-cell specific factors that might influence cardiac wound healing and fibrosis in the heart with consideration of age and sex as important factors in T-cell activity.

Background Macrophages play an important role in the development of cardiac fibrosis. However, the roles of different macrophage subtypes in cardiac fibroblast (CF) activation and cardiac fibrosis are unknown.Methods and Results:Bone marrow-derived macrophages (BMDMs) were treated with different stimuli to induce differentiation into M1, M2a, M2b, and M2c macrophage subtypes. CFs were co-cultured with different subtypes of macrophages or cultured with macrophage supernatants. Results revealed that M2b macrophages significantly suppressed the proliferation and migration of CFs, the expression of fibrosis-related proteins (collagen I [COL-1] and α-smooth muscle actin [α-SMA]), and differentiation into cardiac myofibroblasts (MFs). The opposite effects were observed with M2a macrophages. A rat model of cardiac ischemia/reperfusion (I/R) injury was used to determine the effect of M2b macrophages transplantation. After cardiac I/R injury, transplantation of M2b macrophages improved cardiac function and reduced cardiac fibrosis. The effect of macrophage subtypes on p-ERK, ERK, p-p38, and p38 phosphorylation was examined by Western blotting. The results showed that M2b macrophages significantly inhibited the mitogen-activated protein kinase (MAPK) signaling pathway. Conclusions These study results demonstrate for the first time that different subtypes of macrophages have different roles in regulating CF activation. M2b macrophages inhibit CF activation, and thus can be considered anti-fibrotic macrophages. M2a macrophages promote CF activation, and thus are pro-fibrotic macrophages.

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.

Exercise training has been reported to alleviate cardiac fibrosis and ameliorate heart dysfunction after myocardial infarction (MI), but the molecular mechanism is still not fully clarified. Fibroblast growth factor 21 (FGF21) exerts a protective effect on the infarcted heart. This study investigates whether exercise training could increase FGF21 protein expression and regulate the transforming growth factor-β1 (TGF-β1)-Smad2/3-MMP2/9 signaling pathway to alleviate cardiac fibrosis following MI. Male wild type (WT) C57BL/6J mice and Fgf21 knockout (Fgf21 KO) mice were used to establish the MI model and subjected to five weeks of different types of exercise training. Both aerobic exercise training (AET) and resistance exercise training (RET) significantly alleviated cardiac dysfunction and fibrosis, up-regulated FGF21 protein expression, inhibited the activation of TGF-β1-Smad2/3-MMP2/9 signaling pathway and collagen production, and meanwhile, enhanced antioxidant capacity and reduced cell apoptosis in the infarcted heart. In contrast, knockout of Fgf21 weakened the cardioprotective effects of AET after MI. In vitro, cardiac fibroblasts (CFs) were isolated from neonatal mice hearts and treated with H2O2 (100 μM, 6 h). Recombinant human FGF21 (rhFGF21, 100 ng/mL, 15 h) and/or 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM, 15 h) inhibited H2O2-induced activation of the TGF-β1-Smad2/3-MMP2/9 signaling pathway, promoted CFs apoptosis and reduced collagen production. In conclusion, exercise training increases FGF21 protein expression, inactivates the TGF-β1-Smad2/3-MMP2/9 signaling pathway, alleviates cardiac fibrosis, oxidative stress, and cell apoptosis, and finally improves cardiac function in mice with MI. FGF21 plays an important role in the anti-fibrosis effect of exercise training.

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast-specific Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in their contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

Cardiac fibrosis is involved in myocardial remodeling following acute myocardial infarction (AMI), which can result in heart failure, arrhythmias and even sudden cardiac death. Investigating the molecular mechanisms of cardiac fibrosis in acute myocardial infarction (AMI) is essential for better understanding this pathology. The current study aims to investigate the effect of TUG1 on cardiac fibrosis after AMI and elucidated the underlying molecular mechanism of AMI. Rats were randomly divided into four groups (sham-operation group, myocardial infarction group (AMI group), si-NC treated group and si-TUG1 treated group). The biological behavior of cardiac fibroblasts treated with TGF-β1after being transfected by si-TUG1 or miR-590 mimic or miR-590 inhibitor or FGF1 mimic or a combination was evaluated using the cell counting kit-8 (CCK8) and Transwell assays. SatarBase v2.0 was used to predict the target microRNAs binding site candidates with TUG1 and FGF1. Western blot and recovery experiments were used to explore the potential mechanism. TUG1 expression was up-regulated and knockdown of TUG1 improved cardiac function in AMI rats. Knockdown of TUG1 suppressed cell viability and migration and improved collagen production of TGF-β1 treated cardiac fibroblasts. SatarBase v2.0 showed TUG1 served as a sponge for miR-590 and FGF1 is a direct target of miR-590. TUG1 expression was increased in AMI tissue and cardiac fibroblasts treated with TGF-β1. TUG1 knockdown suppressed the biological process of cardiac fibroblasts treated with TGF-β1 by sponging miR-590.

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.

Myocardial infarction (MI) is one of the most catastrophic diseases threatening human health in the world. Because cardiomyocytes have a minuscule regenerative potential, the natural repair of infarct healing after MI shows fibrotic scar. MicroRNA-143-3p (miR-143-3p) plays a critical regulatory role in various pathophysiological processes in the heart. Sprouty3 (SPRY3) is predicted to be a potential fibrosis-associated target gene of miR-143-3p. The aim was to explore the role and mechanism of miR-143-3p in the infarct healing after MI in vivo and in vitro. Myocardial samples were obtained during autopsy from 12 human patients with or without MI. An increase in miR-143-3p mRNA levels was detected in the infarct zone of human MI samples. Moreover, silencing expression of miR-143-3p by antagomir-143-3p alleviated fibrotic scar in MI model of mice. To assess the mechanism by which miR-143-3p may function in fibrosis, human cardiac fibroblasts (HCFs) were transfected with miR-143-3p mimics and inhibitors. MiR-143-3p overexpression promoted HCFs proliferation, migration, transformation, and extracellular matrix (ECM) excessive accumulation. Additionally, miR-143-3p inhibitors reversed the fibrosis effect of HCFs treated with transforming growth β1 (TGFβ1) in vitro. Importantly, a luciferase reporter assay demonstrated that miR-143-3p could directly bind to the 3'-untranslational region (3'-UTR) of SPRY3 mRNA. Lastly, HCFs transfected with SPRY3 siRNA (si-SPRY3) enhanced the activation of the P38, ERK, and JNK pathways in the process of fibrosis. MiR-143-3p promoted fibrosis along with SPRY3 degradation and the activation of its downstream P38, ERK, and JNK pathways. Our results may contribute to improve the quality of life in MI patients by interfering with the role of miR-143-3p in MI area.

Background Long noncoding RNAs (lncRNAs) are emerging as important mediators of cardiac pathophysiology. The aim of the present study is to investigate the effects of lncR-30245, an lncRNA, on cardiac fibrogenesis and the underlying mechanism. Methods Myocardial infarction (MI) and transforming growth factor (TGF)-β1 were used to induce fibrotic phenotypes. Cardiac fibrosis was detected by Masson’s trichrome staining. Cardiac function was evaluated by echocardiography. Western blot, quantitative reverse transcription-polymerase chain reaction, and pharmacological approaches were used to investigate the role of lncR-30245 in cardiac fibrogenesis. Results Expression of lncR-30245 was significantly increased in MI hearts and TGF-β1–treated cardiac fibroblasts (CFs). LncR-30245 was mainly located in the cytoplasm. Overexpression of lncR-30245 promoted collagen production and CF proliferation. Knockdown of lncR-30245 significantly inhibited TGF-β1–induced collagen production and CF proliferation. LncR-30245 overexpression inhibited the antifibrotic role of peroxisome proliferator-activated receptor (PPAR)-γ and increased connective tissue growth factor (CTGF) expression, whereas lncR-30245 knockdown exerted the opposite effects. Rosiglitazone, a PPAR-γ agonist, significantly inhibited lncR-30245–induced CTGF upregulation and collagen production in CFs. In contrast, T0070907, a PPAR-γ antagonist, attenuated the inhibitory effects of lncR-30245 small interfering RNA (siRNA) on TGF-β1–induced CTGF expression and collagen production. LncR-30245 knockdown significantly enhanced ejection fraction and fractional shortening and attenuated cardiac fibrosis in MI mice. Conclusion Our study indicates that the lncR-30245/PPAR-γ/CTGF pathway mediates MI-induced cardiac fibrosis and might be a therapeutic target for various cardiac diseases associated with fibrosis.

Rationale: As a hallmark of various heart diseases, cardiac fibrosis ultimately leads to end-stage heart failure. Anti-fibrosis is a potential therapeutic strategy for heart failure. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of heart diseases that promise to serve as therapeutic targets. However, few lncRNAs have been directly implicated in cardiac fibrosis. Methods: The lncRNA expression profiles were assessed by microarray in cardiac fibrotic and remote ventricular tissues in mice with myocardial infarction. The mechanisms and functional significance of lncRNA-AK137033 in cardiac fibrosis were further investigated with both in vitro and in vivo models. Results: We identified 389 differentially expressed lncRNAs in cardiac fibrotic and remote ventricular tissues in mice with myocardial infarction. Among them, a lncRNA (AK137033) we named Safe was enriched in the nuclei of fibroblasts, and elevated in both myocardial infarction and TGF-β-induced cardiac fibrosis. Knockdown of Safe prevented TGF-β-induced fibroblast-myofibroblast transition, aberrant cell proliferation and secretion of extracellular matrix proteins in vitro, and mended the impaired cardiac function in mice suffering myocardial infarction. In vitro studies indicated that knockdown of Safe significantly inhibited the expression of its neighboring gene Sfrp2, and vice versa. The Sfrp2 overexpression obviously disturbed the regulatory effects of Safe shRNAs in both the in vitro cultured cardiac fibroblasts and myocardial infarction-induced fibrosis. Dual-Luciferase assay demonstrated that Safe and Sfrp2 mRNA stabilized each other via their complementary binding at the 3'-end. RNA electrophoretic mobility shift assay and RNA immunoprecipitation assay indicated that RNA binding protein HuR could bind to Safe-Sfrp2 RNA duplex, whereas the knockdown of HuR dramatically reduced the stabilization of Safe and Sfrp2 mRNAs, down-regulated their expression in cardiac fibroblasts, and thus inhibited TGF-β-induced fibrosis. The Safe overexpression partially restrained the phenotype change of cardiac fibroblasts induced by Sfrp2 shRNAs, but not that induced by HuR shRNAs. Conclusions: Our study identifies Safe as a critical regulator of cardiac fibrosis, and demonstrates Safe-Sfrp2-HuR complex-mediated Sfrp2 mRNA stability is the underlying mechanism of Safe-regulated cardiac fibrosis. Fibroblast-enriched Safe could represent a novel target for anti-fibrotic therapy in heart diseases.

Rationale: Long non-coding RNA (lncRNAs) has been identified as a pivotal novel regulators in cardiac development as well as cardiac pathogenesis. lncRNA H19 is known as a fetal gene but it is exclusively abundant in the heart and skeletal muscles in adulthood, and is evolutionarily conserved in humans and mice. It has been reported to possess a significant correlation with the risk of coronary artery diseases. However, the function of H19 is not well characterized in heart. Methods: Loss-of-function and gain-of-function mouse models with left anterior descending coronary artery-ligation surgery were utilized to evaluate the functionality of H19 in vivo. For mechanistic studies, hypoxia condition were exerted in in vitro models to mimic cardiac ischemic injury. Chromatin isolation by RNA immunoprecipitation (ChIRP) was performed to reveal the interacting protein of lncRNA H19. Results: lncRNA H19 was significantly upregulated in the infarct area post-surgery day 4 in mouse model. Ectopic expression of H19 in the mouse heart resulted in severe cardiac dilation and fibrosis. Several extracellular matrix (ECM) genes were significantly upregulated. While genetic ablation of H19 by CRISPR-Cas9 ameliorated post-MI cardiac remodeling with reduced expression in ECM genes. Through chromatin isolation by RNA purification (ChIRP), we identified Y-box-binding protein (YB)-1, a suppressor of Collagen 1A1, as an interacting protein of H19. Furthermore, H19 acted to antagonize YB-1 through direct interaction under hypoxia, which resulted in de-repression of Collagen 1A1 expression and cardiac fibrosis. Conclusions: Together these results demonstrate that lncRNA H19 and its interacting protein YB-1 are crucial for ECM regulation during cardiac remodeling.

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.

Aims The renin-angiotensin-aldosterone axis plays a key role in mediating cardiac and kidney injury. Mineralocorticoid receptor antagonism has beneficial effects on cardiac dysfunction, but effects are less well quantified in the cardiorenal syndrome. This study investigated cardiac and kidney pathophysiology following permanent surgical ligation to induce myocardial infarction (MI) in hypertensive animals with or without mineralocorticoid receptor antagonism. Methods Hypertension was induced in adult male Cyp1a1Ren2 rats. Hypertensive animals underwent MI surgery (n = 6), and were then treated daily with spironolactone for 28 days with serial systolic blood pressure measurements, echocardiograms and collection of urine and serum biochemical data. They were compared to hypertensive animals (n = 4), hypertensive animals treated with spironolactone (n = 4), and hypertensive plus MI without spironolactone (n = 6). Cardiac and kidney tissue was examined for histological and immunohistochemical analysis. Results MI superimposed on hypertension resulted in an increase in interstitial cardiac fibrosis (p Conclusion This model of progressive cardiorenal dysfunction more closely replicates the clinical setting. Mineralocorticoid receptor blockade at a clinically relevant dose, blunted progression of cardiac and kidney fibrosis with reduction in cardiac and kidney inflammatory myofibroblast and macrophage infiltration. Further studies are underway to investigate the combined actions of angiotensin blockade with mineralocorticoid receptor blockade.

Ethnopharmacological relevance Myocardial fibrosis after myocardial infarction (MI) leads to cardiac remodeling and loss of function. Taohong siwu decoction (THSWD), a well-known traditional Chinese medicinal prescription, has been clinically used to treat various cardiovascular and cerebrovascular diseases, but its potential functions in myocardial fibrosis after MI remain uncharacterized. Aim of the study The purpose of current study was to explore the potential mechanism action and anti-myocardial fibrosis effects of treatment with THSWD in vivo and in vitro. Materials and methods Mouse underwent ligation of coronary artery to induce MI and divided equally into the sham group, model group and THSWD treatment groups. After 4 weeks, the effects of THSWD treatment on cardiac function were estimated by echocardiography. HE staining was used to detect the pathologic changes and Masson trichrome staining was used to estimate tissue fibrosis. To further explore the regulatory molecular mechanisms of THSWD, transcriptome analysis was performed. Furthermore, in vitro, we investigated the effect of THSWD on cell proliferation and collagen deposition in primary cardiac fibrosis cells and its possible mechanism of action. Overexpression of TGFBR1 was achieved by infection with an adenovirus vector encoding TGFBR1. Results Treatment with THSWD significantly decreased myocardial fibrosis and recovered cardiac function in the post-MI mouse. The transcriptomics data imply that the TGF-β pathway might be a target in the anti-fibrosis effect of THSWD. THSWD inhibits TGF-β1-induced proliferation of primary cardiac fibroblasts. THSWD decreased collagen expression and TGFBR1 and Smad2/3 phosphorylation. Moreover, the inhibitory effect of THSWD on CFs proliferation and collagen deposition, as well as TGFBR1 signaling pathway-associated proteins expression was partially abrogated by overexpression of TGFBR1. Conclusion Collectively, the results implicate that THSWD attenuates myocardial fibrosis by inhibiting fibrosis proliferation and collagen deposition via inhibiting TGFBR1, and might be a potential therapeutic agent for treatment of myocardial fibrosis post-MI.

Background: Cardiac fibrosis after myocardial infarction (MI) is a major cause of heart deterioration. Recently, the roles of microRNAs (miRNAs) in various cardiovascular diseases associated with cardiac fibrosis have been extensively investigated. The present study aimed to investigate the role and mechanism of miR-29b-3p in cardiac fibrosis after MI. Methods: miR-29b-3p expression in TGF-β1-activated cardiac fibroblasts (CFs) was detected by qRT-PCR. Cell Counting Kit-8 (CCK-8) and Trans-well assays were performed to evaluate CFs proliferation and migration ability, respectively. Protein expressions of α-SMA, collagen I, collagen III, MMP2, and MMP9 were examined by Western blot assay. Bioinformatics, luciferase, and RNA immunoprecipitation (RIP) assays were carried out to determine whether FOS was targeted by miR-29b-3p. Results: TGF-β1 treatment dose-dependently curbed miR-29b-3p expression in CFs. miR-29b-3p restrained the promotive impacts of TGF-β1 on CFs proliferation, migration, and differentiation. FOS was affirmed to be a target of miR-29b-3p, elevated expression of FOS reversed the inhibitory effects of miR-29b-3p on cell proliferation, migration, and differentiation in TGF-β1-activated CFs. Conclusion: miR-29b-3p degraded the pro-fibrosis effect induced by TGF-β1 via targeting FOS, providing a prospective therapeutic avenue for cardiac fibrosis after MI.

The adult heart following injury such as a myocardial infarction forms a fibrotic scar associated with transformation of resident cardiac fibroblasts into myofibroblast, accelerating cardiac remodeling and dysfunction. Cell therapies provide a novel direction for the enhancement of cardiac structure and function but remain poorly described in terms of the effect on resident cardiac fibroblasts. We have shown cortical bone derived stem cells (CBSCs) exhibit an ability to repair the heart after myocardial injury together with reduced scar formation. Nevertheless, whether CBSCs possess ability to modulate resident fibroblast response after myocardial injury remains untested. Objective; To determine the effect of secreted factors from CSBCs to attenuate myofibroblast formation in the heart after injury. Methods and Results; CBSCs were injected in mice after myocardial infarction which demonstrated reduced fibrosis as determined by Masson’s trichrome and Picro-Sirius red staining. In parallel, decreased expression of myofibroblast markers such as Acta2 was observed compared to PBS injected mice. To determine the effect of CBSCs on cardiac fibrosis, adult mouse cardiac fibroblasts were isolated from C57BL/6 mice, primed with CBSC pre-conditioned media for 12 hours, and treated with 10ng TGF-β for 48 hours to mimic cardiac injury. Decreased expression of Acta2, periostin and CTGF was observed in adult cardiac fibroblasts cultured in CBSC medium compared to control cells. Additionally, analysis of myofibroblast markers such as vimentin and pSMAD/SMAD was also decreased compared to control cells. To determine the mechanism, we looked for enriched miRNA in CBSCs that can mediate anti fibrotic response after injury. Results showed significantly increased expression of miR-18a in CBSCs. The upregulation of miR-18a was also validated in adult fibroblasts treated with CBSCs compared to control cells. Adult fibroblasts treated with mimic for miR-18a followed by TGF-β showed significant decrease in myofibroblast formation while miR-18a inhibitor completely inhibited the effect of CBSC medium. Conclusion; CBSCs reduce fibroblast to myofibroblast transition and differentiation in adult cardiac fibroblasts via miR-18a-5p. This finding reveals a new avenue for cell therapies to target myocardial scar modulation and provides a resolution for the cardiac repair response after injury in the adult myocardium.

The aim of the present study was to determine the effect of microRNA (miR)-132 on cardiac fibrosis in myocardial infarction (MI)-induced heart failure and angiotensin (Ang) II-treated cardiac fibroblasts (CFs). Experiments were carried out in Sprague-Dawley rat treatment with ligation of left coronary artery to induce heart failure, and in CFs administration of Ang II to induce fibrosis. The level of miR-132 was increased in the heart of rats with MI-induced heart failure and the Ang II-treated CFs. In MI rats, left ventricle (LV) ejection fraction, fractional shortening, the maximum of the first differentiation of LV pressure (LV +dp/dtmax) and decline (LV -dp/dtmax) and LV systolic pressure (LVSP) were reduced, and LV end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD), LV volumes in systole (LVVS) and LV volumes in diastole (LVVD) were increased, which were reversed by miR-132 agomiR but deteriorated by miR-132 antagomiR. The expression levels of collagen I, collagen III, transforming growth factor-β (TGF-β), and α-smooth muscle actin (α-SMA) were increased in the heart of rat with MI-induced heart failure and CFs administration of Ang II. These increases were inhibited by miR-132 agomiR but enhanced by miR-132 antagomiR treatment. MiR-132 inhibited PTEN expression, and attenuated PI3K/Akt signal pathway in CFs. These results indicated that the up-regulation of miR-132 improved the cardiac dysfunction, attenuated cardiac fibrosis in heart failure via inhibiting PTEN expression, and attenuating PI3K/Akt signal pathway. Up-regulation of miR-132 may be a strategy for the treatment of heart failure and cardiac fibrosis.

Ischaemic heart disease is one of the leading causes of death. Protease-activated receptor 2 (PAR2) is widely expressed within the cardiovascular system and is known to mediate inflammatory processes in various immunocytes, such as macrophages, mastocytes and neutrophils. Here, we investigated whether activating macrophage PAR2 modulates cardiac remodelling in a murine model of myocardial infarction. Myocardial infarction was produced by the permanent ligation of the left anterior descending coronary artery (LAD) in C57BL/6J background wild-type (WT) mice transplanted with bone marrow from WT or PAR2 knockout (PAR2 KO) mice. Hematopoietic deficiency of PAR2 had improvement of left ventricular systolic dysfunction and dilatation and decreased fibrosis deposition in remote zone at 1 week after LAD ligation. Inactivation of PAR2 also led to less recruitment of macrophages in myocardium, which was accompanied by decreased expression of pro-inflammatory cytokines. Furthermore, cultured cardiac fibroblasts (CFs) were activated and showed a fibrotic phenotype after being co-cultured in medium containing PAR2-activating macrophage, which enhances interferon-beta (INF-β) expression. The beneficial effects of macrophages with INF-β neutralisation or PAR2-deletion ameliorates the JAK/STAT3 pathway in CFs, which might be attributed to CF activation. These data suggest that macrophage-derived IFN-β plays a crucial role in adverse cardiac remodelling after myocardial infarction, at least in part, through a PAR2-dependent mechanism.

Previous studies have shown that lncRNA small nuclear RNA host gene 7 (lncRNA SNHG7) played an important role in cancer progression. However, the role of lncRNA SNHG7 in cardiac fibrosis is still poorly understood. In this study, the results of quantitative real time polymerase chain reaction (qRT-PCR) analysis showed that lncRNA SNHG7 was over expressed in the infarcted and peri-infarcted area in the left ventricle after MI in mice. Western blot analysis showed that knockdown of SNHG7 decreased the expression of collagen type 1 (Col1)and α-smooth muscle actin (α-SMA). Echocardiographic study suggested that inhibition of SNHG7 improved cardiac function after MI in mice. Luciferase assay indicated SNHG7 could act as a competing endogenous RNA (ceRNA) by sponging miR-34-5p. The MTT cell proliferation assay and 5-ethynyl-2'-deoxyuridine (EdU) labelling assay revealed that co-transfection of SNHG7 and miR-34-5p inhibited cell viability and proliferation of cardiac fibroblasts (CF). All the results indicated that lncRNA SNHG7 could promote cardiac fibrosis via targeting miR-34-5p through acting as a ceRNA in mice after MI. Silencing of SNHG7 could attenuate deposition of collagens and improve cardiac function. miR-34-5p could suppress the fibrogenesis of CF by targeting ROCK1 and abolish SNHG7-induced CF proliferation and fibroblast-to-myofibroblast transition.

The present study aimed to determine whether apelin-13 could attenuate cardiac fibrosis via inhibiting the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway to inhibit reactive oxygen species in heart failure (HF) rats. HF models were established by inducing ischemia myocardial infarction (MI) through ligation of the left anterior descending artery in Sprague–Dawley (SD) rats. MI-induced changes in hemodynamics and cardiac function were reversed by apelin-13 administration. The increases in the levels of collagen I, collagen III, α-smooth muscle actin (SMA), and transforming growth factor-β (TGF-β) in the heart of MI rats and cardiac fibroblasts (CFs) treated with angiotensin (Ang) II were inhibited by apelin-13. The levels of PI3K and p-Akt increased in Ang II-treated CFs, and these increases were blocked by apelin-13. The PI3K overexpression reversed the effects of apelin-13 on Ang II-induced increases in collagen I, collagen III, α-SMA, and TGF-β, NADPH oxidase activity and superoxide anions in CFs. Apelin-13 reduced the increases in the levels of NADPH oxidase activity and superoxide anions in the heart of MI rats and CFs with Ang II treatment. The results demonstrated that apelin-13 improved cardiac dysfunction, impaired cardiac hemodynamics, and attenuated fibrosis of CFs induced by Ang II via inhibiting the PI3K/Akt signaling pathway to inhibit oxidative stress.

Myocardial infarction (MI) will inevitably result in cardiac fibrosis. In this study, we investigated the effect of microRNA-145 (miR-145) and transcription factor sex-determining region Y box 9 (SOX9) in the production of cardiac fibrosis induced by MI. MI rat models were established by left anterior descending coronary artery (LAD) occlusion. Four weeks after LAD, the cardiac fibrosis level was assessed by Masson's trichrome staining. Cardiac fibroblasts (CFs) exposed to hypoxia were used to simulate MI-induced fibrosis. Flow cytometry, cell counting kit-8, and transwell assays were used to examine changes in CF apoptosis, proliferation, and migration, respectively. miR-145 expression was measured by quantitative real-time polymerase chain reaction. Immunofluorescence and Western blot analysis were performed to determine the relative expression of proteins. In comparison to the sham-operated group, the expression of miR-145 was significantly downregulated in the infarction peripheral area, whereas, SOX9 was upregulated. In the infarcted heart, the overexpression of miR-145 significantly ameliorated cardiac fibrosis and cardiac function, and there was a negative correlation between miR-145 and SOX9 expressions in hypoxic CFs in vitro. In addition, SOX9 was verified to be a functional target of miR-145. Overexpression of miR-145 or inhibition of SOX9 decreased CF proliferation, migration, and fibrosis, but augmented their apoptotic rate. Moreover, the upregulation of miR-145 or suppression of SOX9 inhibited AKT and β-catenin signaling in hypoxic CFs. Taken together, this study highlights a potential treatment for cardiac fibrosis through the targeted regulation of SOX9 by miR-145, and our findings indicate that miR-145 exerts anti-fibrotic effects in MI via the negative regulation of SOX9 and its downstream AKT/GSK-3β/β-catenin pathways.

Cardiac fibrosis caused by adverse cardiac remodeling following myocardial infarction can eventually lead to heart failure. Although the role of soluble factors such as TGF-β is well studied in cardiac fibrosis following myocardial injury, the physiological role of mechanotransduction is not fully understood. Here, we investigated the molecular mechanism and functional role of TRPV4 mechanotransduction in cardiac fibrosis. TRPV4KO mice, 8 weeks following myocardial infarction (MI), exhibited preserved cardiac function compared to WT mice. Histological analysis demonstrated reduced cardiac fibrosis in TRPV4KO mice. We found that WT CF exhibited hypotonicity-induced calcium influx and extracellular matrix (ECM)-stiffness-dependent differentiation in response to TGF-β1. In contrast, TRPV4KO CF did not display hypotonicity-induced calcium influx and failed to differentiate on high-stiffness ECM gels even in the presence of saturating amounts of TGF-β1. Mechanistically, TRPV4 mediated cardiac fibrotic gene promoter activity and fibroblast differentiation through the activation of the Rho/Rho kinase pathway and the mechanosensitive transcription factor MRTF-A. Our findings suggest that genetic deletion of TRPV4 channels protects heart from adverse cardiac remodeling following MI by modulating Rho/MRTF-A pathway-mediated cardiac fibroblast differentiation and cardiac fibrosis.

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

[Background]: Macrophages and fibroblasts are 2 major cell types involved in healing after myocardial infarction (MI), contributing to myocardial remodeling and fibrosis. Post-MI fibrosis progression is characterized by a decrease in cardiac macrophage content. [Objectives]: This study explores the potential of macrophages to express fibroblast genes and the direct role of these cells in post-MI cardiac fibrosis. [Methods]: Prolonged in vitro culture of human macrophages was used to evaluate the capacity to express fibroblast markers. Infiltrating cardiac macrophages was tracked in vivo after experimental MI of LysM(Cre/+);ROSA26(EYFP/+) transgenic mice. The expression of Yellow Fluorescent Protein (YFP) in these animals is restricted to myeloid lineage allowing the identification of macrophage-derived fibroblasts. The expression in YFP-positive cells of fibroblast markers was determined in myocardial tissue sections of hearts from these mice after MI. [Results]: Expression of the fibroblast markers type I collagen, prolyl-4-hydroxylase, fibroblast specific protein-1, and fibroblast activation protein was evidenced in YFP-positive cells in the heart after MI. The presence of fibroblasts after MI was evaluated in the hearts of animals after depletion of macrophages with clodronate liposomes. This macrophage depletion significantly reduced the number of Mac3+Col1A1+ cells in the heart after MI. [Conclusions]: The data provide both in vitro and in vivo evidence for the ability of macrophages to transition and adopt a fibroblast-like phenotype. Therapeutic manipulation of this macrophage-fibroblast transition may hold promise for favorably modulating the fibrotic response after MI and after other cardiovascular pathological processes., This work was supported by grants SAF2017-82436R and RTC2017-6283 from MINEICO, S2017/BMD-3686 from Comunidad de Madrid, CIVP18A3864 from Fundación Ramón Areces and CIBERCV (funded by the Instituto de Salud Carlos III), Fondos FEDER, and the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Award IK2BX003922 (to Dr. DeLeon-Pennell).

Long non-coding RNAs (lncRNAs) account for a large proportion of genomic transcripts and are critical regulators in various cardiac diseases. Though lncRNAs have been reported to participate in the process of diverse cardiac diseases, the contribution of lncRNAs in cardiac fibrosis remains to be fully elucidated. Here, we identified a novel anti-fibrotic lncRNA, SAIL (scaffold attachment factor B interacting lncRNA). SAIL was reduced in cardiac fibrotic tissue and activated cardiac fibroblasts. Gain- and loss-of-function studies showed that knockdown of SAIL promoted proliferation and collagen production of cardiac fibroblasts with or without TGF-β1 (transforming growth factor beta1) treatment, while overexpression of SAIL did the opposite. In mouse cardiac fibrosis induced by myocardial infarction, knockdown of SAIL exacerbated, whereas overexpression of SAIL alleviated cardiac fibrosis. Mechanically, SAIL inhibited the fibrotic process by directly binding with SAFB via 23 conserved nucleotide sequences, which in turn blocked the access of SAFB to RNA pol II (RNA polymerase II) and reduced the transcription of fibrosis-related genes. Intriguingly, the human conserved fragment of SAIL (hSAIL) significantly suppressed the proliferation and collagen production of human cardiac fibroblasts. Our findings demonstrate that SAIL regulates cardiac fibrosis by regulating SAFB-mediated transcription of fibrotic related genes. Both SAIL and SAFB hold the potential to become novel therapeutic targets for cardiac fibrosis.

Background Cardiac fibrosis is an important cause of heart failure (HF) after myocardial infarction (MI). Cyclin-dependent kinase inhibitor 2b (CDKN2b) regulates the cell cycle by encoding the p15 protein and participates in the development of various tumours. However, the role of CDKN2b/p15 in cardiac fibrosis and HF after MI remains unclear. Methods Lentivirus was used to induce the silence and overexpression of CDKN2b. Cardiac function was detected with the use of echocardiography. Immunohistochemistry, immunofluorescence, Western blotting, Cell Counting Kit 8, and wound healing assay were used to illustrate the potential mechanism associated with CDKN2b. Results The p15 protein expression was significantly down-regulated in both human and mouse failing hearts. Cardiac down-regulation of CDKN2b promoted myocardial fibrosis and worsened cardiac function in MI mice, while systemic CDKN2b silencing induced diastolic dysfunction in vivo. In addition, cardiac overexpression of CDKN2b ameliorated cardiac fibrosis and improved cardiac function in MI mice. Mechanistically, silencing CDKN2b gene enhanced the phosphorylation of retinoblastoma (Rb) protein and reinforced the migration and proliferation capabilities of cardiac fibroblasts. B Lymphoma Mo-MLV insertion region 1 homolog (BMI1) was up-regulated in failing heart and inversely regulated the expression of CDKN2b/p15 and the phosphorylation of Rb protein. The BMI1-p15-Rb signalling pathway is a potential mechanism of ischemia-induced cardiac fibrosis and HF. Conclusions Cardiac fibrosis and heart function could be worsened by the down-regulation and relieved by the up-regulation of CDKN2b/p15 in ischemia-induced HF via regulating the proliferation and migration capabilities of cardiac fibroblasts. These effects could be partially explained by the regulation of the BMI1-p15-Rb signalling pathway.

Cardiac fibrosis is a pathological remodeling response to myocardial infarction (MI) and impairs cardiac contractility. Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is increased in patients with MI. However, the functions of MALAT1 in cardiac fibrosis have not been elucidated. This study elucidates the roles of MALAT1 in MI and the underlying mechanisms. The MI model was established by artificial coronary artery occlusion in mice. Western blot analysis and quantitative reverse transcription-polymerase chain reaction were performed to analyze protein expression and RNA expression, respectively. Cardiac function was measured by echocardiography. Masson's trichrome staining was used to exhibit the fibrotic area in MI hearts. Cardiac fibroblasts were isolated from newborn pups, and cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Upregulation of MALAT1 and downregulation of microRNA-145 (miR-145) were induced in MI heart and angiotensin II (AngII)-treated cardiac fibroblasts, and the inhibition of miR-145 expression was reversed by MALAT1 depletion. Knockdown MALAT1 ameliorated MI-impaired cardiac function and prevented AngII-induced fibroblast proliferation, collagen production, and α-SMA expression in cardiac fibroblasts. MALAT1 stability and transforming growth factor-β1 (TGF-β1) activity were regulated by miR-145. AngII-induced TGF-β1 activity in cardiac fibroblasts was blocked by MALAT1 knockdown. Based on these results, we concluded that lncRNA MALAT1 promotes cardiac fibrosis and deteriorates cardiac function post-MI by regulating TGF-β1 activity via miR-145.

BACKGROUND Abnormally expressed long noncoding RNAs (lncRNAs) are recognized as one of the key causes of cardiac diseases. However, the role of lncRNA in cardiac fibrosis remains largely unknown. MATERIAL AND METHODS The experiment was divided into 4 groups: a sham operation group, a myocardial infarction (MI) group, a lentivirus group (LV-si-n379519), and a lentivirus control (LV-NC) group. The adenovirus expression vectors LV-si-n379519 and LV-NC were constructed and transfected into mice. Echocardiography, HE staining, and Masson staining were performed to detect the heart function and collagen volume fraction in each group. RT-PCR was used to detect the expression level of n379519, miR-30, collagen I, and collagen III. In vitro, cardiac fibroblasts (CFs) were cultured and the relationship between n379519 and miR-30 was verified using luciferase reporter vector, n379519 siRNA, and miR-30 inhibitor. RESULTS The expression of n379519 was markedly upregulated in the hearts of mice with MI and in the fibrotic CFs. Knockdown of endogenous n379519 by its siRNA improved the heart function and reduced collagen deposition and the process of cardiac fibrosis. Further experiments showed the opposite trend of expression between n379519 and miR-30. Bioinformatics analysis and luciferase reporter assay indicated that n379519 directly binds to miR-30. Moreover, miR-30 inhibitor abrogated the collagen synthesis inhibition induced by n379519. CONCLUSIONS These findings reveal a novel function of n379519-miR-30 axis as a negative regulator for the treatment of MI-induced cardiac fibrosis and the associated cardiac dysfunction.

Cardiac fibrosis is an independent risk factor for heart failure and even the leading cause of death in myocardial infarction patients. However, molecular mechanisms associated with the pathogenesis of cardiac fibrosis following myocardial infarction are not yet fully understood. Nogo-C protein ubiquitously expresses in tissues including in the heart. Our previous study found that Nogo-C regulated cardiomyocyte apoptosis during myocardial infarction. In the present study, we found that Nogo-C was upregulated in fibrotic hearts after myocardial infarction and in Ang II- or TGF-β1-stimulated cardiac fibroblasts. Overexpression of Nogo-C in cardiac fibroblasts increased expression of pro-fibrogenic proteins, while knockdown of Nogo-C inhibited the fibrotic responses of cardiac fibroblasts to Ang II- or TGF-β1 stimulation. Functionally, Nogo-C deficiency suppressed pro-fibrogenic proteins in post-myocardial infarction hearts and ameliorated post-myocardial infarction cardiac function. Mechanistically, we found that Nogo-C increased intracellular Ca2+ concentration and buffering Ca2+ totally abolished Nogo-C-induced fibrotic responses. Moreover, overexpression of Nogo-C caused increased Sec61α, the Ca2+ leakage channel on endoplasmic reticulum membrane. Nogo-C interacted with Sec61α on endoplasmic reticulum and stabilized Sec61α protein by inhibiting its ubiquitination. Inhibition or knockdown of Sec61α blocked Nogo-C-induced increase of cytosolic Ca2+ concentration and inhibited Nogo-C- and TGF-β1-induced fibrotic responses in cardiac fibroblasts, suggesting that Nogo-C regulates cardiac fibrosis through interacting with Sec61α to mediate the Ca2+ leakage from endoplasmic reticulum. Thus, our results reveal a novel mechanism underlying cardiac fibrosis following myocardial infarction, and provide a therapeutic strategy for cardiac remodeling related heart diseases.

Background/Aims: Percutaneous coronary intervention reduces acute myocardial infarction (MI)-induced mortality to a great extent, but effective treatments for MI-induced cardiac fibrosis and heart failure are still lacking. MicroRNAs (miRNAs) play a variety of roles in cells and have thus been investigated extensively. MicroRNA-223 (miR-223) expression has been reported to be altered in post-MI heart failure in humans; however, the roles of miR-223 in MI remain unknown. Our study aimed to elucidate the roles of miR-223 in cardiac fibrosis. Methods: Cultured cardiac fibroblasts (CFs) were activated by TGF-β1 stimulation. Gain and loss of miR-223 and RAS p21 protein activator 1 (RASA1) knockdown in CFs were achieved by transfecting the cells with miR-223 mimics and inhibitors, as well as small interfering RNA-RASA1 (siRASA1), respectively. Quantitative real-time reverse transcriptase-polymerase chain reactions (qRT-PCR) was used to determine miR-223-3p and RASA1 expression levels, and Cell Counting Kit-8 (CCK-8), transwell migration and scratch assays were performed to assess CFs viability and migration, respectively. Western blotting was used to detect collagen I, collagen III, alpha-smooth muscle actin (a-SMA), RASA1, p-Akt/t-Akt, p-MEK1/2/t-MEK1/2, and p-ERK1/2/t-ERK1/2 protein expressions, and immunofluorescence assays were used to detect the expression of α-actin, vimentin and α-SMA. Luciferase assays were carried out to determine whether miR-223 binds to RASA1. Rat models of MI were established by the ligation of the left anterior descending (LAD) coronary artery. MiR-223 inhibition in vivo was achieved via intramyocardial injections of the miR-223 sponge carried by adeno-associated virus 9 (AAV9). The cardiac function was detected by echocardiography, and cardiac fibrosis was shown by Masson’s trichrome staining. Results: miR-223 was increased in CFs compared to cardiomypcytes, and TGF-β1 treatment increased miR-223 expression in CFs. The miR-223 mimics enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs, while the miR-223 inhibitors had contrasting effects and partially prevented the promoting effects of TGF-β1. qRT-PCR and western blotting revealed that miR-223 negatively regulated RASA1 expression, and the luciferase assays showed that miR-223 suppressed the luciferase activity of the RASA1 3’ untranslated region (3'UTR), indicating that miR-223 binds directly to RASA1. Similar to transfection with the miR-223 mimics, RASA1 knockdown enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs. Moreover, RASA1 knockdown partially reversed the inhibitory effects of the miR-223 inhibitor on cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression, indicating that the effects of miR-223 in CFs are partially mediated by the regulation of RASA1 expression. Further exploration showed that miR-223 mimics and siRASA1 promoted MEK1/2, ERK1/2 and AKT phosphorylation, while the miR-223 inhibitors had contrasting effects. The in vivo experiments confirmed the results of the in vitro experiments and showed that miR-223 inhibition prevented cardiac functional deterioration and cardiac fibrosis. Conclusions: miR-223 enhanced cell proliferation, migration, and differentiation in CFs, thus mediated cardiac fibrosis after MI partially via the involvement of RASA1.

Background/Aims: Cardiac fibrosis is an important cardiac remodeling event that can ultimately lead to the development of severe arrhythmia and heart failure. MicroRNAs (miRNAs) are involved in the pathogenesis of many cardiovascular diseases. Here, we aimed to investigate the effects of caveolin-3 (Cav3) on the pathogenesis of cardiac fibrosis and the underlying molecular mechanisms. Methods: Cav3 expression was decreased in cardiac fibrosis in vivo and in vitro model. To investigate the role of Cav3 in cardiac fibrosis, we transfected cardiac fibroblasts (CFs) with the siRNA of Cav3 and Cav3-overexpressing plasmid. The collagen content and proliferation of CFs were detected by qRT-PCR, western blot, MTT, and immunofluorescence. A luciferase reporter gene assay and gain/loss of function were used to detect the relationship between miR-22 and Cav3. Results: Cav3 depletion in CFs induced an increase in collagen content, cell proliferation, and phenotypic conversion of fibroblasts to myofibroblasts. Conversely, Cav3 overexpression in CFs was shown to inhibit angiotensin II-mediated excessive collagen deposition through protein kinase C (PKC)ε inactivation. Cav3 was experimentally confirmed as a direct target of miR-22, containing two seed binding sites in its 3′-untranslated region, and miR-22 was demonstrated to be significantly upregulated in the ischemic border zone in mice after myocardial infarction and in neonatal rat CFs pretreated with angiotensin II. miR-22 overexpression increased CFs proliferation, and collagen and α-smooth muscle actin levels in CFs, while the knockdown of endogenous miR-22 decreased CFs numbers. Conclusions: Our findings demonstrate that miR-22 accelerates cardiac fibrosis through the miR-22-Cav3-PKCε pathway, which, therefore, may represent a new therapeutic target for treatment of excessive fibrosis-associated cardiac diseases.

Myocardial infarction (MI) is accompanied by cardiac fibrosis, which contributes to cardiac dysfunction. Mineralocorticoid receptor (MR) antagonists have beneficial effects in patients with left ventricular (LV) dysfunction after MI. We herein investigated the role of the MR target NGAL (neutrophil gelatinase–associated lipocalin) in post-MI cardiac damages. Both higher baseline NGAL and a greater increase in serum NGAL levels during follow-up were significantly associated with lower 6-month LV ejection fraction recovery in a cohort of 119 post-MI patients, as assessed by cardiac magnetic resonance imaging. NGAL protein levels increased in the LV at 7 days post-MI in wild-type mice with MI. This effect was prevented by treatment with the nonsteroidal MR antagonist finerenone (1 mg/kg per day). NGAL knockout mice with MI had lower LV interstitial fibrosis and inflammation, better LV contractility and compliance, and greater stroke volume and cardiac output than wild-type mice with MI at 3 months post-MI. Aldosterone (10 −8 mol/L) increased NGAL expression in cultured human cardiac fibroblasts. Cells treated with aldosterone or NGAL (500 ng/mL) showed increased production of collagen type I. The effects of aldosterone were abolished by finerenone (10 −6 mol/L) or NGAL knockdown. This NGAL-mediated activity relied on NFκB (nuclear factor-κB) activation, confirmed by the use of the NFκB-specific inhibitor BAY11-7082, which prevented the effect of both aldosterone and NGAL on collagen type I production. In conclusion, NGAL, a downstream MR activation target, is a key mediator of post-MI cardiac damage. NGAL may be a potential therapeutic target in cardiovascular pathological situations in which MR is involved.

To evaluate the role of ghrelin in cardiac fibrosis after myocardial infarction (MI) and to investigate the underlying mechanisms of ghrelin-regulated Nrf2/NADPH/ROS pathway-mediated cardioprotection, the profile of Nrf2, fibrosis markers, and oxidative stress markers were characterized in a rat model of MI and Angiotensin II (Ang II)-stimulated cardiac fibroblasts (CFs). The effects of ghrelin on cardiac function, fibrosis and oxidative stress were investigated after MI in vivo. The role of ghrelin in CF migration and proliferation was evaluated in Ang II-stimulated CFs in vitro. Inhibition of ghrelin receptors using the antagonist, d-Lys3-GHRP-6, in addition to ghrelin was employed in MI and CFs to investigate the direct effect of ghrelin on cardiac fibrosis. Loss function of Nrf2 in CFs was performed to investigate the effect of ghrelin-regulated Nrf2 on oxidative stress and cardiac fibrosis. Ghrelin improved the post-MI cardiac function and reduced cardiac fibrosis. This phenotype is associated with the upregulation of Nrf2 and downregulation of fibrotic proteins, NADPH oxidase and ROS production. In line with in vivo findings, ghrelin attenuated Ang II-stimulated CF migration, proliferation, and oxidative stress in vitro. Inhibition of the ghrelin receptor or knockdown of Nrf2 abolished the beneficial effects of ghrelin on MI or Ang II-stimulated cardiac fibroblasts. In conclusion, ghrelin ameliorates post-MI and Ang II-induced cardiac fibrosis by activating Nrf2, which in turn inhibits the NADPH/ROS pathway.

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 after myocardial infarction (MI) has been identified as an important factor in the deterioration of heart function. Previous studies have demonstrated that miR-21 plays an important role in various pathophysiological processes in the heart. However, the role of miR-21 in fibrosis regulation after MI remains unclear. Methods: To induce cardiac infarction, the left anterior descending coronary artery was permanently ligated of mice. First, we explored the expression of miR-21 in the infarcted zone in mice model of MI via RT-qPCR. Next, we examined the effects of TGF-β1 on miR-21 expression in cardiac fibroblasts (CFs). Then, CFs were infected with miR-21 mimics or miR-21 inhibitors to investigate the effects of miR-21 on the process of CFs activation in vitro. Further, bioinformatics analysis and luciferase reporter assay were performed to identify and validate the target gene of miR-21. At last, in-vivo study was done to confirm MiR-21 regulated myocardial fibrosis after MI in mice. Results: MiR-21 was up-regulated in the infarcted zone after MI in vivo. TGF-β1 treatment increased miR-21 expression in CFs. Overexpression of miR-21 promoted the effects of TGF-β1-induced activation of CFs, evidenced by increased expression of Col-1, α-SMA and F-actin, whereas inhibition of miR-21 attenuated the process of fibrosis. Bioinformatics, Western blot analysis and luciferase reporter assay demonstrated that Smad7 is a direct target of miR-21. In addition, in-vivo study revealed that MiR-21 regulated myocardial fibrosis after MI in mice. Conclusion: These findings suggested that miR-21 has a critical role in CF activation and cardiac fibrosis after MI through via TGF-β/Smad7 signaling pathway. Thus, miR-21 promises to be a potential therapy in treatment of cardiac fibrosis after MI.

Background/Aims: Cardiac fibrosis after myocardial infarction (MI) has been identified as a key factor in the development of heart failure, but the mechanisms undelying cardiac fibrosis remained unknown. microRNAs (miRNAs) are novel mechanisms leading to fibrotic diseases, including cardiac fibrosis. Previous studies revealed that miR-22 might be a potential target. However, the roles and mechanisms of miR-22 in cardiac fibrosis remained ill defined. The present study thus addressed the impact of miR-22 in cardiac fibrosis. Methods: After seven days following coronary artery occlusion in mice, tissues used for histology were collected and processed for Masson's Trichrome staining. In addition, cardiac fibroblasts were transfected with mimics and inhibitors of miR-22 using Lipofectamin 2000, and luciferase activity was measured in cell lysates using a luciferase assay kit. Western blotting was used to detect the expression of collagen1, α-SMA and TGFβRI proteins levels, and real time-PCR was employed to measure the Col1α1, Col3α1, miR-22 and TGFβRI mRNA levels. Results: In this study, we found that miR-22 was dynamically downregulated following MI induced by permanent ligation of the left anterior descending coronary artery for 7 days, an effect paralleled by significant collagen deposition. Inhibition of miR-22 with AMO-22 resulted in increased expression of Col1α1, Col3α1 and fibrogenesis in cultured cardiac fibroblasts. Conversely, overexpression of miR-22 in cultured cardiac fibroblasts significantly abrogated angiotensin II-induced collagen formation and fibrogenesis. Furthermore, we found that TGFβRI is a direct target for miR-22, and downregulation of TGFβR may have mediated the antifibrotic effect of miR-22. Conclusion: Our data clearly demonstrate that miR-22 acts as a novel negative regulator of angiotensin II-induced cardiac fibrosis by suppressing the expression of TGFβRI in the heart and may represent a new potential therapeutic target for treating cardiac fibrosis.

Secreted Frizzled-related protein 2 (sFRP2) plays a key role in chronic fibrosis after myocardial infarction and in heart failure. The aim of this study was to elucidate the mechanisms through which sFRP2 may regulate the growth and extracellular matrix (ECM) remodeling of adult mouse cardiac fibroblasts (CFs). We found that sFRP2 activates CFs in part through canonical Wnt/β-catenin signaling, as evidenced by increased expression of Axin2 and Wnt3a, but not Wnt5a, as well as accumulation of nuclear β-catenin. In response to sFRP2, CFs exhibited robust cell proliferation associated with increased glucose consumption and lactate production, a phenomenon termed “the Warburg effect” in oncology. The coupling between CF expansion and anaerobic glycolysis is marked by upregulation of glyceraldehyde-3-phosphate dehydrogenase and tissue-nonspecific alkaline phosphatase. In conjunction with these phenotypic changes, CFs accelerated ECM remodeling through upregulation of expression of the matrix metalloproteinase (MMP) 1 and MMP13 genes, two members of the collagenase subfamily, and enzyme activities of MMP2 and MMP9, two members of the gelatinase subfamily. Consistent with the induction of multiple MMPs possessing collagenolytic activities, the steady-state level of collagen type 1 in CF-spent medium was reduced by sFRP2. Analysis of non-CF cell types revealed that the multifaceted effects of sFRP2 on growth control, glucose metabolism, and ECM regulation are largely restricted to CFs and highly sensitive to Wnt signaling perturbation. The study provides a molecular framework on which the functional versatility and signaling complexity of sFRP2 in cardiac fibrosis may be better defined.

Tissue nonspecific alkaline phosphatase (TNAP) is expressed widely in different tissues, modulating functions of metabolism and inflammation. However, the effect of TNAP on cardiac fibrosis remains controversial and needs to be further studied. The present study aims to investigate the role of TNAP on myocardial infarction (MI)-induced fibrosis and its mechanism. TNAP was upregulated in patients with MI, both in serum and injured hearts, and predicted in-hospital mortality. TNAP was also significantly upregulated after MI in rats, mostly in the border zone of the infarcted hearts combined with collagen synthesis. Administration of TNAP inhibitor, tetramisole, markedly improved cardiac function and fibrosis after MI. In the primary cultures of neonatal rat cardiac fibroblasts (CFs), TNAP inhibition significantly attenuated migration, differentiation, and expression of collagen-related genes. The TGF-β1/Smads signaling suppression, and p-AMPK and p53 upregulation were involved in the process. When p53 inhibitor was administered, the antifibrotic effect of TNAP inhibition can be blocked. This study provides a direct evidence that inhibition of TNAP might be a novel regulator in cardiac fibrosis and exert an antifibrotic effect mainly through AMPK-TGF-β1/Smads and p53 signals.

Fibrotic scarring drives the progression of heart failure after myocardial infarction (MI). Therefore, the development of specific treatment regimens to counteract fibrosis is of high clinical relevance. The transcription factor sex-determining region Y box 9 (SOX9) functions as an important regulator during embryogenesis, but recent data point toward an additional causal role in organ fibrosis. We show here that SOX9 is upregulated in the scar after MI in mice. Fibroblast-specific deletion of Sox9 ameliorated MI-induced left ventricular dysfunction, dilatation, and myocardial scarring in vivo. Unexpectedly, deletion of Sox9 also potently eliminated persisting leukocyte infiltration of the scar in the chronic phase after MI. RNA-Seq from the infarct scar revealed that Sox9 deletion in fibroblasts resulted in strongly downregulated expression of genes related to extracellular matrix, proteolysis, and inflammation. Importantly, Sox9 deletion in isolated cardiac fibroblasts in vitro similarly affected gene expression as in the cardiac scar and reduced fibroblast proliferation, migration, and contraction capacity. Together, our data demonstrate that fibroblast SOX9 functions as a master regulator of cardiac fibrosis and inflammation and might constitute a novel therapeutic target during MI.

Myocardial infarction (MI) occurs when the heart muscle is severely damaged due to a decrease in blood flow from the coronary arteries. During recovery from an MI, cardiac fibroblasts become activated and produce extracellular matrices, contributing to the wound healing process in the damaged heart. Inappropriate activation of the fibroblasts leads to excessive fibrosis in the heart. However, the molecular pathways by which cardiac fibroblasts are activated have not yet been fully elucidated.Here we show that serum deprivation, which recapitulates the cellular microenvironment of the MI area, strikingly induces collagen production in C3H/10T1/2 cells. Based on transcriptomic and pharmacological studies, we found that cell cycle perturbation is directly linked to collagen production in fibroblasts. Importantly, collagen synthesis is increased independently of the transcriptional levels of type I collagen genes. These results reveal a novel mode of fibroblast activation in the ischemic area, which will allow us to gain insights into the molecular mechanisms underlying cardiac fibrosis and establish a basis for anti-fibrotic therapy.