Cardiac Fibroblast Proliferation Research Articles

4260Loss of Pnn in cardiomyocytes results in impairment of intercalated discs and arrhythmogenic cardiomyopathy in mice

Abstract Background Pnn, a multiple functional protein that localizes within nucleus and in the cytoplasmic region of desmosome. Although recent studies have characterized the physiological role of nuclear Pnn in regulating pre-mRNA alternative splicing and gene transcription, the function of Pnn in desmosome, particularly in cardiomyocytes, remains unidentified. Purpose and methods In the present study, we applied an inducible cardiomyocyte-specific Pnn depletion mouse model (Myh6-CreERT2, Pnnflox/flox) to observe the impact of loss of Pnn on cardiac structure and function in adult mice. Results Six weeks after tamoxifen injection to induce cardiac Pnn depletion, the electrocardiographic abnormalities and decrease of left ventricular ejection fraction (LVEF) were observed. Histopathological examination showed that the proliferation of cardiac fibroblasts and expression of α-smooth muscle actin were increased with Pnn depletion, while the cell-cell connection among cardiomyocytes were impaired. In addition, cellular hypertrophy and decrease of capillary density were also found. Results from Immunofluorescent staining further showed that the distribution pattern of desmosomal proteins and adherent junctional proteins, including desmoglein, desmocollin, plakoglobin, plakophllin, desmoplakin and β-catenin were altered in the cardiomyocytes with Pnn depletion. Both desmosomal and adherent junctional proteins were translocated form cell-cell junction to cytoplasm, indicating the disruption of intercalated discs in Pnn-depleted cardiomyocytes. Microarray data also indicated that loss of cardiac Pnn regulated the mRNA expression of genes responsible for extracellular matrix-receptor interaction, ribosome, and integrin. In addition, expression levels of oxidative stress-associated proteins were significantly regulated by Pnn depletion. Impairment of intercalated disc Conclusion Results from present study indicated that Pnn plays an essential role in the maintenance of intercalated disc integrity of cardiomyocytes, while cardiomyocyte-specific loss of Pnn leads to arrhythmogenic cardiomyopathy in mice. Acknowledgement/Funding Ministry of Science and Technology Taiwan, MOST-106-2320-B-182A-010-MY3

P4475Vasodilation- and blood pressure normalization-independent cardioprotective effects of endogenous, physical activity-induced alpha calcitonin gene-related peptide in chronic hypertension

Abstract Background Alpha calcitonin gene-related peptide (αCGRP) is one of the strongest vasodilators and, as such, is cardioprotective in chronic hypertension when reducing the associated elevated blood pressure. However, we hypothesize that endogenous, physical activity-induced αCGRP has blood pressure independent cardioprotective effects in chronic hypertension. Methods Chronic hypertension was induced in WT and αCGRP−/− mice by one-kidney one-clip surgery. Chronic hypertensive WT and αCGRP−/− mice lived sedentarily or performed voluntary wheel running and were treated simultaneously with either vehicle, αCGRP or αCGRP receptor antagonist CGRP8–37. Cardiac function and tissue phenotype were evaluated echocardiographically and by ddPCR, Western blotting and histology, respectively. Results Blood pressure was similar among all hypertensive experimental groups. Endogenous αCGRP limited pathological cardiac remodeling and symptomatic heart failure already in sedentary, chronic hypertensive WT mice. In these mice, voluntary wheel running significantly improved cardiac tissue phenotype and function, that was abolished by CGRP8–37 treatment. In αCGRP−/− mice, αCGRP treatment, in contrast to voluntary wheel running, improved cardiac tissue phenotype and function. Specific inhibition of proliferation and myofibroblast differentiation of primary murine cardiac fibroblasts by αCGRP suggests involvement of these cells in αCGRP-mediated blunting of pathological cardiac remodeling. Conclusion Endogenous, physical activity-induced αCGRP has blood pressure independent cardioprotective effects and is crucial for maintaining cardiac function in chronic hypertension. Consequently, permanently inhibiting endogenous αCGRP signaling, as currently approved for migraine prophylaxis, could endanger hypertensive patients. Acknowledgement/Funding Swiss National Science Foundation, Novartis Foundation for Medical-biological Research

P2556TXNDC5 is a novel therapeutic target of atrial fibrosis and fibrillation

Abstract Background Atrial fibrillation (AF), one of the most common cardiac arrhythmias, increases the risk of stroke, systemic embolization and cardiovascular mortality. Atrial fibrosis, a hallmark of chronic AF, provides substrates to initiate/propagate fibrillation waves in the atria. There, however, lacks effective and specific therapeutics targeting atrial fibrosis. We have recently identified an endoplasmic reticulum (ER) protein thioredoxin domain containing 5 (TXNDC5) as a critical mediator of cardiac ventricular fibrosis. We hypothesized that TXNDC5 could also play an important role in the pathogenesis of atrial fibrosis and fibrillation. Purpose To determine the role of TXNDC5 in atrial fibrosis and fibrillation. Methods and results TXNDC5 transcript and protein levels were both significantly upregulated in the atrial tissue from patients with AF. In addition, TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 (TGFβ1) and extracellular matrix (ECM) proteins in human atrial tissue. Knockdown of TXNDC5 in human atrial fibroblasts (hAF) attenuated TGFβ1–induced hAF activation, proliferation and ECM protein upregulation, whereas overexpression of TXNDC5 was sufficient to trigger hAF activation, proliferation and ECM protein production. Further experiments revealed that the fibrogenic effects of TXNDC5 were dependent on c-Jun N-terminal kinase (JNK) signaling. Furthermore, using α-MHC-TGFβcys33ser mice, a transgenic mouse model with cardiac-specific overexpression of constitutively active TGFβ, which develop extensive atrial fibrosis and inducible AF, we showed that TXNDC5 was strongly upregulated in the fibrotic atria of α-MHC-TGFβcys33ser mice and specifically enriched in collagen-secreting atrial fibroblasts. Targeted deletion of TXNDC5 (Txndc5−/−) in α-MHC-TGFβcys33ser mice considerably mitigated the extent of atrial fibrosis. In addition, transesophageal atrial burst pacing induced AF in 75% (3 out of 4) α-MHC-TGFβcys33ser mice, whereas knockout of Txndc5 markedly reduced the inducibility of AF (25%, 3 out of 12) in α-MHC-TGFβcys33ser mice (Figure). TXNDC5 KO Reduces AF Inducibility Conclusion The present study revealed that ER protein TXNDC5 augments atrial fibrosis by promoting cardiac fibroblast proliferation and ECM protein production via JNK signaling activation. Targeted deletion of Txndc5 protects against TGFβ induced atrial fibrosis and AF. Targeting TXNDC5, therefore, could be a promising new therapeutic approach to treat or prevent atrial fibrosis and AF.

Hippo pathway deletion in adult resting cardiac fibroblasts initiates a cell state transition with spontaneous and self-sustaining fibrosis.

Cardiac fibroblasts (CFs) respond to injury by transitioning through multiple cell states, including resting CFs, activated CFs, and myofibroblasts. We report here that Hippo signaling cell-autonomously regulates CF fate transitions and proliferation, and non-cell-autonomously regulates both myeloid and CF activation in the heart. Conditional deletion of Hippo pathway kinases, Lats1 and Lats2, in uninjured CFs initiated a self-perpetuating fibrotic response in the adult heart that was exacerbated by myocardial infarction (MI). Single cell transcriptomics showed that uninjured Lats1/2 mutant CFs spontaneously transitioned to a myofibroblast cell state. Through gene regulatory network reconstruction, we found that Hippo-deficient myofibroblasts deployed a network of transcriptional regulators of endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) consistent with elevated secretory activity. We observed an expansion of myeloid cell heterogeneity in uninjured Lats1/2 CKO hearts with similarity to cells recovered from control hearts post-MI. Integrated genome-wide analysis of Yap chromatin occupancy revealed that Yap directly activates myofibroblast cell identity genes, the proto-oncogene Myc, and an array of genes encoding pro-inflammatory factors through enhancer-promoter looping. Our data indicate that Lats1/2 maintain the resting CF cell state through restricting the Yap-induced injury response.

The deficiency of miR-214-3p exacerbates cardiac fibrosis via miR-214-3p/NLRC5 axis.

Cardiac fibrosis is a common pathological feature of many cardiovascular diseases. The regulatory mechanisms of miRNAs in cardiac fibrosis are still unknown. Previous studies on miR-214-3p in cardiac fibroblasts reached contradictory conclusions. Thus the role of miR-214-3p in cardiac fibrosis deserves further exploration. Using a combination of in vitro and in vivo studies, we identified miR-214-3p as an important regulator of cardiac fibrosis, and the proliferation and activation of cardiac fibroblasts. We demonstrated that the expression of miR-214-3p is down-regulated in TGF-β1-treated myofibroblasts and transverse aortic constriction (TAC)-induced murine model. Additionally, miR-214-3pflox/flox/FSP1-cre mice and miR-214-3pwt/wt/FSP1-cre mice were subjected to TAC operation or sham operation, and the conditional knockout of miR-214-3p in cardiac fibroblasts aggravates TAC-induced cardiac fibrosis. In vitro, our results indicate that miR-214-3p is an important repressor for fibroblasts proliferation and fibroblast-to-myofibroblast transition by functionally targeting NOD-like receptorfamilyCARD domaincontaining 5 (NLRC5). In conclusion, our findings show that the deficiency of miR-214-3p exacerbates cardiac fibrosis and reveal a novel miR-214-3p/NLRC5 axis in the regulation of cardiac fibrosis.

Upregulation of lncRNA RMRP promotes the activation of cardiac fibroblasts by regulating miR‑613.

Long non-coding RNAs (lncRNAs) have been reported to serve a key role in a variety of cardiovascular diseases, including in cardiac fibrosis. The present study aimed to investigate the biological role and underlying mechanisms of the induction of cardiac fibroblasts by the lncRNA, RNA component of mitochondrial RNA processing endoribonuclease (RMRP). The results demonstrated that RMRP expression was upregulated in the presence of cardiac fibrosis in an abdominal aortic banding-treated rat model. Treatment with angiotensin II increased RMRP expression in cardiac fibroblasts, while the knockdown of RMRP by small-interfering RNA inhibited cardiac fibroblast proliferation, differentiation and collagen accumulation. To further investigate the underlying mechanisms of this interaction, microRNA (miR)-613 was predicted to be a target miR of RMRP and sequence alignment, luciferase activity and MS2 RNA immunoprecipitation were performed to detect the interaction between RMRP and miR-613. The results suggested that RMRP negatively regulated miR-613 in cardiac fibroblasts. Furthermore, miR-613 was indicated to mediate the promoting effect of RMRP on cardiac fibroblast activation. The current study suggested that RMRP promoted cardiac fibroblast activation by acting as a competing endogenous RNA for miR-613. Therefore, RMRP may be a novel target for the prevention or treatment of cardiac fibrosis.

MiR-32-5p influences high glucose-induced cardiac fibroblast proliferation and phenotypic alteration by inhibiting DUSP1

BackgroundThe current study aimed to investigate the effects of miR-32-5p on cardiac fibroblasts (CFs) that were induced with high levels of glucose; we also aimed to identify the potential mechanisms involved in the regulation of DUSP1 expression.MethodsHuman CFs were transfected with a miR-32-5p inhibitor or mimic and were treated with a normal concentration or a high concentration of glucose. Flow cytometry analysis was performed to identify cardiac fibroblasts by examining vimentin, fibronectin (FN) and α-actin expression in human CFs. qRT-PCR and western blot assays were performed to confirm the expression of miR-32-5p, DUSP1 and cardiac fibrosis relevant proteins. The proliferation of CFs was assessed by using MTT assay. An immunocytofluorescent staining assay was performed to determine the protein level of α-SMA and to investigate the degree of phenotypic changes in human CFs. The specific relationship between miR-32-5p and DUSP1 was investigated by a dual luciferase reporter assay. Cell apoptosis rates were measured with flow cytometry and the annexin V-FITC and propidine iodide (PI) staining method.ResultsA luciferase reporter assay indicated that miR-32-5p could directly target DUSP1. High glucose levels resulted in the overexpression of miR-32-5p, which downregulated DUSP1 expression. Both the upregulation of miR-32-5p and the downregulation of DUSP1 promoted cell apoptosis, proliferation and phenotypic changes in human CFs.ConclusionsAll findings in this study provide further evidence for the positive effects of miR-32-5p on cell proliferation and the phenotypic changes in CFs by inhibiting DUSP1 expression, and reveal that miR-32-5p could serve as prognostic diagnostic target for cardiac fibrosis.

Sophora alopecuroides L.: An ethnopharmacological, phytochemical, and pharmacological review

Sophora alopecuroides L., which is called Kudouzi in China, is a medicinal plant distributed in Western and Central Asia, especially in China, and has been used for decades to treat fever, bacterial infection, heart disease, rheumatism, and gastrointestinal diseases. This review aims to provide up-to-date information on S. alopecuroides, including its botanical characterization, medicinal resources, traditional uses, phytochemistry, pharmacological research, and toxicology, in exploring future therapeutic and scientific potentials. The information related to this article was systematically collected from the scientific literature databases including PubMed, Google Scholar, Web of Science, Science Direct, Springer, China National Knowledge Infrastructure, published books, PhD and MS dissertations, and other web sources, such as the official website of Flora of China and Yao Zhi website (https://db.yaozh.com/). A total of 128 compounds, such as alkaloids, flavonoids, steroids, and polysaccharides, were isolated from S. alopecuroides. Among these compounds, the effects of alkaloids, such as matrine and oxymatrine, were extensively studied and developed into new drugs. S. alopecuroides and its active components had a wide range of pharmacological activities, such as anticancer, antiviral, anti-inflammatory, antimicrobial, analgesic, and neuroprotective functions, as well as protective properties against pulmonary fibrosis and cardiac fibroblast proliferation. As an important traditional Chinese medicine, modern pharmacological studies have demonstrated that S. alopecuroides has prominent bioactivities, especially on gynecological inflammation and hepatitis B, and anticancer activities. These activities provide prospects for novel drug development for cancer and some chronic diseases. Nevertheless, the comprehensive evaluation, quality control, understanding of the multitarget network pharmacology, long-term in vivo toxicity, and clinical efficacy of S. alopecuroides require further detailed research.

MeCP2 triggers diabetic cardiomyopathy and cardiac fibroblast proliferation by inhibiting RASSF1A

Diabetes causes cardiomyopathy and increases the risk of heart failure independent of hypertension and cardiac fibrosis disease. However, the molecular mechanism of cardiomyopathy caused by diabetic (DCM) is currently unknown. Here we explore the role of the Methyl CpG binding protein 2 (MeCP2) in DCM patients and a type 1 DM (T1DM) rat model. In this study, we employed streptozotocin (STZ)-induced rats DCM and DCM patient and found that MeCP2 triggers cardiac fibroblast proliferation in DCM by inhibiting of RASSF1A expression. Moreover, the in vitro study demonstrated that high glucose inhibited RASSF1A expression, accompanied by the increases of MeCP2 expression and DNA hypermethylation in RASSF1A promoter region. MeCP2 inhibition or knockdown reversed the decrease of RASSF1A transcription induced by high glucose in cardiac fibroblasts. MeCP2 triggers cardiac fibroblasts proliferation through the activation of RASSF1A/ERK1/2 signaling pathways. Our results demonstrated that MeCP2 plays a key role in RASSF1A mediated ERK1/2 activation in DCM. Taken together, these indicate that MeCP2 acts as a key regulator of DCM and cardiac fibroblasts proliferation.

Abstract 251: Mitochondrial Calcium Uniporter Regulates Proliferative Activity of Cardiac Fibroblasts Under Angiotensin II Stimulation

Introduction: Cardiac fibrosis persists in patients with heart failure (HF) even during treatment with conventional HF therapies designed to control volume/pressure overload and restore cardiomyocyte function. This suggests a critical need to develop novel and effective anti-fibrotic therapies specifically targeting cardiac fibroblasts (CFs). CFs contribute to pathophysiological remodeling such as cardiac fibrosis after cardiac injury and/or during repair in response to various profibrotic stimuli in vivo such as angiotensin II (Ang II). Oxidative stress promotes pathological CF proliferation, but the detailed molecular mechanisms linking mitochondria and CF proliferation remain unclear. Hypothesis: Ang II stimulation promotes mitochondrial ROS (mROS) generation via mitochondrial Ca 2+ (mCa 2+ ) uptake, which subsequently activates mROS-dependent proliferative signaling in CFs. Methods: In CFs isolated from rat hearts, mCa 2+ uptake, mROS levels and mitochondrial morphology were measured using confocal microscopy with a mitochondria-targeted Ca 2+ biosensor mtRCamp1h, a mitochondrial superoxide-sensitive dye MitoSOX, and mitochondria-targeted GFP, respectively.. Results: Ang II (≥1 μM) stimulation induces Ca 2+ release from the endoplasmic reticulum (ER) followed by increased mCa 2+ , mROS, and mitochondrial fragmentation. In addition, Ang II activates ERK1/2- and p38-mediated proliferative pathways, which was inhibited by pretreatment with losartan, an Ang II receptor antagonist. Overexpression of a dominant-negative mCa 2+ uniporter (MCU) with pore domain mutations abolished Ang II-mediated mCa 2+ uptake and mROS generation, and prevented the activation of proliferative pathways. Pretreatment with a mitochondria-targeted antioxidant, mitoTEMPO, also significantly inhibited Ang II-mediated activation of the proliferative pathways without affecting ER-Ca 2+ release or mCa 2+ -uptake. Conclusion: The mROS generation via mCa 2+ accumulation and mitochondrial fragmentation resulting from Ang II stimulation activates mROS-dependent proliferative signaling pathways in CFs. The MCU-dependent mROS generation may serve as an important molecular switch for CF proliferation during cardiac stress.

Abstract 521: Cardiac Fibroblasts are Activated During Postnatal Extracellular Matrix Remodeling

Objectives: During the postnatal period, P0 to P30 in the mouse, the myocardial extracellular matrix (ECM) transitions to a mature profile necessary for increased cardiac output. We hypothesize that, during the postnatal period, cardiac fibroblasts (CF) become activated, as indicated by Periostin (Postn) expression, and remodel the ECM, followed by quiescence in the mature heart. Our goal is to study CF activation and its role in postnatal heart development. Methods: CF activation was monitored by Postn MerCreMer(MCM) ;Rosa GFP lineage analysis and the resident CF were studied using a TCF21 MCM ;R26 GFP model. Tamoxifen induction of Cre MCM was initiated in the postnatal period and lineage-specific CF were quantified. Cardiac ECM maturation and CF proliferation, together with bone marrow-derived and endothelial cells, were assessed by Immunofluorescence (IF). TCF21 , Postn , Col1a1 , TnnI and CD31 transcripts were examined by RNAscope, and Fibronectin (FN) compartmentalization, an indicator of ECM maturation, also was examined. Results: FN, Postn, and Postn MCM R26 GFP lineage cells were more prominent at P0-7 than at P30. Postn -expressing cells also co-express Tcf21 and Col1a1 , but not TnnI (myocytes) or PECAM (endothelial cells), as determined by RNAscope. In agreement, Postn MCM R26 GFP CF do not express CD31, CD45, or the myofibroblast marker alphaSMA at P7. At P30, Postn, FN, and Col1a1 expression is reduced in CF, but TCF21 MCM ;R26 GFP is maintained, suggesting a transient period of CF activation followed by quiescence. Similarly, proliferation rates of the Postn MCM R26 GFP cells are higher than TCF21 MCM ;R26 GFP CF at P7, followed by decreased proliferation at P30. Conclusions: Activation and proliferation of CFs and ECM gene expression is increased in the week after birth relative to quiescent CFs at P30. Postn+ cells also peak at P7 but are not detected in appreciable numbers at P30, suggesting a critical role in ECM remodeling and myocardial maturation in the postnatal period. Studies are ongoing to determine the codependence of postnatal activated CF, ECM maturation and myocardial regeneration.

Abstract 846: The Effects and Mechanisms of Pyridostigmine on Cell Proliferation and Fibrosis of Human Cardiac Fibroblasts

Background: Heart failure (HF) is a common, costly, and potentially fatal condition. Cardiac fibrosis plays a key role in the process of HF. Pyridostigmine (PYR) could improve the cardiac function by inhibiting collagen deposition in cardiac tissue, but the molecular mechanisms are unknown. Objective: To investigate the effects and mechanisms of Pyridostigmine on cell proliferation and fibrosis in human cardiac fibroblasts (HCFs). Methods: The model of fibrosis in HCFs was induced by transforming growth factor beta. After the intervention of PYR, the content of collagen I(COL1) was observed by measuring the hydroxyproline concentration. The cell cycle of HCFs was screened by Flow cytometry. The mRNA levels of Connective tissue growth factor (CTGF) and Smad3 were examined by real-time qPCR analysis. The expressions of CTGF, Smad3, pSmad3 were examined by Western blot. Results: Compared with the TGF-β group, the TGF-β+PYR group had significantly lower proportion of S/G2 phased cells[(30.8±1.4) % vs (48.6±3.5)%], less synthesis of COL1 [(0.95±0.15) vs (1.25±0.18)]and fewer expressions of CTGF mRNA [(1.73±0.10) vs (2.92±0.12)]and CTGF protein[(1.23±0.42) vs (2.36±0.62)]. However, the expressions of Smad3 mRNA [(1.50±0.06) vs (1.55±0.08)], Smad3 protein [(2.85±0.45) vs (2.98±0.47)] and pSmad3 protein [(2.39±0.32) vs (2.51±0.52)] between these two groups showed no difference (P>0.05). Conclusion: Pyridostigmine significantly alleviated cell proliferation and collagen synthesis in HCFs induced by TGF-β, and its mechanism was related to the inhibition of CTGF expression rather than blocking the TGF-β/Smads signaling pathway.

Abstract 511: Regulation of p53 Protein Levels Drives Activation of Cardiac Fibroblasts in Response to Pressure Overload

Heart disease is accompanied by the accumulation of resident cardiac fibroblasts (CF) that subsequently become myofibroblasts and secrete copious amounts of extracellular matrix (ECM), impeding cardiac function and driving the progression of heart failure. Understanding the mechanisms coordinating CF accumulation and myofibroblast activation may reveal novel therapeutic strategies to block pathologic fibrosis. We recently found that s mall pr oline r ich protein 2b (SPRR2B) drives CF proliferation in response to pathological cues by facilitating MDM2-dependent proteasomal degradation of p53. Surprisingly, although Sprr2b gene deletion or targeted mutation of the USP7/MDM2 interacting domain in mice ( Sprr2b-KO and Sprr2b-USP m , respectively) stimulated the expression of p53-dependent cell cycle arrest genes, mutant animals also developed excessive fibrosis in LV pressure overload. The development of fibrosis in Sprr2b mutant mice was traced primarily to a more robust myofibroblast activation response, providing evidence that CF accumulation and myofibroblast activation may be mutually antagonistic. To investigate the contribution p53 to CF accumulation and fibrosis in mouse heart disease models, we deleted floxed p53 alleles specifically in adult CF using the tamoxifen-inducible Tcf21 MerCreMer mouse line (called p53-CKO). Surprisingly, while p53-CKO animals display exaggerated accumulation of Tcf21 + /PDGFRα + CF in response to left ventricle pressure overload, we observed a biphasic physiological response; initially, p53-CKO animals are resistant to systolic functional decline, only developing more severe fibrosis and functional decline than littermate controls at later time points. Time course studies using primary adult mouse CF revealed that p53 positively correlates with myofibroblast activation, while reduction in p53 levels correlates with accelerated cell cycle and the suppression of myofibroblast activation until the subsequent induction of p16/19 - Rb-mediated cell cycle arrest. Taken together, this study offers detailed insight into the transition of CF from a proliferative to an activated state that may accelerate the development of anti-fibrotic strategies.

Calhex231 Alleviates High Glucose-Induced Myocardial Fibrosis via Inhibiting Itch-Ubiquitin Proteasome Pathway in Vitro.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes, and features myocardial fibrosis as its main pathological feature. Calcium sensing receptor (CaSR) is a G protein-coupled receptor, which involves in myocardial fibrosis by regulation of calcium homeostasis. Calhex231, the CaSR inhibitor, is not clear whether it regulates myocardial fibrosis in DCM. In the present study, type 1 diabetic (T1D) rats and primary neonatal rat cardiac fibroblasts were used to observe the role of Calhex231. In vivo experiments showed that in the T1D group, contractile dysfunction and the deposition of collagen I and III were obvious after 12 weeks. In vitro experiments, we found that high glucose (HG) could increase the expression of CaSR, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) collagen I/III, matrix metalloproteinase-2 (MMP-2), MMP9, along with cardiac fibroblast migration and proliferation. We further demonstrated that CaSR activation increased intracellular Ca2+ concentration and upregulated the expression of Itch (atrophin-1 interacting protein 4), which resulted in increasing the ubiquitination levels of Smad7 and upregulating the expression of p-Smad2, p-Smad3. However, treatment with Calhex231 clearly inhibited the above-mentioned changes. Collectively these results suggest that Calhex231 could inhibit Itch-ubiquitin proteasome and TGF-β1/Smads pathways, and then depress the proliferation of cardiac fibroblasts, along with the reduction deposition of collagen, alleviate glucose-induced myocardial fibrosis. Our findings indicate an important new mechanism for myocardial fibrosis, and suggest Calhex231 would be a new therapeutic agent for the treatment of DCM.

Liraglutide protects high-glucose-stimulated fibroblasts by activating the CD36-JNK-AP1 pathway to downregulate P4HA1

BackgroundDiabetic cardiomyopathy (DCM) is a serious complication of diabetes mellitus. It’s known that glucagon-like peptide-1 (GLP-1) and prolyl 4-hydroxylase subunit alpha-1 (P4HA1) have significant effect on cardiovascular function, but their interaction in cardiac fibroblasts (CFs) is still being unraveled. Methods and resultsThe present study demonstrated that glucose promotes CFs proliferation and cardiac fibrosis. Using qRT-PCR, Western blot, CCK-8, EdU, flow cytometry, wound healing and Transwell assays to explore the functions of liraglutide and P4HA1 in high-glucose (HG)-induced CFs, we proved that liraglutide as well as silencing of P4HA1 inhibited cell proliferation, migration and invasion, and promoted cell cycle arrest and apoptosis in HG-induced CFs. In addition, liraglutide downregulated P4HA1 expression, upregulated CD36 and P-JNK expression levels, and enhanced the DNA binding activity of AP-1 on P4HA1. Inhibition of CD36 or p--JNK promoted P4HA1 expression. ConclusionsLiraglutide may down-regulate P4HA1 expression at least partly though CD36-JNK-AP1 pathway, thereby reducing myocardial fibrosis. Therefore, our study provides novel insight into the molecular mechanism and function of liraglutide in HG-mediated CFs.

Inhibitory Effects of Oxymatrine on Transdifferentiation of Neonatal Rat Cardiac Fibroblasts to Myofibroblasts Induced by Aldosterone via Keap1/Nrf2 Signaling Pathways In Vitro.

BackgroundOxymatrine (OMT), a quinolizidine alkaloid derived from the traditional Chinese herb Radix Sophorae flavescentis, has widely reported pharmacological efficacy in treating cardiovascular dysfunction-related diseases. However, the underlying mechanism has been unclear. Here, we investigated the potential inhibitory effects and mechanism of OMT on transdifferentiation of cardiac fibroblast to myofibroblasts induced by aldosterone in vitro.Material/MethodsThe cardiac fibroblasts (CFBs) proliferation and migration capacity were evaluated by MTT assay, cell cycle assay, and scratch analysis, respectively. The protein expression of the Nrf2/Keap1 signal pathway, FN, Collagen I, Collagen III, α-SMA, CTGF, and mineralocorticoid receptor (MR) protein was detected by Western blot analysis. The mRNA expression of Nrf2 was detected by qRT-PCR. Immunofluorescence staining was used to observe the expression of α-SMA protein. Nrf2 siRNA was used to explore the role of Nrf2 in OMT-treated CFBs. GSH, SOD, and MDA levels and hydroxyproline content were measured by colorimetric assay with commercial kits. The DCFH-DA fluorescent probe was used to assess cellular ROS levels.ResultsOMT and Curcumin (an Nrf2 agonist) attenuated aldosterone (ALD)-induced proliferation and migration in CFBs, as well as the fibrosis-associated protein expression levels. Moreover, OMT activated Nrf2 and promoted the nucleus translocation of Nrf2. OMT alleviated the elevated levels of α-SMA, Collagen I, Collagen III, and CTGF, which were abrogated by the Nrf2 siRNA transfection. We also found that OMT decreased oxidative stress levels.ConclusionsOur results confirm that OMT alleviates transdifferentiation of cardiac fibroblasts to myofibroblasts induced by aldosterone via activating the Nrf2/Keap1 pathway in vitro.

Activation of DNA Damage Response and Cellular Senescence in Cardiac Fibroblasts Limit Cardiac Fibrosis After Myocardial Infarction.

Cardiac fibrosis plays an important role in cardiac remodeling after myocardial infarction (MI). The molecular mechanisms that promote cardiac fibrosis after MI are well studied; however, the mechanisms by which the progression of cardiac fibrosis becomes attenuated after MI remain poorly understood. Recent reports show the role of cellular senescence in limiting tissue fibrosis. In the present study, we tested whether cellular senescence of cardiac fibroblasts (CFs) plays a role in attenuating the progression of cardiac fibrosis after MI. We found that the number of γH2AX-positive CFs increased up to day 7, whereas the number of proliferating CFs peaked at day 4 after MI. Senescent CFs were also observed at day 7, suggesting that attenuation of CF proliferation occurred simultaneously with the activation of the DNA damage response (DDR) system and the appearance of senescent CFs. We next cultured senescent CFs with non-senescent CFs and showed that senescent CFs suppressed proliferation of the surrounding non-senescent CFs in a juxtacrine manner. We also found that the blockade of DDR by Atm gene deletion sustained the proliferation of CFs and exacerbated the cardiac fibrosis at the early stage after MI. Our results indicate the role of DDR activation and cellular senescence in limiting cardiac fibrosis after MI. Regulation of cellular senescence in CFs may become one of the therapeutic strategies for preventing cardiac remodeling after MI.

LncRNA PCFL promotes cardiac fibrosis via miR-378/GRB2 pathway following myocardial infarction

Long noncoding RNAs (lncRNAs) are a class of novel molecular regulators in cardiac development and diseases. However, the role of specific lncRNAs in cardiac fibrosis remains to be fully explored. The aim of the present study was to investigate the effects and underlying mechanisms of lncRNA PCFL (pro-cardiac fibrotic lncRNA) on cardiac fibrosis after myocardial infarction (MI).Cardiac fibroblasts (CFs) with gain and loss of function of PCFL and mice with global knockout or overexpression of PCFL were used to explore the effects of PCFL on cardiac fibrosis. The data showed that PCFL was significantly increased in hearts of mice subjected to MI and CFs treated with transforming growth factor-β1 (TGF-β1). Overexpression of PCFL promoted collagen production and CF proliferation, while silencing PCFL exhibited the opposite effects. Compared with wild type MI mice, heterozygous knockout of PCFL (PCFL+/−) in mice significantly improved heart function and reduced cardiac fibrosis after MI. While overexpression of PCFL impaired cardiac function and aggravated MI-induced cardiac fibrosis. The mechanistic data demonstrated that PCFL functioned as a sponge of miR-378. Luciferase reporter assay confirmed the interaction of PCFL with miR-378. MiR-378 inhibited collagen production by suppressing its target gene, GRB2 (growth factor receptor bound protein 2). Knockdown of PCFL led to an increase of miR-378. Silencing of miR-378 reserved the inhibitory effects of PCFL knockdown on collagen production, cell proliferation and GRB2 expression. In conclusion, the study identifies a novel pro-fibrotic lncRNA, PCFL, and the mechanism involves the direct interaction of PCFL with miR-378, which in turn relieves the inhibition effect of miR-378 on GRB2 and promotes cardiac fibrosis.

Calcium‑sensing receptor promotes high glucose‑induced myocardial fibrosis via upregulation of the TGF‑β1/Smads pathway in cardiac fibroblasts.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes and myocardial fibrosis is its major pathological feature. Calcium-sensing receptor (CaSR) is a G protein-coupled receptor and participates in the regulation of calcium homeostasis; it is implicated in a range of diseases, including myocardial ischemia/reperfusion injury, myocardial infarction and pulmonary hypertension. However, whether CaSR is associated with myocardial fibrosis in DCM has remained elusive. In the present study, type 1 diabetic (T1D) rats and primary neonatal rat cardiac fibroblasts (CFs) were used to observe changes in CaSR to assess its potential as an indicator of myocardial fibrosis. The in vivo experiments revealed that in the T1D and CaSR agonist (R568) groups, evident collagen (Col)-I and -III deposition was present after 12 weeks. Furthermore, the in vitro experiment indicated that the levels of transforming growth factor (TGF)-β1, phosphorylated (p-) protein kinase C, p-p38, p-Smad2, TβRI, TβRII, along with the intracellular Ca2+ levels and the content of TGF-β1 in the culture medium were significantly increased in a high-glucose (HG) group and an R568-treated group. Treatment with the CaSR inhibitor Calhex231 significantly inhibited the abovementioned changes. Collectively, the results indicated that the increase of CaSR expression in CFs may induce intracellular Ca2+ increases and the activation of TGF-β1/Smads, and enhance the proliferation of CFs, along with the excessive deposition of Col, resulting in myocardial fibrosis. The present results indicate an important novel mechanism for HG-induced myocardial fibrosis and suggest that CaSR may be a promising potential therapeutic target for DCM.

The protective effects of Berberine and Hesperidin on inflammatory factor-stimulating cardiac fibroblasts.

The previous work has shown that Berberine and Hesperidin have beneficial effects on cardiovascular diseases. However, the underlying mechanisms remain unknown. This study aimed to investigate the effect of Berberine and Hesperidin on inflammatory cytokine secretion, proliferation, differentiation, and collagen synthesis of cardiac fibroblasts stimulated by the transforming growth factor-β1 (TGF-β1), and the potential of these drugs to regulate the Notch1 signaling pathway. Neonatal rat primary cardiac fibroblasts were stimulated with 5 ng/mL TGF-β1 as model (TGF) group. In the Berberine (TGF+B) group cells were given TGF-β1, along with 1.25/2.5/5/10 mg/L Berberine, while the Hesperidin (TGF+H) group was treated with TGF-β1 and 12.5/25/50/100 µmmol/L Hesperidin. Cellular proliferation, differentiation, and collagen synthesis were evaluated. The role of the Notch1 signaling pathway in the protective effects of Berberine and Hesperidin was analyzed by using γ-secretase inhibitor (DAPT) to block the Notch1 pathway. 5/10 mg/L Berberine intervention could noticeably decrease both TGF-β1 and IL-1β levels, 25/50/100 µmol/L Hesperidin could reduce IL-1β secretion from TGF-β1 stimulated cardiac fibroblasts. Both Berberine and Hesperidin decreased the expression of α-SMA and cell viability in a concentration-dependent manner; however, the apoptosis of cardiac fibroblasts was not influenced. 10 mg/L Berberine or at least 50 µmol/L Hesperidin could noticeably decrease MMP-1 expression, and at least 5 mg/L Berberine or 100 µmol/L Hesperidin could markedly reduce MMP-9 expression. Using DAPT to block Notch1 signaling could reverse the protective effects of Berberine and Hesperidin. Berberine and Hesperidin can reduce the secretion of inflammatory cytokines, differentiation, and proliferation, and increase the collagen synthesis of cardiac fibroblasts stimulated by TGF-β1 via the Notch1 signaling pathway.