Cardiac Fibrosis In Mice Research Articles

Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

Rationale: Cardiac fibrosis is an important feature of cardiac remodeling and is a hallmark of heart failure. Recent studies indicate that elevated IgE plays a causal role in pathological cardiac remodeling. However, the underlying mechanism of how IgE promotes cardiac fibrosis has not been fully elucidated.Methods and Results: To explore the function of IgE in cardiac fibrosis, we stimulated mouse primary cardiac fibroblasts (CFs) with IgE and found that both IgE receptor (FcεR1) and fibrosis related proteins were increased after IgE stimulation. Specific deletion of FcεR1 in CFs alleviated angiotensin II (Ang II)-induced cardiac fibrosis in mice. To investigate the mechanisms underlying the IgE-mediated cardiac fibrosis, deep miRNA-seq was performed. Bioinformatics and signaling pathway analysis revealed that IgE upregulated Col1a1 and Col3a1 expression in CFs by repressing miR-486a-5p, with Smad1 participating downstream of miR-486a-5p in this process. Lentivirus-mediated overexpression of miR-486a-5p was found to alleviate Ang II-induced myocardial interstitial fibrosis in mice. Moreover, miR-486-5p serum levels were lower in patients with heart failure than in healthy controls, and were negatively correlated with NT-proBNP levels.Conclusions: Our study demonstrates that elevated IgE promotes pathological cardiac fibrosis by modulating miR-486a-5p and downstream factors, such as Smad1. These findings suggest new targets for pathological cardiac fibrosis intervention.

MicroRNA-99b-3p promotes angiotensin II-induced cardiac fibrosis in mice by targeting GSK-3β.

Cardiac fibrosis is a typical pathological change in various cardiovascular diseases. Although it has been recognized as a crucial risk factor responsible for heart failure, there is still a lack of effective treatment. Recent evidence shows that microRNAs (miRNAs) play an important role in the development of cardiac fibrosis and represent novel therapeutic targets. In this study we tried to identify the cardiac fibrosis-associated miRNA and elucidate its regulatory mechanisms in mice. Cardiac fibrosis was induced by infusion of angiotensin II (Ang II, 2 mg·kg-1·d-1) for 2 weeks via osmotic pumps. We showed that Ang II infusion induced cardiac disfunction and fibrosis accompanied by markedly increased expression level of miR-99b-3p in heart tissues. Upregulation of miR-99b-3p and fibrotic responses were also observed in cultured rat cardiac fibroblasts (CFs) treated with Ang II (100 nM) in vitro. Transfection with miR-99b-3p mimic resulted in the overproduction of fibronectin, collagen I, vimentin and α-SMA, and facilitated the proliferation and migration of CFs. On the contrary, transfection with specific miR-99b-3p inhibitor attenuated Ang II-induced fibrotic responses. Similarly, intravenous injection of specific miR-99b-3p antagomir could prevent Ang II-infused mice from cardiac dysfunction and fibrosis. We identified glycogen synthase kinase-3 beta (GSK-3β) as a direct target of miR-99b-3p. In CFs, miR-99b-3p mimic significantly reduced the expression of GSK-3β, leading to activation of its downstream profibrotic effector Smad3, whereas miR-99b-3p inhibitor caused anti-fibrotic effects. GSK-3β knockdown ameliorated the anti-fibrotic role of miR-99b-3p inhibitor. These results suggest that miR-99b-3p contributes to Ang II-induced cardiac fibrosis at least partially through GSK-3β. The modulation of miR-99b-3p may provide a new approach for tackling fibrosis-related cardiomyopathy.

Microsomal Prostaglandin E2 Synthase-1 Deletion Attenuates Isoproterenol-Induced Myocardial Fibrosis in Mice.

Deletion of microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibits inflammation and protects against atherosclerotic vascular diseases but displayed variable influence on pathologic cardiac remodeling. Overactivation of β-adrenergic receptors (β-ARs) causes heart dysfunction and cardiac remodeling, whereas the role of mPGES-1 in β-AR-induced cardiac remodeling is unknown. Here we addressed this question using mPGES-1 knockout mice, subjecting them to isoproterenol, a synthetic nonselective agonist for β-ARs, at 5 or 15 mg/kg per day to induce different degrees of cardiac remodeling in vivo. Cardiac structure and function were assessed by echocardiography 24 hours after the last of seven consecutive daily injections of isoproterenol, and cardiac fibrosis was examined by Masson trichrome stain in morphology and by real-time polymerase chain reaction for the expression of fibrosis-related genes. The results showed that deletion of mPGES-1 had no significant effect on isoproterenol-induced cardiac dysfunction or hypertrophy. However, the cardiac fibrosis was dramatically attenuated in the mPGES-1 knockout mice after either low-dose or high-dose isoproterenol exposure. Furthermore, in vitro study revealed that overexpression of mPGES-1 in cultured cardiac fibroblasts increased isoproterenol-induced fibrosis, whereas knocking down mPGES-1 in cardiac myocytes decreased the fibrogenesis of fibroblasts. In conclusion, mPGES-1 deletion protects against isoproterenol-induced cardiac fibrosis in mice, and targeting mPGES-1 may represent a novel strategy to attenuate pathologic cardiac fibrosis, induced by β-AR agonists. SIGNIFICANCE STATEMENT: Inhibitors of microsomal prostaglandin E2 synthase-1 (mPGES-1) are being developed as alternative analgesics that are less likely to elicit cardiovascular hazards than cyclooxygenase-2 selective nonsteroidal anti-inflammatory drugs. We have demonstrated that deletion of mPGES-1 protects inflammatory vascular diseases and promotes post-myocardial infarction survival. The role of mPGES-1 in β-adrenergic receptor-induced cardiomyopathy is unknown. Here we illustrated that deletion of mPGES-1 alleviated isoproterenol-induced cardiac fibrosis without deteriorating cardiac dysfunction. These results illustrated that targeting mPGES-1 may represent an efficacious approach to the treatment of inflammatory cardiovascular diseases.

Matrine attenuates pathological cardiac fibrosis via RPS5/p38 in mice.

Pathological cardiac fibrosis is a common feature in multiple cardiovascular diseases that contributes to the occurrence of heart failure and life-threatening arrhythmias. Our previous study demonstrated that matrine could attenuate doxorubicin-induced oxidative stress and cardiomyocyte apoptosis. In this study, we investigated the effect of matrine on cardiac fibrosis. Mice received aortic banding (AB) operation or continuous injection of isoprenaline (ISO) to generate pathological cardiac fibrosis and then were exposed to matrine lavage (200 mg·kg-1·d-1) or an equal volume of vehicle as the control. We found that matrine lavage significantly attenuated AB or ISO-induced fibrotic remodeling and cardiac dysfunction. We also showed that matrine (200 μmol/L) significantly inhibited the proliferation, migration, collagen production, and phenotypic transdifferentiation of cardiac fibroblasts. Mechanistically, matrine suppressed p38 activation in vivo and in vitro, and overexpression of constitutively active p38 completely abolished the protective effects of matrine. We also demonstrated that ribosomal protein S5 (RPS5) upregulation was responsible for matrine-mediated inhibition on p38 and fibrogenesis. More importantly, matrine was capable of ameliorating preexisting cardiac fibrosis in mice. In conclusion, matrine treatment attenuates cardiac fibrosis by regulating RPS5/p38 signaling in mice, and it might be a promising therapeutic agent for treating pathological cardiac fibrosis.

Protective effect of trimetazidine in radiation-induced cardiac fibrosis in mice.

Radiation-induced heart damage is a serious side effect caused by radiotherapy, especially during the treatment of cancer near the chest. Trimetazidine is effective at reducing inflammation in the heart, but how it affects radiation-induced cardiac fibrosis (RICF) is unknown. To investigate the potential effect and molecular mechanism, we designed this project with a C57BL6 male mouse model supposing trimetazidine could inhibit RICF in mice. During the experiment, mice were randomly divided into six groups including a control group (Con), radiation-damaged model group (Mod) and four experimental groups receiving low-dose (10 mg/kg/day) or high-dose (20 mg/kg/day) trimetazidine before or after radiation treatment. Apart from the control group, all mice chests were exposed to 6 MV X-rays at a single dose of 20 Gy to induce RICF, and tissue analysis was done at 8 weeks after irradiation. Fibroblast or interstitial tissues and cardiac fibrosis-like characteristics were determined using haematoxylin and eosin and Masson staining, which can be used to assess myocardial fibrosis. Immunohistochemical analysis and RT-PCR were used to determine gene expression and study the molecular mechanism. As a result, this study suggests that trimetazidine inhibits RICF by reducing gene expression related to myocyte apoptosis and fibrosis formation, i.e. connective tissue growth factor (CTGF), transforming growth factor (TGF)-β1, smad2 and smad3. In conclusion, by regulating the CTGF/TGF-β1/Smad pathway, trimetazidine could be a prospective drug for clinical treatment of RICF.

Therapeutic Targeting of Interleukin-11 Signalling Reduces Pressure Overload-Induced Cardiac Fibrosis in Mice.

There are currently no specific treatments for cardiac fibrosis. We tested the efficacy of a neutralising anti-IL11 antibody (X203) to reduce cardiac fibrosis in two preclinical models: transverse aortic constriction (TAC) and chronic angiotensin II infusion (AngII). In the first model, male C57BL/6J mice were subjected to TAC for 2weeks. In the second model, mice received continuous angiotensin II for 4weeks via subcutaneous pump. In both models, mice received either 20mg/kg of X203 or isotype-control antibody twice-weekly, starting 24h after surgery. Cardiac fibrosis and extracellular matrix gene expression were assessed by RT-qPCR, Western blot, histology and collagen (hydroxyproline) assays. In both models, X203 significantly reduced pro-fibrotic gene expression and myocardial fibrosis (TAC: 51% reduction in total collagen, P< 0.001, 39% in perivascular fibrosis, P< 0.001; AngII: 17% reduction in total collagen, P= 0.04, 83% in perivascular fibrosis, P< 0.001). Pharmacological targeting of IL11 reduces cardiac fibrosis in preclinical models. Figa Graphical Abstract.

Exosome‐associated ELAVL1 adversely affects cardiac macrophage‐fibroblast crosstalk in diabetes

BackgroundMacrophage dysfunction, inflammation and excessive fibroblast activation has been implicated in the pathogenesis of diabetes‐associated heart disease. However, the paracrine mechanisms are not completely understood. We sought to unravel the influence of diabetes on exosome (Exo) transfer of Human antigen R (HuR or ELAVL1, an mRNA stabilizing protein) and its implications on cardiac fibrosis and dysfunction.Methods and ResultsWe evaluated the invitro and invivo effects of macrophage cellular‐ and exosome‐HuR or its deficiency on paracrine mechanisms of diabetic milieu‐induced inflammatory and fibrosis‐related signaling. Human failing hearts show higher HuR levels as compared to non‐failing hearts. Diabetic milieu (db/db mice) show increase in HuR expression in cardiac‐ and cultured bone marrow derived‐macrophages (BMMac). High glucose (HG, 30mM) stimulates HuR nuclear‐to‐cytoplasmic translocation and transfer of HuR mRNA and protein into exosomes. PKC‐delta inhibition blocks HG‐induced HuR translocation. Exosome from macrophages exposed to diabetic milieu (HG or db/db mice) significantly increases proinflammatory and profibrogenic responses in fibroblast (invitro) or cardiac fibrosis in mice. Exo‐HuR deficiency (HuR knockdown in macrophage) abrogates the above effects. In diabetic mice, macrophage depletion followed by reconstitution with HuR‐deficient‐BMMac decreases ANG II‐induced cardiac fibrosis and improves LV dysfunction as compared to BMMacWT administered mice.ConclusionsTo the best of our knowledge, this is the first study to demonstrate that diabetes‐mediated increase in BMMac‐HuR expression, its cytoplasmic translocation and exosome transfer activates fibrogenesis signaling invitro and invivo, and HuR deficiency attenuates these deleterious effects. The data suggests that HuR might be targeted to alleviate macrophage dysfunction and pathological fibrosis in diabetes mellitus.Support or Funding InformationNational Institutes of Health (NIH) grants HL116729 (to P.K.), HL138023 (to P.K. and J.Z.), and American Heart Association Grant‐in‐aid GRNT 25860041 (to P.K.) and Transformational Project Award 19TPA34850100 (to P.K.).

Moderate Loss of the Extracellular Matrix Proteoglycan Lumican Attenuates Cardiac Fibrosis in Mice Subjected to Pressure Overload

Introduction: The heart undergoes myocardial remodeling during progression to heart failure following pressure overload. Myocardial remodeling is associated with structural and functional changes in cardiac myocytes, fibroblasts, and the extracellular matrix (ECM) and is accompanied by inflammation. Cardiac fibrosis, the accumulation of ECM molecules including collagens and collagen cross-linking, contributes both to impaired systolic and diastolic function. Insufficient mechanistic insight into what regulates cardiac fibrosis during pathological conditions has hampered therapeutic so­lutions. Lumican (LUM) is an ECM-secreted proteoglycan known to regulate collagen fibrillogenesis. Its expression in the heart is increased in clinical and experimental heart failure. Furthermore, LUM is important for survival and cardiac remodeling following pressure overload. We have recently reported that total lack of LUM increased mortality and left ventricular dilatation, and reduced collagen expression and cross-linking in LUM knockout mice after aortic banding (AB). Here, we examined the effect of LUM on myocardial remodeling and function following pressure overload in a less extreme mouse model, where cardiac LUM level was reduced to 50% (i.e., moderate loss of LUM). Methods and Results: mRNA and protein levels of LUM were reduced to 50% in heterozygous LUM (LUM+/–) hearts compared to wild-type (WT) controls. LUM+/– mice were subjected to AB. There was no difference in survival between LUM+/– and WT mice post-AB. Echocardiography revealed no striking differences in cardiac geometry between LUM+/– and WT mice 2, 4, and 6 weeks post-AB, although markers of diastolic dysfunction indicated better function in LUM+/– mice. LUM+/– hearts revealed reduced cardiac fibrosis assessed by histology. In accordance, the expression of collagen I and III, the main fibrillar collagens in the heart, and other ECM molecules central to fibrosis, i.e. including periostin and fibronectin, was reduced in the hearts of LUM+/– compared to WT 6 weeks post-AB. We found no differences in collagen cross-linking between LUM+/– and WT mice post-AB, as assessed by histology and qPCR. Conclusions: Moderate lack of LUM attenuated cardiac fibrosis and improved diastolic dysfunction following pressure overload in mice, adding to the growing body of evidence suggesting that LUM is a central profibrotic molecule in the heart that could serve as a potential therapeutic target.

Intermittent hypoxia mediated by TSP1 dependent on STAT3 induces cardiac fibroblast activation and cardiac fibrosis.

Intermittent hypoxia (IH) is the predominant pathophysiological disturbance in obstructive sleep apnea (OSA), known to be independently associated with cardiovascular diseases. However, the effect of IH on cardiac fibrosis and molecular events involved in this process are unclear. Here, we tested IH in angiotensin II (Ang II)-induced cardiac fibrosis and signaling linked to fibroblast activation. IH triggered cardiac fibrosis and aggravated Ang II-induced cardiac dysfunction in mice. Plasma thrombospondin-1 (TSP1) content was upregulated in both IH-exposed mice and OSA patients. Moreover, both in vivo and in vitro results showed IH-induced cardiac fibroblast activation and increased TSP1 expression in cardiac fibroblasts. Mechanistically, phosphorylation of STAT3 at Tyr705 mediated the IH-induced TSP1 expression and fibroblast activation. Finally, STAT3 inhibitor S3I-201 or AAV9 carrying a periostin promoter driving the expression of shRNA targeting Stat3 significantly attenuated the synergistic effects of IH and Ang II on cardiac fibrosis in mice. This work suggests a potential therapeutic strategy for OSA-related fibrotic heart disease.

Toll-like receptor 5 deficiency diminishes doxorubicin-induced acute cardiotoxicity in mice.

Rationale: Clinical application of doxorubicin (DOX) is limited by its toxic cardiovascular side effects. Our previous study found that toll-like receptor (TLR) 5 deficiency attenuated cardiac fibrosis in mice. However, the role of TLR5 in DOX-induced cardiotoxicity remains unclear.Methods: To further investigate this, TLR5-deficient mice were subjected to a single intraperitoneal injection of DOX to mimic an acute model.Results: Here, we reported that TLR5 expression was markedly increased in response to DOX injection. Moreover, TLR5 deficiency exerted potent protective effects against DOX-related cardiac injury, whereas activation of TLR5 by flagellin exacerbated DOX injection-induced cardiotoxicity. Mechanistically, the effects of TLR5 were largely attributed to direct interaction with spleen tyrosine kinase to activate NADPH oxidase (NOX) 2, increasing the production of superoxide and subsequent activation of p38. The toxic effects of TLR5 activation in DOX-related acute cardiac injury were abolished by NOX2 deficiency in mice. Our further study showed that neutralizing antibody-mediated TLR5 depletion also attenuated DOX-induced acute cardiotoxicity.Conclusion: These findings suggest that TLR5 deficiency attenuates DOX-induced cardiotoxicity in mice, and targeting TLR5 may provide feasible therapies for DOX-induced acute cardiotoxicity.

Up-regulation of paired-related homeobox 2 promotes cardiac fibrosis in mice following myocardial infarction by targeting of Wnt5a.

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.

Targeting exosome-associated human antigen R attenuates fibrosis and inflammation in diabetic heart.

RNA-binding proteins like human antigen R (HuR) are key regulators in post-transcriptional control of gene expression in several pathophysiological conditions. Diabetes adversely affects monocyte/macrophage biology and function. It is not known whether diabetic milieu affects cellular/exosome-HuR and its implications on cardiac inflammation and fibrosis. Here, we evaluate in vitro and in vivo effects of diabetic milieu on macrophage cellular/exosome-HuR, alterations in intercellular cross talk with fibroblasts, and its impact on cardiac remodeling. Human failing hearts show higher HuR levels. Diabetic milieu activates HuR expression in cardiac- and cultured bone marrow-derived macrophages (BMMØ) and stimulates HuR nuclear-to-cytoplasmic translocation and exosome transfer. Exosomes from macrophages exposed to diabetic milieu (high glucose or db/db mice) significantly increase inflammatory and profibrogenic responses in fibroblast (in vitro) and cardiac fibrosis in mice. Intriguingly, Exo-HuR deficiency (HuR knockdown in macrophage) abrogates the above effects. In diabetic mice, macrophage depletion followed by reconstitution with BMMØ-derived HuR-deficient exosomes inhibits angiotensin II-induced cardiac fibrosis response and preserves left ventricle function as compared to control-exosome administration. To the best of our knowledge, this is the first study to demonstrate that diabetes activates BMMØ HuR expression and its transfer into exosome. The data suggest that HuR might be targeted to alleviate macrophage dysfunction and pathological fibrosis in diabetes.

P1625A pivotal role for cytotoxic CD8+ T cells in development of cardiac fibrosis

Abstract Background/Introduction Pressure overload-induced cardiac fibrosis increases myocardial stiffness leading to reductions in cardiac performances and cardiac failure. CD8+ T cells have been shown to accumulate during development of fibrosis but their role has not been defined. Purpose We examined the role and significance of CD8+ T cells in development of cardiac fibrosis. Methods Trans-aortic constriction (TAC) or 2-kidney-1-clip (2K1C) procedures were used to generate pressure overload-induced cardiac fibrosis in mice. Rat anti-mouse CD8β (lyt-3) monoclonal antibody (clone YTS 156.7) was used to deplete CD8+ T cells. A mixed bone marrow chimera strategy was used to specifically delete innate receptor Natural Killer Group 2D (NKG2D) or cytotoxin perforin from CD8+ T cells. Results Depleting CD8+ T cells in mice subjected to TAC or 2K1C-renal hypertension attenuated left ventricular fibrosis by 93% and 84% without affecting blood pressure. In TAC mice this was associated with a 68% reduction in apoptotic cardiomyocytes, a 74% reduction in macrophage accumulation, a 65% reduction in TGF-beta positive cells and a 95% reduction in TGF-beta positive macrophages, whilst CD4+ T cells were unaffected. Cardiomyocytes in regions of developing cardiac fibrosis contained cytoplasmic DNA and expressed the NKG2D ligand, Rae-1, indicative of activation of a DNA damage response; CD8+ T cells expressed the NKG2D receptor. Deletion of the NKG2D receptor from CD8+ T cells attenuated cardiac fibrosis by 82%; deletion of cytotoxin perforin has similar effects. Conclusion(s) We conclude that CD8+ T cells contribute to development of cardiac fibrosis by targeting stressed/damaged cardiomyocytes via an NKG2D-Rae-1 cytotoxic mechanism inducing their apoptosis. Macrophages then accumulate in the heart in response to increased numbers of apoptotic cardiomyocytes, clearing the apoptotic cells through engulfment and increasing their expression of the pro-fibrotic factor TGF-beta1. Acknowledgement/Funding The National Health and Medical Research Council of Australia

Effect of dietary fat and sucrose consumption on cardiac fibrosis in mice and rhesus monkeys.

Calorie restriction (CR) improved health span in 2 longitudinal studies in nonhuman primates (NHPs), yet only the University of Wisconsin (UW) study demonstrated an increase in survival in CR monkeys relative to controls; the National Institute on Aging (NIA) study did not. Here, analysis of left ventricle samples showed that CR did not reduce cardiac fibrosis relative to controls. However, there was a 5.9-fold increase of total fibrosis in UW hearts, compared with NIA hearts. Diet composition was a prominent difference between the studies; therefore, we used the NHP diets to characterize diet-associated molecular and functional changes in the hearts of mice. Consistent with the findings from the NHP samples, mice fed a UW or a modified NIA diet with increased sucrose and fat developed greater cardiac fibrosis compared with mice fed the NIA diet, and transcriptomics analysis revealed diet-induced activation of myocardial oxidative phosphorylation and cardiac muscle contraction pathways.

RASSF1-AS1, an antisense lncRNA of RASSF1A, inhibits the translation of RASSF1A to exacerbate cardiac fibrosis in mice.

Cardiac fibrosis is associated with various cardiovascular diseases and can eventually lead to heart failure. Dysregulation of long non-coding RNAs (lncRNAs) are recognized as one of the key mechanisms of cardiac diseases. However, the roles and underlying mechanisms of lncRNAs in cardiac fibrosis have not been explicitly defined. Here, we investigated the role of an antisense (AS) lncRNA from the Ras association domain-containing protein 1 isoform A (RASSF1A) gene locus, named RASSF1-AS1, in the development of cardiac fibrosis. Cardiac fibrosis mouse model was established by isoproterenol injection. We found that RASSF1A protein was downregulated, whereas RASSF1-AS1 was markedly upregulated during cardiac fibrosis. Overexpression and knockdown of mouse primary cardiac fibroblasts showed that RASSF1-AS1 negatively regulated RASSF1A expression at the post-transcriptional level. According to the landscape analysis and sense-AS binding evaluation, RASSF1-AS1 partially overlaps with RASSF1A messenger RNA (mRNA) at the exon2 region. RNA pull-down and luciferase activity assays confirmed that RASSF1-AS1 directly bound to RASSF1A mRNA and suppressed its translation. Furthermore, wild-type RASSF1-AS1 had a promoting effect on nuclear factor-κB activation and cardiac fibrosis, but mutated RASSF1-AS1, in which the binding region was deleted, had no effect. In conclusion, RASSF1-AS1 inhibits the translation of RASSF1A to exacerbate cardiac fibrosis in mice, indicating a potential application of RASSF1-AS1 as a therapy target for cardiac fibrosis.

Upregulation of Paired-Related Homeobox 2 Promotes Cardiac Fibrosis in Mice Following Myocardial Infarction by Targeting of Wnt5a

Backgrounds: Cardiac fibrosis is a key factor to determine the prognosis in patient with myocardial infarction (MI). Activation of Wnt signaling by transforming growth factor β (TGF-β) contributes to cell differentiation of fibroblasts in cardiac remodeling. However, the molecular mechanisms by how Wnt protein family 5a (Wnt5a) is upregulated in post-ischemic heart are still mostly unclear. The aim of this study is to investigate whether paired-related homeobox 2 (Prrx2), as a transcriptional factor, regulates Wnt5a gene expression in myocardial fibrosis, and, if so, to identify the role of Prrx2 in the recovery of cardiac functions in mice following MI. Methods: The MI surgery was performed by ligation of left anterior descending coronary artery. Cardiac remodeling was assessed by measuring interstitial fibrosis using Masson staining. Cell differentiation was examined by immunofluorescence analysis of alpha smooth muscle actin (α-SMA). Results: Both Prrx2 and Wnt5a gene expressions were upregulated in Apoe-/- mice following MI, accompanied with increased mRNA and protein levels of α-SMA, collagen I, and collagen III in hearts, compared to mice with sham surgery. Adenovirus-mediated gene knockdown of Prrx2 increased survival rate, alleviates cardiac fibrosis, decreased infarction sizes in hearts, and improved cardiac functions in mice with MI after 30 post-ischemia days. Importantly, inhibition of Prrx2 suppressed ischemia-induced Wnt5a gene expression and Wnt5a signaling in mice hearts. 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 upregulate Wnt5a gene expression. Conclusion: Upregulation of Prrx2 increases Wnt5a gene expression to induce cell differentiations, which contributes to ischemia-induced cardiac fibrosis. In perspective, targeting Prrx2-Wnt5a signaling should be considered to improve cardiac remodeling in patients with ischemic heart diseases. Funding: This project was supported by National Natural Science Foundation of China (81874312, 81770576, 81770493, U1704168, 81673423, and 81570723) and the Natural Science Foundation of Shandong Province (ZR2019PH007, ZR2019QH011, ZR2016HM04). S.X.W. is an exceptional young scholar of Shandong University, a distinguished professor of Henan province, a recipient of Zhong-Yuan-Qian-Ren Program of Henan province (194200510005), an adjunct Tai-Hang Professional Scholarship of Xinxiang Medical University (505067), and an adjunct Lan-Yue Professional Scholar of Hubei University of Science and Technology (2016-18XZ07). L.Z. was supported by general program of Department of Health (B20180875) and clinical medical technology innovation guidance project from Department of Science and Technology (2017SK50504) of Hunan Province. W.D.Q. was sponsored by China Postdoctoral Science Foundation (2018M630788). Declaration of Interest: None. Ethical Approval: The animal protocol was reviewed and approved by the University of Shandong, Animal Care and Use Committee. This animal study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Androgen receptor regulates cardiac fibrosis in mice with experimental autoimmune myocarditis by increasing microRNA-125b expression

Cardiac fibrosis is an important cardiac remodeling event in the development of inflammation dilated cardiomyopathy （iDCM）. We have previously observed that degradation enhancer of androgen receptor (ASC-J9®) could improve cardiac inflammation and fibrosis. Using Primary CFs, we demonstrated that ASC-J9® attenuates the expression of miR-125b, which subsequently inhibits the generation of collagen. In contrast, overexpressed AR in CFs induced collagen production, increases mir-125b.We also found that inhibition of miR-125b attenuates fibrosis which induced by the overexpression of AR. Our results indentify the functional role for AR as a regulator of cardiac fibrosis due to myocarditis and further show that AR exerts its effect by increasing microRNA-125b expression. Treatment with degradation enhancer of AR limits cardiac fibrosis in iDCM, thereby providing potentially a therapeutic approach for patients with iDCM.

A9871 Insulin regulated aminopeptidase (irap) inhibition completely reverses age-induced cardiac fibrosis and improves cardiac function

Objectives: There is an urgent need to discover targets that can reverse established fibrosis that occurs with advanced ageing and in many disease states thus improving organ function. The aim of this study was to determine whether inhibition or deficiency of IRAP would reverse age-induced cardiac fibrosis and improve left ventricular function in 2 year old mice. Methods: Cardiovascular fibrosis and function (using echocardiography) was assessed in young (∼4–5 months old) and aged (∼22–24 months old) WT (C57BL/6J) or IRAP KO mice. Aged WT mice were chronically treated with either the synthetic IRAP inhibitor (HFI-419; 500 ng/kg/min) or vehicle for 4 or 8 weeks. Results: Ageing resulted in marked interstitial cardiac fibrosis and increased IRAP expression that was co-localised with myofibroblasts. Global IRAP gene deletion protected against age-induced fibrosis and resulted in improved left ventricular function. Importantly chronic treatment with 3 structurally-unrelated specific IRAP inhibitors for 4 weeks reversed established cardiac fibrosis in aged mice. Mechanisms involved down-regulation of collagen synthesis, increased collagen degradation and reduced tissue inflammation. In separate animals, we examined if HFI-419 treatment for 8 weeks improved left ventricular function assessed by serial echocardiography at weeks 0, 4 and 8. Our preliminary data suggest improvement of left ventricular function within 4 weeks of HFI-419 treatment and was associated with reduced cardiac fibrosis in these mice. Conclusion: Our data provides compelling evidence that inhibition of IRAP represents a novel therapeutic strategy for treatment of established cardiac fibrosis leading to improved organ function.

Tanshinone IIA protects against subclinical lipopolysaccharide induced cardiac fibrosis in mice through inhibition of NADPH oxidase

Myocardial fibrosis plays a central role in the development of heart failure. It has been shown that recurrent exposure to subclinical lipopolysaccharide (LPS) increases mortality and induces cardiac fibrosis in mice, which is not mediated by the common renin-angiotensin system. LPS increased NADPH oxidase2 (NOX2) in isolated adult mouse cardiac fibroblasts and NOX2 may mediate LPS-induced cardiac fibrosis. Therefore, the current study was designed to delineate the role of NOX2 in LPS-induced fibrosis model and to investigate the preventive role of Tanshinone IIA (TIIA) on the development of cardiac fibrosis. The protective mechanism of TIIA was determined to be associated with the inhibition of NOX2, by comparing its effects with the NADPH oxidase inhibitor, apocynin. The results revealed remarkable effects of apocynin and TIIA on attenuating the development of myocardial fibrosis and fibrosis-related genes and mediators. Furthermore, TIIA and apocynin decreased the expression of NADPH oxidase subunits (NOX2 and P67phox) expression and the ROS levels. The anti-fibrotic effect of apocynin suggested that NOX2 inhibition may be a potential preventive strategy for attenuating the progression of LPS-induced cardiac fibrosis. Our results demonstrate that TIIA may be a potent agent against subclinical LPS-induced cardiac fibrosis in mice partially via inhibition of NADPH oxidase 2.

Dopamine Receptor D3 Agonist (Pramipexole) Abolishes Morphine‐Induced Cardiac Fibrosis in Mice

Prolonged morphine exposure leads to desensitization of the G protein‐coupled mu‐opioid receptor (MOR). Additionally, extended morphine exposure has been shown to associate with cardiac dysfunction. This is problematic for patients undergoing withdrawal from extended morphine pain management, especially those with pre‐existing heart conditions. Recent literature suggests mechanistic interactions between MOR and the dopamine receptor D3 (DRD3). The purpose of this study was to investigate whether a DRD3 agonist (pramipexole, 0.5 mg/kg/day, i.p.; DRD3ag) could reduce morphine‐induced cardiac dysfunction in mice. Thirty mice were randomly divided into 5 groups (G1–G5, Figure 1) and treated with morphine (2 mg/kg/day, i.p.) for 7 days (D7). Two groups were euthanized after morphine tolerance was established at D7 (G1, morphine only; G2, morphine + DRD3ag). The other 3 groups (D14) underwent a subsequent 7‐day period of morphine withdrawal (G3, withdrawal no treatment; G4, morphine + DRD3ag until D7 followed by withdrawal until D14; G5, morphine until D7 followed by withdrawal + DRD3ag until D14). Echocardiography was performed on each animal at D0 to establish a baseline for cardiac function, on D7, and again on D14. Histology and immunoblotting were conducted on the left ventricle (LV) to measure tissue fibrosis, myocyte hypertrophy, and DA receptor expression. Our data show that morphine treatment for 7 days followed by withdrawal induced cardiomyocyte hypertrophy and significantly increased myocardial fibrosis (both at D7 and D14). Picrosirius red staining showed that DRD3ag treatment completely abolished morphine‐induced fibrosis, independent of treatment time. Moreover, significantly decreased cardiomyocyte hypertrophy was noted in all groups receiving DRD3ag in the morphine withdrawal phase. Immunoblotting shows that morphine withdrawal significantly reduced DRD3 expression independent of the presence of DRD3ag. Our results suggest that using a DRD3ag as an adjunctive therapy with morphine could decrease morphine‐induced cardiac fibrosis, and subsequently blunt morphine‐dependent cardiac dysfunction. Further, our data suggest that the DRD3 agonist pramipexole has cardioprotective potential when administered during morphine withdrawal.Support or Funding InformationThe Brody School of Medicine Research Distinction Track, the Department of Physiology, and the Research and Graduate School at East Carolina University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.