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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.

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.

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.

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.

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.

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 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.

Background/Aims: Deregulated myocardial fibrosis is associated with a wide spectrum of cardiac conditions, being considered one of the major causes for heart disease. Our study was designed to investigate the role of microRNA-328 (miR-328) in regulating cardiac fibrosis. Methods: We induced cardiac fibrosis following MI by occlusion of the left coronary artery in C57BL/6 mice. Real-time PCR was employed to evaluate the level of miR-328. Masson's Trichrome stain was used to evaluate the development of fibrosis. Luciferase activity assay was performed to confirm the miRNA's binding site in the TGFβRIII gene. Western blot analysis was used to examine TGFβRIII, p-smad2/3 and TGF-β1 at protein level. Results: In this study, we found that miR-328 was significantly upregulated in the border zone of infarcted myocardium of wild type (WT) mice; TGFβRIII was downregulated whereas TGF-β1 was upregulated along with increased cardiac fibrosis. And miR-328 stimulated TGF-β1 signaling and promoted collagen production in cultured fibroblasts. We further found that the pro-fibrotic effect of miR-328 was mediated by targeting TGFβRIII. Additionally, cardiac fibrosis was significantly reduced in infarcted heart when treated with miR-328 antisense. Conclusions: These data suggest that miR-328 is a potent pro-fibrotic miRNA and an important determinant of cardiac fibrosis in diseased heart.

Background: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. Methods: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. Results: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor β-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. Conclusions: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor β-1–SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

Right ventricular failure predicts adverse outcome in patients with pulmonary hypertension (PH), and in subjects with left ventricular heart failure and is associated with interstitial fibrosis. This review manuscript discusses the cellular effectors and molecular mechanisms implicated in right ventricular fibrosis. The right ventricular interstitium contains vascular cells, fibroblasts, and immune cells, enmeshed in a collagen-based matrix. Right ventricular pressure overload in PH is associated with the expansion of the fibroblast population, myofibroblast activation, and secretion of extracellular matrix proteins. Mechanosensitive transduction of adrenergic signalling and stimulation of the renin-angiotensin-aldosterone cascade trigger the activation of right ventricular fibroblasts. Inflammatory cytokines and chemokines may contribute to expansion and activation of macrophages that may serve as a source of fibrogenic growth factors, such as transforming growth factor (TGF)-β. Endothelin-1, TGF-βs, and matricellular proteins co-operate to activate cardiac myofibroblasts, and promote synthesis of matrix proteins. In comparison with the left ventricle, the RV tolerates well volume overload and ischemia; whether the right ventricular interstitial cells and matrix are implicated in these favourable responses remains unknown. Expansion of fibroblasts and extracellular matrix protein deposition are prominent features of arrhythmogenic right ventricular cardiomyopathies and may be implicated in the pathogenesis of arrhythmic events. Prevailing conceptual paradigms on right ventricular remodelling are based on extrapolation of findings in models of left ventricular injury. Considering the unique embryologic, morphological, and physiologic properties of the RV and the clinical significance of right ventricular failure, there is a need further to dissect RV-specific mechanisms of fibrosis and interstitial remodelling.

Background/Aims: After myocardial infarction (MI), cardiac fibrosis greatly contributes to left ventricular remodeling and heart failure. The intermediate-conductance calcium-activated potassium Channel (KCa3.1) has been recently proposed as an attractive target of fibrosis. The present study aimed to detect the effects of KCa3.1 blockade on ventricular remodeling following MI and its potential mechanisms. Methods: Myocardial expression of KCa3.1 was initially measured in a mouse MI model by Western blot and real time-polymerase chain reaction. Then after treatment with TRAM-34, a highly selective KCa3.1 blocker, heart function and fibrosis were evaluated by echocardiography, histology and immunohistochemistry. Furthermore, the role of KCa3.1 in neonatal mouse cardiac fibroblasts (CFs) stimulated by angiotensin II (Ang II) was tested. Results: Myocardium expressed high level of KCa3.1 after MI. Pharmacological blockade of KCa3.1 channel improved heart function and reduced ventricular dilation and fibrosis. Besides, a lower prevalence of myofibroblasts was found in TRAM-34 treatment group. In vitro studies KCa3.1 was up regulated in CFs induced by Ang II and suppressed by its blocker.KCa3.1 pharmacological blockade attenuated CFs proliferation, differentiation and profibrogenic genes expression and may regulating through AKT and ERK1/2 pathways. Conclusion: Blockade of KCa3.1 is able to attenuate ventricular remodeling after MI through inhibiting the pro-fibrotic effects of CFs.

Background/Aims: Hypoxia is a basic pathological challenge that is associated with numerous cardiovascular disorders including aberrant cardiac remodeling. Transforming growth factor beta (TGF-β) signaling pathway plays a pivotal role in mediating cardiac fibroblast (CF) function and cardiac fibrosis. Recent data suggested that microRNA-101a (miR-101a) exerted anti-fibrotic effects in post-infarct cardiac remodeling and improved cardiac function. This study aimed to investigate the potential relationship between hypoxia, miR-101a and TGF-β signaling pathway in CFs. Methods and Results: Two weeks following coronary artery occlusion in rats, the expression levels of both TGFβ1 and TGFβRI were increased, but the expression of miR-101a was decreased at the site of the infarct and along its border. Cultured rat neonatal CFs treated with hypoxia were characterized by the up-regulation of TGFβ1 and TGFβRI and the down-regulation of miR-101a. Delivery of miR-101a mimics significantly suppressed the expression of TGFβRI and p-Smad 3, CF differentiation and collagen content of CFs. These anti-fibrotic effects were abrogated by co-transfection with AMO-miR-101a, an antisense inhibitor of miR-101a. The repression of TGFβRI, a target of miR-101a, was validated by luciferase reporter assays targeting the 3'UTR of TGFβRI. Additionally, we found that overexpression of miR-101a reversed the improved migration ability of CFs and further reduced CF proliferation caused by hypoxia. Conclusion: Our study illustrates that miR-101a exerts anti-fibrotic effects by targeting TGFβRI, suggesting that miR-101a plays a multi-faceted role in modulating TGF-β signaling pathway and cardiac fibrosis.

The phenylephrine-induced complex-1 (PEX1) transcription factor, also known as zinc-finger protein 260 (Zfp260), is an effector of endothelin-1 and alpha(1)-adrenergic signaling in cardiac hypertrophy. However, the role of PEX1 in transcriptional regulation of myocardial remodeling remains largely unknown. In the present study, we used PEX1 gain- and loss-of-function to examine the effects of PEX1 on left ventricular remodeling. Adenoviral constructs expressing PEX1, antisense PEX1, or LacZ were delivered by local injection into the anterior wall of the left ventricle in Sprague-Dawley rats. PEX1 overexpression led to induction of hypertrophic gene program and increased fibrosis. In agreement with this, the expression of genes involved in the fibrotic process, such as collagens I and III, matrix metalloproteinases (MMPs), fibronectin-1, transforming growth factor beta-1 and connective tissue growth factor, were significantly up-regulated following PEX1 overexpression, whereas silencing of PEX1 significantly inhibited the expression of pro-fibrotic genes and increased left ventricular ejection fraction and fractional shortening. In vitro luciferase reporter assays showed that PEX1 regulates the expression of MMP-9 by activating promoter. Furthermore, PEX1 gain- and loss-of-function experiments in rat neonatal cardiac fibroblasts and myocytes revealed that MMP-9 gene expression was affected by PEX1 predominantly in fibroblasts. Our results indicate that PEX1 is involved in regulating cardiac fibrosis and extracellular matrix turnover, particularly fibroblasts being responsible for the fibrosis-associated changes in gene expression. Furthermore, PEX1 activation of the MMP-9 promoter triggers the pro-fibrotic response directed by PEX1.

Jie Xiao, Xi Sheng, Xinyu Zhang, Mengqi Guo, Xiaoping JiKey Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital of Shandong University, Jinan, Shandong, People’s Republic of ChinaAbstract: Curcumin, a polyphenolic compound derived from turmeric, protects against myocardial injury by alleviating oxidative stress, inflammation, apoptosis, and fibrosis. However, the role of curcumin and its mechanism of action on interstitial fibrosis after myocardial infarction (MI) are poorly understood. To clarify, MI was induced by a permanent ligation of the left anterior descending coronary artery in adult mice, and the effects of curcumin were evaluated 4 weeks after the MI event. In vitro, we treated cardiac fibroblasts (CFs) with Ang II, and investigated the anti-fibrotic mechanism of curcumin. Our results showed that curcumin significantly attenuated collagen deposition in vivo and inhibited CF proliferation and migration, and MMP expression. In addition, we found that the down-regulation of SIRT1 after MI was attenuated by curcumin pretreatment, which indicated that the activation of SIRT1 might be involved in the protective action of curcumin. This hypothesis was confirmed by genetic inhibition of SIRT1 (siRNA-SIRT1) in Ang II-treated CFs. Our results provide new insights into the mechanism underlying the anti-fibrotic effects of curcumin in the heart.Keywords: curcumin, myocardial infarction, angiotensin II, cardiac fibroblasts, fibrosis, SIRT1

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.

The majority of cardiac fibroblasts in a mature mammalian heart are derived from the epicardium during prenatal development and reactivate developmental programs during the progression of fibrotic disease. In addition, epicardial activation, proliferation, and fibrosis occur with ischemic, but not hypertensive injury. Here we review cellular and molecular mechanisms that control epicardium-derived cell lineages during development and disease with a focus on cardiac fibroblasts. This article is part of a special issue entitled "Fibrosis and Myocardial Remodeling".

Senescence is a recognized mechanism of cardiovascular diseases; however, its contribution to myocardial fibrosis and rupture after infarction and the underlying mechanisms remain unclear. Here we showed that senescent cardiac fibroblasts markedly accumulated in heart after myocardial infarction. The expression of key senescence regulators, especially p53, was significantly up-regulated in the infarcted heart or hypoxia-treated fibroblasts. Furthermore, knockdown of endogenous p53 by siRNA in fibroblasts markedly reduced hypoxia-induced cell senescence, cytokine expression but increased collagen expression, whereas increased expression of p53 protein by adenovirus infection had opposite effects. Consistent with in vitro results in cardiac fibroblasts, p53 deficiency in vivo significantly decreased the accumulation of senescent fibroblasts, the infiltration of macrophages and matrix metalloproteinases, but enhanced collagen deposition after myocardial infarction. In conclusion, these results suggest that the p53-mediated fibroblast senescence limits cardiac collagen production, and inhibition of p53 activity could represent a novel therapeutic target to increase reparative fibrosis and to prevent heart rupture after myocardial infarction.

Heart failure is strongly associated with aging. Elderly patients with heart failure often have preserved systolic function exhibiting left ventricular hypertrophy accompanied by a decline in diastolic function. Experimental studies have demonstrated that age-related cardiac fibrosis plays an important role in the pathogenesis of diastolic heart failure in senescent hearts. Reactive oxygen species and angiotensin II are critically involved in fibrotic remodeling of the aging ventricle; their fibrogenic actions may be mediated, at least in part, through transforming growth factor (TGF)-beta. The increased prevalence of heart failure in the elderly is also due to impaired responses of the senescent heart to cardiac injury. Aging is associated with suppressed inflammation, delayed phagocytosis of dead cardiomyocytes, and markedly diminished collagen deposition following myocardial infarction, due to a blunted response of fibroblasts to fibrogenic growth factors. Thus, in addition to a baseline activation of fibrogenic pathways, senescent hearts exhibit an impaired reparative reserve due to decreased responses of mesenchymal cells to stimulatory signals. Impaired scar formation in senescent hearts is associated with accentuated dilative remodeling and worse systolic dysfunction. Understanding the pathogenesis of interstitial fibrosis in the aging heart and dissecting the mechanisms responsible for age-associated healing defects following cardiac injury are critical in order to design new strategies for prevention of adverse remodeling and heart failure in elderly patients.